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1.
GTP cyclohydrolase I (GCYH-I) is an essential Zn2+-dependent enzyme that catalyzes the first step of the de novo folate biosynthetic pathway in bacteria and plants, the 7-deazapurine biosynthetic pathway in Bacteria and Archaea, and the biopterin pathway in mammals. We recently reported the discovery of a new prokaryotic-specific GCYH-I (GCYH-IB) that displays no sequence identity to the canonical enzyme and is present in ∼25% of bacteria, the majority of which lack the canonical GCYH-I (renamed GCYH-IA). Genomic and genetic analyses indicate that in those organisms possessing both enzymes, e.g., Bacillus subtilis, GCYH-IA and -IB are functionally redundant, but differentially expressed. Whereas GCYH-IA is constitutively expressed, GCYH-IB is expressed only under Zn2+-limiting conditions. These observations are consistent with the hypothesis that GCYH-IB functions to allow folate biosynthesis during Zn2+ starvation. Here, we present biochemical and structural data showing that bacterial GCYH-IB, like GCYH-IA, belongs to the tunneling-fold (T-fold) superfamily. However, the GCYH-IA and -IB enzymes exhibit significant differences in global structure and active-site architecture. While GCYH-IA is a unimodular, homodecameric, Zn2+-dependent enzyme, GCYH-IB is a bimodular, homotetrameric enzyme activated by a variety of divalent cations. The structure of GCYH-IB and the broad metal dependence exhibited by this enzyme further underscore the mechanistic plasticity that is emerging for the T-fold superfamily. Notably, while humans possess the canonical GCYH-IA enzyme, many clinically important human pathogens possess only the GCYH-IB enzyme, suggesting that this enzyme is a potential new molecular target for antibacterial development.The Zn2+-dependent enzyme GTP cyclohydrolase I (GCYH-I; EC 3.5.4.16) is the first enzyme of the de novo tetrahydrofolate (THF) biosynthesis pathway (Fig. (Fig.1)1) (38). THF is an essential cofactor in one-carbon transfer reactions in the synthesis of purines, thymidylate, pantothenate, glycine, serine, and methionine in all kingdoms of life (38), and formylmethionyl-tRNA in bacteria (7). Recently, it has also been shown that GCYH-I is required for the biosynthesis of the 7-deazaguanosine-modified tRNA nucleosides queuosine and archaeosine produced in Bacteria and Archaea (44), respectively, as well as the 7-deazaadenosine metabolites produced in some Streptomyces species (33). GCYH-I is encoded in Escherichia coli by the folE gene (28) and catalyzes the conversion of GTP to 7,8-dihydroneopterin triphosphate (55), a complex reaction that begins with hydrolytic opening of the purine ring at C-8 of GTP to generate an N-formyl intermediate, followed by deformylation and subsequent rearrangement and cyclization of the ribosyl moiety to generate the pterin ring in THF (Fig. (Fig.1).1). Notably, the enzyme is dependent on an essential active-site Zn2+ that serves to activate a water molecule for nucleophilic attack at C-8 in the first step of the reaction (2).Open in a separate windowFIG. 1.Reaction catalyzed by GCYH-I, and metabolic fate of 7,8-dihydroneopterin triphosphate.A homologous GCYH-I is found in mammals and other higher eukaryotes, where it catalyzes the first step of the biopterin (BH4) pathway (Fig. (Fig.1),1), an essential cofactor in the biosynthesis of tyrosine and neurotransmitters, such as serotonin and l-3,4-dihydroxyphenylalanine (3, 52). Recently, a distinct class of GCYH-I enzymes, GCYH-IB (encoded by the folE2 gene), was discovered in microbes (26% of sequenced Bacteria and most Archaea) (12), including several clinically important human pathogens, e.g., Neisseria and Staphylococcus species. Notably, GCYH-IB is absent in eukaryotes.The distribution of folE (gene product renamed GCYH-IA) and folE2 (GCYH-IB) in bacteria is diverse (12). The majority of organisms possess either a folE (65%; e.g., Escherichia coli) or a folE2 (14%; e.g., Neisseria gonorrhoeae) gene. A significant number (12%; e.g., B. subtilis) possess both genes (a subset of 50 bacterial species is shown in Table Table1),1), and 9% lack both genes, although members of the latter group are mainly intracellular or symbiotic bacteria that rely on external sources of folate. The majority of Archaea possess only a folE2 gene, and the encoded GCYH-IB appears to be necessary only for the biosynthesis of the modified tRNA nucleoside archaeosine (44) except in the few halophilic Archaea that are known to synthesize folates, such as Haloferax volcanii, where GCYH-IB is involved in both archaeosine and folate formation (13, 44).

TABLE 1.

Distribution and candidate Zur-dependent regulation of alternative GCYH-I genes in bacteriaa
OrganismcPresence of:
folEfolE2
Enterobacteria
    Escherichia coli+
    Salmonella typhimurium+
    Yersinia pestis+
    Klebsiella pneumoniaeb++a
    Serratia marcescens++a
    Erwinia carotovora+
    Photorhabdus luminescens+
    Proteus mirabilis+
Gammaproteobacteria
    Vibrio cholerae+
    Acinetobacter sp. strain ADP1++a
    Pseudomonas aeruginosa++a
    Pseudomonas entomophila L48++a
    Pseudomonas fluorescens Pf-5++a
    Pseudomonas syringae++a
    Pseudomonas putida++a
    Hahella chejuensis KCTC 2396++a
    Chromohalobacter salexigens DSM 3043++a
    Methylococcus capsulatus++a
    Xanthomonas axonopodis++a
    Xanthomonas campestris++a
    Xylella fastidiosa++a
    Idiomarina loihiensis+
    Colwellia psychrerythraea++
    Pseudoalteromonas atlantica T6c++a
    Pseudoalteromonas haloplanktis TAC125++
    Alteromonas macleodi+
    Nitrosococcus oceani++
    Legionella pneumophila+
    Francisella tularensis+
Betaproteobacteria
    Chromobacterium violaceum+
    Neisseria gonorrhoeae+
    Burkholderia cepacia R18194++
    Burkholderia cenocepacia AU 1054++
    Burkholderia xenovorans+
    Burkholderia mallei+
    Bordetella pertussis+
    Ralstonia eutropha JMP134+
    Ralstonia metallidurans++
    Ralstonia solanacearum+
    Methylobacillus flagellatus+
    Nitrosomonas europaea+
    Azoarcus sp.++
Bacilli/Clostridia
    Bacillus subtilisd++
    Bacillus licheniformis++
    Bacillus cereus+
    Bacillus halodurans++
    Bacillus clausii+
    Geobacillus kaustophilus+
    Oceanobacillus iheyensis+
    Staphylococcus aureus+
Open in a separate windowaGenes that are preceded by candidate Zur binding sites.bZur-regulated cluster is on the virulence plasmid pLVPK.cExamples of organisms with no folE genes are in boldface type.dZn-dependent regulation of B. subtilis folE2 by Zur was experimentally verified (17).Expression of the Bacillus subtilis folE2 gene, yciA, is controlled by the Zn2+-dependent Zur repressor and is upregulated under Zn2+-limiting conditions (17). This led us to propose that the GCYH-IB family utilizes a metal other than Zn2+ to allow growth in Zn2+-limiting environments, a hypothesis strengthened by the observation that an archaeal ortholog from Methanocaldococcus jannaschii has recently been shown to be Fe2+ dependent (22). To test this hypothesis, we investigated the physiological role of GCYH-IB in B. subtilis, an organism that contains both isozymes, as well as the metal dependence of B. subtilis GCYH-IB in vitro. To gain a structural understanding of the metal dependence of GCYH-IB, we determined high-resolution crystal structures of Zn2+- and Mn2+-bound forms of the N. gonorrhoeae ortholog. Notably, although the GCYH-IA and -IB enzymes belong to the tunneling-fold (T-fold) superfamily, there are significant differences in their global and active-site architecture. These studies shed light on the physiological significance of the alternative folate biosynthesis isozymes in bacteria exposed to various metal environments, and offer a structural understanding of the differential metal dependence of GCYH-IA and -IB.  相似文献   

2.
The magnitude and character of adenovirus serotype 5 (Ad5)-specific T cells were determined in volunteers with and without preexisting neutralizing antibodies (NAs) to Ad5 who received replication-defective Ad5 (rAd5)-based human immunodeficiency virus vaccines. There was no correlation between T-cell responses and NAs to Ad5. There was no increase in magnitude or activation state of Ad5-specific CD4+ T cells at time points where antibodies to Ad5 and T-cell responses to the recombinant gene products could be measured. These data indicate that rAd5-based vaccines containing deletions in the E1, E3, and E4 regions do not induce appreciable expansion of vector-specific CD4+ T cells.Replication-defective adenoviruses (rAd) have been engineered to provide high levels of expression of foreign inserts with minimum expression of adenovirus proteins, making them excellent candidates for vaccine and gene therapy applications (3, 16). Despite promising immunogenicity, a prophylactic vaccine trial of a serotype 5 rAd (rAd5) vector expressing human immunodeficiency virus (HIV) Gag, Pol, and Nef genes (Step trial) was recently halted due to an increase in HIV infections among volunteers who had preexisting neutralizing antibodies (NAs) to Ad5 (7). This finding raises the possibility that the presence of Ad5-specific T-cell responses (specifically CD4+ T-cell responses) in subjects with preexisting Ad5 NAs could be boosted by rAd5 vaccines, thereby providing an expanded susceptible target cell population that could be more easily infected by HIV. If this mechanism were operative, it would have broad implications for the future use of rAd viruses, and indeed other virus vectors, as vaccines or therapeutic agents within HIV-susceptible populations (2, 12, 15). We therefore measured the frequency, magnitude, and activation status of rAd5-specific T cells in HIV-uninfected volunteers who had received rAd5-based HIV vaccines in the presence or absence of preexisting NAs to Ad5.We studied 31 volunteers enrolled in two NIAID Institutional Review Board-approved phase I clinical trials of rAd5-based HIV vaccines. VRC 006 was a dose escalation study evaluating a single inoculation of a rAd5 mixture expressing EnvA, EnvB, EnvC, and fusion protein Gag/PolB at 109, 1010, and 1011 total particle units (10). VRC 008 evaluated DNA priming by needle and syringe or Biojector, followed by rAd5 boosting. Both studies enrolled healthy, HIV-uninfected adults; used the same rAd5 products; and evaluated immunogenicity on the day of and 4 weeks after rAd5 immunization. Both of these trials involved rAd5 products that contained deletions in the E1, E3, and E4 regions (8, 10).NAs to Ad5 were determined for all volunteers as previously described (19). A 90% NA titer of 12 or more was considered positive and taken as evidence of preexisting humoral immunity to Ad5. Volunteers were chosen for assessment of Ad5-specific T-cell responses based upon the availability of peripheral blood mononuclear cell samples at key time points and the presence or absence of preexisting NAs to Ad5. Only volunteers who received the vaccine (not the placebo) were included. Table Table11 lists the volunteers who were tested for Ad5-specific T-cell responses and their NA titers to Ad5 before and after rAd5 vaccination. All volunteers, except for one (volunteer 12) who had a less-than-maximum NA titer to Ad5 before vaccination, had an increase in titer by 4 weeks after vaccination, indicating the successful “take” of the rAd5-based vaccine. There was no correlation between rAd5 dose and increase in Ad5 NA titer.

TABLE 1.

Ad5 serostatus before and after vaccination
VolunteerPrior DNA immunizationrAd5 dose (PUa)Ad5 NA titer
PrevaccinePostvaccine
1No1011<12739
2No1011<12834
3No1011<124,787
4No1011<12806
5No1011<121,033
6No1010<12130
7No1010<121,354
8Yes1010<121,387
9Yes1010<12575
10Yes1010<12170
11Yes1010<12>8,748
12Yes1010<12<12
13No101130>8,748
14No10946>8,748
15No10970328
16No1010176>8,748
17No10104786,198
18No1092,472>8,748
19No1093,502>8,748
20No10104,820>8,748
21No1095,078>8,748
22No10116,162>8,748
23No109>8,748>8,748
24No1011>8,748>8,748
25Yes1010643>8,748
26Yes1010942>8,748
27Yes10101,510>8,748
28Yes10101,611>8,748
29Yes10102,934>8,748
30Yes1010>8,748>8,748
31Yes1010>8,748>8,748
Open in a separate windowaPU, particle units.HIV-specific T-cell responses were measured by multiparameter flow cytometry after 6 h of stimulation with peptides (15-mers overlapping by 11) corresponding to the HIV EnvA protein (one of the vaccine inserts expressed in the Ad5 vectors), as previously described (13). Overlapping peptides corresponding to the major Ad5 surface protein (hexon), the Ad5 early regulatory protein (E2A), and Ad5 ORF1, -2, and -3 proteins were used to assess Ad5-specific T-cell responses, and additional markers of cell viability (ViViD), T-cell memory (CD45RO and CD27), and activation/division (CCR5, CD38, HLA-DR, and Ki67) were added to the panel for these assessments. Antibodies and fluorochromes used in this panel were CCR5-Cy7-phycoerythrin (PE), CD38-allophycocyanin, Ki67-fluorescein isothiocyanate, and CD3-Cy7-allophycocyanin, all from BD PharMingen; CD8-Cy55-PE from BD Biosciences; CD27-Cy5-PE and CD45RO-Texas Red-PE, both from Beckman Coulter; CD4-Cy5.5-PE from Caltag; CD14- and CD19-PacificBlue, CD57-QDot545, and HLA-DR-Alexa680, conjugated according to standard protocols [http://drmr.com/abcon/index.html]); gamma interferon-PE and interleukin-2-PE from BD Biosciences; and a violet amine dye from Invitrogen. Cells were analyzed on an LSRII instrument (Becton Dickinson), and data analysis was performed using FlowJo, version 8.1.1 (TreeStar). The gating strategy is shown in Fig. Fig.1A1A.Open in a separate windowFIG. 1.CD4+ and CD8+ T-cell responses to Ad5. (A) Gating tree used to determine antigen-specific T-cell frequencies. Single CD3+ ViViD CD14 CD19 cells were gated on CD4 or CD8 cells. Naïve CD27+ CD45RO cells were gated out, and the frequency of cells expressing gamma interferon (IFNg) and/or interleukin-2 (IL2) was determined. FSC-A, forward scatter area; FSC-H, forward scatter height; SSC-A, side scatter area. (B) Frequencies of CD4+ and CD8+ T-cell responses after stimulation with Ad5 hexon or E2A peptides were plotted against the prevaccination Ad5 NA titer. The prevaccine T-cell response was used. (C) Frequencies of CD4+ and CD8+ T-cell responses to Ad5 hexon, E2A, and HIV EnvA before and 4 weeks after rAd5 vaccination are shown for subjects with (Ad5 NA titer of >12) and without (Ad5 NA titer of <12) preexisting NAs to Ad5. Boxed areas represent interquartile ranges, and horizontal lines represent medians.Previously, we had found no T-cell responses to Ad5 ORF1, -2, or -3, so data from these antigen stimulations are not shown. As shown in Fig. Fig.1B,1B, T-cell responses to Ad5 hexon and E2A were detected, but there was no association between the NA response to Ad5 and the T-cell responses to these Ad5 proteins. Volunteers with an absence of NAs to Ad5 often had very strong CD4+ and CD8+ T-cell responses to Ad5 proteins. This probably reflects the degree of protein sequence homology between different adenovirus serotypes (11) and suggests that T-cell responses to adenoviruses may be significantly cross-reactive, while NAs are serotype specific. It also indicates that the NA response to Ad5 cannot be used as a surrogate for either a CD4+ or a CD8+ T-cell response to that adenovirus serotype.We next asked whether Ad5-specific T-cell responses were boosted by a single rAd5 vaccination in subjects with or without preexisting NAs to Ad5. At the time point 4 weeks after vaccination, there was clear evidence of boosting of the insert-specific (EnvA) CD4+ and CD8+ T-cell responses in volunteers with and without preexisting NAs to Ad5 (Fig. (Fig.1C).1C). The results of the Ad5-specific responses were consistent across volunteers who had received prior DNA immunization (VRC 008) and those who had not (VRC 006), so the results are combined in Fig. Fig.1C1C and show no increase in Ad5 hexon- or E2A-specific CD4+ T-cell responses after rAd5 immunization irrespective of Ad5 NA status. There was evidence of an increase in the CD8+ T-cell response to Ad5 hexon (P = 0.004 by paired t test), but not that to E2A, after rAd5 vaccination. These results, while showing evidence of adenovirus-specific CD8+ T-cell boosting by rAd5 vaccination, do not indicate an expansion of Ad5-specific CD4+ T cells that could serve as a substrate for HIV infection in subjects with or without NAs to Ad5.Having failed to demonstrate an expansion of Ad5-specific CD4+ T cells after vaccination, we assessed whether the activation profile of the unexpanded Ad5-specific CD4+ T cells was changed by vaccination. The gating tree is shown in Fig. Fig.2A.2A. Ad5 hexon- and E2A-specific CD4+ T cells expressed activation markers CCR5, CD38, and HLA-DR and a marker of recent cell division, Ki67, more frequently than did total memory CD4+ T cells (Fig. (Fig.2B).2B). However, none of these markers were significantly increased on total or Ad5-specific CD4+ T cells after vaccination in volunteers with or without preexisting NAs to Ad5.Open in a separate windowFIG. 2.Vaccine-induced activation of Ad5-specific CD4+ T cells. (A) Total CD4+ memory cells or Ad5-specific CD4+ memory cells (as gated in Fig. Fig.1A)1A) were further defined by expression of Ki67, CD38, CCR5, and HLA-DR. (B) Percentages of Ad5 hexon-specific cells, E2A-specific cells, or total memory CD4+ T cells that express CCR5, CD38, HLA-DR, or Ki67 before and 4 weeks after rAd5 vaccination are shown for subjects with (Ad5 NA titer of >12) (left) and without (Ad5 NA titer of >12) (right) preexisting NAs to Ad5. The phenotype was assessed only for those responders for whom at least 10 cytokine-positive events were counted. None of the comparisons of pre- and postvaccination marker expression were significant at a P value of 0.02 by paired t test. Boxed areas represent interquartile ranges, and horizontal lines represent medians.Expansion of Ad5-specific T cells after rAd5-based vaccination or gene therapy has been reported by others (14, 20, 21). Those studies evaluated Ad5-specific responses to rAd5 vectors with only the adenovirus E1 gene deleted (as used in the Step trial vaccines). The vectors used here contained deletions of the adenovirus E1, E3, and E4 genes (8, 10). While adenovirus gene deletions can render the vectors replication defective (6, 9), they do not necessarily completely shut off all adenovirus protein expression (20, 21). To demonstrate the importance of E4 deletions in limiting expression of adenovirus gene products, we measured the level of adenovirus protein synthesis in infected A549 cells as previously described (1, 4, 5). Cells were infected with adenovirus vectors with E1 and E3 deletions or with E1, E3, and E4 deletions at the same multiplicity of infection (10 focus-forming units per cell). At 24 h postinfection, [35S]methionine was added for 1 h. Levels of total and adenovirus protein synthesis in the infected and mock-infected cells were compared (Fig. (Fig.3).3). Adenovirus early protein single-stranded DNA binding protein, as well as late gene products hexon, penton, and fiber, was immunoprecipitated, fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and resolved by autoradiography. The results show that the amount of newly synthesized adenovirus proteins in cells infected with adenovirus with E1, E3, and E4 deletions is significantly lower than that for an adenovirus vector with E1 and E3 deletions. Therefore, our inability to detect a vaccine-induced increase in the frequency and character of the Ad5-specific T-cell response could relate to the very low levels of adenovirus proteins that were probably expressed in vivo by the rAd5 vectors with multiple deletions.Open in a separate windowFIG. 3.Ad5 protein expression in vitro after infection with different Ad5 vectors. A549 cells were infected with adenovirus vectors with E1 and E3 deletions or with E1, E3, and E4 deletions and [35S]methionine labeled, and levels of total and adenovirus protein synthesis in the infected and mock-infected cells were compared after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Markers for the adenovirus early protein single-stranded DNA binding protein (DBP) and capsid proteins hexon, penton base, and fiber are shown.We were therefore unable to demonstrate (i) that Ad5-specific CD4+ T cells were restricted to subjects with preexisting Ad5 NAs, (ii) that rAd5 vaccination expanded or increased the activation of Ad5-specific CD4+ T cells, or (iii) that there was a substantial effect on the magnitude or character of the Ad5-specific CD4+ T-cell response to vaccination based upon preexisting NAs to Ad5. While the kinetics of Ad5-specific T-cell responses after rAd5-based vaccination are not known, it is clear that insert-specific responses are increased at 4 weeks after vaccination and subsequently contract (10). It is therefore reasonable to assume that if Ad5-specific responses were similarly affected, they would be detected at the 4-week-postvaccination time point.It is possible that rAd5 vaccines expand a preexisting mucosal T-cell response to Ad5 that is not reflected within the blood. While we do not have mucosal samples from our vaccine volunteers to directly address this possibility, it is likely that expansion of a mucosal response would be reflected to some degree within the blood.The mechanism underlying the increase in HIV infections in vaccinees with NAs to Ad5 in the Step trial is yet to be determined (2, 7, 12, 15, 17). Confounding factors and alternative hypotheses have recently been proposed to account for the increased acquisition (7, 12, 15, 18). Until there is a better understanding of the processes involved, future studies of rAd5-based products should proceed with appropriate safety considerations and monitoring of adenovirus-specific responses. In addition, the use of vaccine regimens involving single injections of vectors with multiple deletions may help mitigate risk.  相似文献   

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Short-chain alcohol dehydrogenase, encoded by the gene Tsib_0319 from the hyperthermophilic archaeon Thermococcus sibiricus, was expressed in Escherichia coli, purified and characterized as an NADPH-dependent enantioselective oxidoreductase with broad substrate specificity. The enzyme exhibits extremely high thermophilicity, thermostability, and tolerance to organic solvents and salts.Alcohol dehydrogenases (ADHs; EC 1.1.1.1.) catalyze the interconversion of alcohols to their corresponding aldehydes or ketones by using different redox-mediating cofactors. NAD(P)-dependent ADHs, due to their broad substrate specificity and enantioselectivity, have attracted particular attention as catalysts in industrial processes (5). However, mesophilic ADHs are unstable at high temperatures, sensitive to organic solvents, and often lose activity during immobilization. In this relation, there is a considerable interest in ADHs from extremophilic microorganisms; among them, Archaea are of great interest. The representatives of all groups of NAD(P)-dependent ADHs have been detected in genomes of Archaea (11, 12); however, only a few enzymes have been characterized, and the great majority of them belong to medium-chain (3, 4, 14, 16, 19) or long-chain iron-activated ADHs (1, 8, 9). Up to now, a single short-chain archaeal ADH from Pyrococcus furiosus (10, 18) and only one archaeal aldo-keto reductase also from P. furiosus (11) have been characterized.Thermococcus sibiricus is a hyperthermophilic anaerobic archaeon isolated from a high-temperature oil reservoir capable of growth on complex organic substrates (15). The complete genome sequence of T. sibiricus has been recently determined and annotated (13). Several ADHs are encoded by the T. sibiricus genome, including three short-chain ADHs (Tsib_0319, Tsib_0703, and Tsib_1998) (13). In this report, we describe the cloning and expression of the Tsib_0319 gene from T. sibiricus and the purification and the biochemical characterization of its product, the thermostable short-chain ADH (TsAdh319).The Tsib_0319 gene encodes a protein with a size of 234 amino acids and the calculated molecular mass of 26.2 kDa. TsAdh319 has an 85% degree of sequence identity with short-chain ADH from P. furiosus (AdhA; PF_0074) (18). Besides AdhA, close homologs of TsAdh319 were found among different bacterial ADHs, but not archaeal ADHs. The gene flanked by the XhoI and BamHI sites was PCR amplified using two primers (sense primer, 5′-GTTCTCGAGATGAAGGTTGCTGTGATAACAGGG-3′, and antisense primer, 5′-GCTGGATCCTCAGTATTCTGGTCTCTGGTAGACGG-3′) and cloned into the pET-15b vector. TsAdh319 was overexpressed, with an N-terminal His6 tag in Escherichia coli Rosetta-gami (DE3) and purified to homogeneity by metallochelating chromatography (Hi-Trap chelating HP column; GE Healthcare) followed by gel filtration on Superdex 200 10/300 GL column (GE Healthcare) equilibrated in 50 mM Tris-HCl (pH 7.5) with 200 mM NaCl. The homogeneity and the correspondence to the calculated molecular mass of 28.7 kDa were verified by SDS-PAGE (7). The molecular mass of native TsAdh319 was 56 to 60 kDa, which confirmed the dimeric structure in solution.The standard ADH activity measurement was made spectrophotometrically at the optimal pH by following either the reduction of NADP (in 50 mM Gly-NaOH buffer; pH 10.5) or the oxidation of NADPH (in 0.1 M sodium phosphate buffer; pH 7.5) at 340 nm at 60°C. The enzyme exhibited a strong preference for NADP(H) and broad substrate specificity (Table (Table1).1). The highest oxidation rates were found with pentoses d-arabinose (2.0 U mg−1) and d-xylose (2.46 U mg−1), and the highest reduction rates were found with dimethylglyoxal (5.9 U mg−1) and pyruvaldehyde (2.2 U mg−1). The enzyme did not reduce sugars which were good substrates for the oxidation reaction. The kinetic parameters of TsAdh319 determined for the preferred substrates are shown in Table Table2.2. The enantioselectivity of the enzyme was estimated by measuring the conversion rates of 2-butanol enantiomers. TsAdh319 showed an evident preference, >2-fold, for (S)-2-butanol over (RS)-2-butanol. The enzyme stereoselectivity is confirmed by the preferred oxidation of d-arabinose over l-arabinose (Table (Table1).1). The fact that TsAdh319 is metal independent was supported by the absence of a significant effect of TsAdh319 preincubation with 10 mM Me2+ for 30 min before measuring the activity in the presence of 1 mM Me2+ or EDTA (Table (Table3).3). TsAdh319 also exhibited a halophilic property, so the enzyme activity increased in the presence of NaCl and KCl and the activation was maintained even at concentration of 4 M and 3 M, respectively (Table (Table33).

TABLE 1.

Substrate specificity of TsAdh319
SubstrateaRelative activity (%)
Oxidation reactionb
    Methanol0
    2-Methoxyethanol0
    Ethanol36
    1-Butanol80
    2-Propanol100
    (RS)-(±)-2-Butanol86
    (S)-(+)-2-Butanol196
    2-Pentanol67
    1-Phenylmethanol180
    1.3-Butanediol91
    Ethyleneglycol0
    Glycerol16
    d-Arabinose*200
    l-Arabinose*17
    d-Xylose*246
    d-Ribose*35
    d-Glucose*146
    d-Mannose*48
    d-Galactose*0
    Cellobiose*71
Reduction reactionc
    Pyruvaldehyde100
    Dimethylglyoxal270
    Glyoxylic acid36
    Acetone0
    Cyclopentanone0
    Cyclohexanone4
    3-Methyl-2-pentanone*13
    d-Arabinose*0
    d-Xylose*0
    d-Glucose*0
    Cellobiose*0
Open in a separate windowaSubstrates were present in 250 mM or 50 mM (*) concentrations.bRelative rates, measured under standard conditions, were calculated by defining the activity for 2-propanol as 100%, which corresponds to 1.0 U mg−1. Data are averages from triplicate experiments.cRelative rates, measured under standard conditions, were calculated by defining the activity for pyruvaldehyde as 100%, which corresponds to 2.2 U mg−1. Data are averages from triplicate experiments.

TABLE 2.

Apparent Km and Vmax values for TsAdh319
Coenzyme or substrateApparent Km (mM)Vmax (U mg−1)kcat (s−1)
NADPa0.022 ± 0.0020.94 ± 0.020.45 ± 0.01
NADPHb0.020 ± 0.0033.16 ± 0.111.51 ± 0.05
2-Propanol168 ± 291.10 ± 0.090.53 ± 0.04
d-Xylose54.4 ± 7.41.47 ± 0.090.70 ± 0.04
Pyruvaldehyde17.75 ± 3.384.26 ± 0.402.04 ± 0.19
Open in a separate windowaActivity was measured under standard conditions with 2-propanol. Data are averages from triplicate experiments.bActivity was measured under standard conditions with pyruvaldehyde. Data are averages from triplicate experiments.

TABLE 3.

Effect of various ions and EDTA on TsAdh319a
CompoundConcn (mM)Relative activity (%)
None0100
NaCl400206
600227
4,000230
KCl600147
2,000200
3,000194
MgCl21078
CoCl210105
NiSO410100
ZnSO41079
FeSO41074
EDTA1100
580
Open in a separate windowaThe activity was measured under standard conditions with 2-propanol; relative rates were calculated by defining the activity without salts as 100%, which corresponds to 0.9 U mg−1. Data are averages from duplicate experiments.The most essential distinctions of TsAdh319 are the thermophilicity and high thermostability of the enzyme. The optimum temperature for the 2-propanol oxidation catalyzed by TsAdh319 was not achieved. The initial reaction rate of oxidation increased up to 100°C (Fig. (Fig.1).1). The Arrhenius plot is a straight line, typical of a single rate-limited thermally activated process, but there is no obvious transition point due to the temperature-dependent conformational changes of the protein molecule. The activation energy for the oxidation of 2-propanol was estimated at 84.0 ± 5.8 kJ·mol−1. The thermostability of TsAdh319 was calculated from residual TsAdh319 activity after preincubation of 0.4 mg/ml enzyme solution in 50 mM Tris-HCl buffer (pH 7.5) containing 200 mM NaCl at 70, 80, 90, or 100°C. The preincubation at 70°C or 80°C for 1.5 h did not cause a decrease in the TsAdh319 activity, but provoked slight activation. The residual TsAdh319 activities began to decrease after 2 h of preincubation at 70°C or 80°C and were 10% and 15% down from the control, respectively. The determined half-life values of TsAdh319 were 2 h at 90°C and 1 h at 100°C.Open in a separate windowFIG. 1.Temperature dependence of the initial rate of the 2-propanol reduction by TsAdh319. The reaction was initiated by enzyme addition to a prewarmed 2-propanol-NADP mixture. The inset shows the Arrhenius plot of the same data.Protein thermostability often correlates with such important biotechnological properties as increased solvent tolerance (2). We tested the influence of organic solvents at a high concentration (50% [vol/vol]) on TsAdh319 by using either preincubation of the enzyme at a concentration of 0.2 mg/ml with solvents for 4 h at 55°C or solvent addition into the reaction mixture to distinguish the effect of solvent on the protein stability and on the enzyme activity. TsAdh319 showed significant solvent tolerance in both cases (Table (Table4),4), and the effects of solvents could be modulated by salts, acting apparently as molecular lyoprotectants (17). Furthermore, TsAdh319 maintained 57% of its activity in 25% (vol/vol) 2-propanol, which could be used as the cosubstrate in cofactor regeneration (6).

TABLE 4.

Influence of various solvents on TsAdh319 activitya
SolventRelative activity (%)bRelative activity (%)c
Buffer without NaClBuffer with 600 mM NaCl
None100100100
DMSOd98040
DMFAe1011341
Methanol98259
Acetonitrile9500
Ethyl acetate470*33*
Chloroform10579*81*
n-Hexane10560*118*
n-Decane3691*107*
Open in a separate windowaThe activity measured at the standard condition with 2-propanol as a substrate. Data are averages from triplicate experiments.bPreincubation for 4 h at 55°C in the presence of 50% (vol/vol) of solvent prior the activity assay.cWithout preincubation, solvent addition to the reaction mixture up to 50% (vol/vol) or using the buffer saturated by a solvent (*).dDMSO, dimethyl sulfoxide.eDMFA, dimethylformamide.From all the aforesaid we may suppose TsAdh319 or its improved variant to be interesting both for the investigation of structural features of protein tolerance and for biotechnological applications.  相似文献   

10.
11.
Twelve cluster groups of Escherichia coli O26 isolates found in three cattle farms were monitored in space and time. Cluster analysis suggests that only some O26:H11 strains had the potential for long-term persistence in hosts and farms. As judged by their virulence markers, bovine enterohemorrhagic O26:H11 isolates may represent a considerable risk for human infection.Shiga toxin (Stx)-producing Escherichia coli (STEC) strains comprise a group of zoonotic enteric pathogens (42). In humans, infections with some STEC serotypes result in hemorrhagic or nonhemorrhagic diarrhea, which can be complicated by hemolytic-uremic syndrome (HUS) (49). These STEC strains are also designated “enterohemorrhagic E. coli” (EHEC). Consequently, EHEC strains represent a subgroup of STEC with a high pathogenic potential for humans. Strains of the E. coli serogroup O26 were originally classified as enteropathogenic E. coli due to their association with outbreaks of infantile diarrhea in the 1940s. In 1977, Konowalchuk et al. (37) recognized that these bacteria produced Stx, and 10 years later, the Stx-producing E. coli O26:H11/H− strains were classified as EHEC. EHEC O26 strains constitute the most common non-O157 EHEC group associated with diarrhea and HUS in Europe (12, 21, 23, 24, 26, 27, 55, 60). Reports on an association between EHEC O26 and HUS or diarrhea from North America including the United States (15, 30, 33), South America (51, 57), Australia (22), and Asia (31, 32) provide further evidence for the worldwide spread of these organisms. Studies in Germany and Austria (26, 27) on sporadic HUS cases between 1996 and 2003 found that EHEC O26 accounted for 14% of all EHEC strains and for ∼40% of non-O157 EHEC strains obtained from these patients. A proportion of 11% EHEC O26 strains was detected in a case-control study in Germany (59) between 2001 and 2003. In the age group <3 years, the number of EHEC O26 cases was nearly equal to that of EHEC O157 cases, although the incidence of EHEC O26-associated disease is probably underestimated because of diagnostic limitations in comparison to the diagnosis of O157:H7/H− (18, 34). Moreover, EHEC O26 has spread globally (35). Beutin (6) described EHEC O26:H11/H−, among O103:H2, O111:H, O145:H28/H−, and O157:H7/H−, as the well-known pathogenic “gang of five,” and Bettelheim (5) warned that we ignore the non-O157 STEC strains at our peril.EHEC O26 strains produce Stx1, Stx2, or both (15, 63). Moreover, these strains contain the intimin-encoding eae gene (11, 63), a characteristic feature of EHEC (44). In addition, EHEC strains possess other markers associated with virulence, such as a large plasmid that carries further potential virulence genes, e.g., genes coding for EHEC hemolysin (EHEC-hlyA), a catalase-peroxidase (katP), and an extracellular serine protease (espP) (17, 52). The efa1 (E. coli factor for adherence 1) gene was identified as an intestinal colonization factor in EHEC (43). EHEC O26 represents a highly dynamic group of organisms that rapidly generate new pathogenic clones (7, 8, 63).Ruminants, especially cattle, are considered the primary reservoir for human infections with EHEC. Therefore, the aim of this study was the molecular characterization of bovine E. coli field isolates of serogroup O26 using a panel of typical virulence markers. The epidemiological situation in the beef herds from which the isolates were obtained and the spatial and temporal behavior of the clonal distribution of E. coli serogroup O26 were analyzed during the observation period. The potential risk of the isolates inducing disease in humans was assessed.In our study, 56 bovine E. coli O26:H11 isolates and one bovine O26:H32 isolate were analyzed for EHEC virulence-associated factors. The isolates had been obtained from three different beef farms during a long-term study. They were detected in eight different cattle in farm A over a period of 15 months (detected on 10 sampling days), in 3 different animals in farm C over a period of 8 months (detected on 3 sampling days), and in one cow on one sampling day in farm D (Table (Table1)1) (28).

TABLE 1.

Typing of E. coli O26 isolates
Sampling day, source, and isolateSerotypeVirulence profile by:
fliC PCR-RFLPstx1 genestx2 geneStx1 (toxin)Stx2 (toxin)Subtype(s)
efa1 genebEHEC-hlyA genekatP geneespP genePlasmid size(s) in kbCluster
stx1/stx2eaetirespAespB
Day 15
    Animal 6 (farm A)
        WH-01/06/002-1O26:H11H11++stx1ββββ+/++++110, 127
        WH-01/06/002-2O26:H11H11++stx1ββββ+/++++110, 127
        WH-01/06/002-3O26:H11H11++stx1ββββ+/++++110, 127
    Animal 8 (farm A)
        WH-01/08/002-2O26:H11H11++stx1ββββ+/++++110, 127
    Animal 26 (farm A)
        WH-01/26/001-2O26:H11H11++stx1ββββ+/++++130, 127
        WH-01/26/001-5O26:H11H11++stx1ββββ+/++++110, 127
        WH-01/26/001-6O26:H11H11++stx1ββββ+/++++110, 127
        WH-01/26/001-7O26:H11H11++stx1ββββ+/−+++110, 127
Day 29
    Animal 2 (farm A)
        WH-01/02/003-1O26:H11H11++stx1ββββ+/++++110, 126
        WH-01/02/003-2O26:H11H11++stx1ββββ+/++++110, 126
        WH-01/02/003-5O26:H11H11++stx1ββββ+/++++110, 126
        WH-01/02/003-6O26:H11H11++stx1ββββ+/+++110, 126
        WH-01/02/003-7O26:H11H11++stx1ββββ+/++++110, 126
        WH-01/02/003-8O26:H11H11++stx1ββββ−/++++110, 126
        WH-01/02/003-9O26:H11H11++stx1ββββ+/++++1106
        WH-01/02/003-10O26:H11H11++stx1ββββ+/++++1106
    Animal 26 (farm A)
        WH-01/26/002-2O26:H11H11++stx1ββββ+/++++130, 125
        WH-01/26/002-5O26:H11H11++stx1ββββ+/++++130, 125
        WH-01/26/002-8O26:H11H11++stx1ββββ+/++++130, 125
        WH-01/26/002-9O26:H11H11++stx1ββββ+/++110, 125
        WH-01/26/002-10O26:H11H11++stx1ββββ+/++++130, 125
Day 64
    Animal 20 (farm A)
        WH-01/20/005-3O26:H11H11++stx1ββββ+/+130, 2.52
Day 78
    Animal 29 (farm A)
        WH-01/29/002-1O26:H11H11++stx1ββββ+/−+130, 12, 2.54
        WH-01/29/002-2O26:H11H11++stx1ββββ+/++++130, 12, 2.54
        WH-01/29/002-3O26:H11H11++stx1ββββ+/++++130, 12, 2.54
        WH-01/29/002-4O26:H11H11++stx1ββββ+/++++130, 12, 2.54
        WH-01/29/002-5O26:H11H11++stx1ββββ+/++130, 12, 2.54
Day 106
    Animal 27 (farm A)
        WH-01/27/005-2O26:H11H11++stx1ββββ+/−+++145, 110, 123
        WH-01/27/005-5O26:H11H11++stx1ββββ+/++++130, 12, 2.55
        WH-01/27/005-6O26:H11H11++stx1ββββ+/+130, 12, 2.55
Day 113
    Animal 7 (farm C)
        WH-04/07/001-2O26:H11H11++++stx1/stx2ββββ+/+++55, 35, 2.511
        WH-04/07/001-4O26:H11H11++++stx1/stx2ββββ+/++++5512
        WH-04/07/001-6O26:H11H11++++stx1/stx2ββββ+/++++5512
Day 170
    Animal 22 (farm C)
        WH-04/22/001-1O26:H11H11++stx1ββββ+/++++110, 12, 6.312
        WH-04/22/001-4O26:H11H11++stx1ββββ+/++++110, 12, 6.312
        WH-04/22/001-5O26:H11H11++stx1ββββ+/++++110, 12, 6.312
Day 176
    Animal 14 (farm D)
        WH-03/14/004-8O26:H11H11++stx1ββββ+/+++11010
Day 218
    Animal 27 (farm A)
        WH-01/27/009-1O26:H11H11++++stx1/stx2ββββ+/++++110, 129
        WH-01/27/009-2O26:H11H11++++stx1/stx2ββββ+/++++110, 129
        WH-01/27/009-3O26:H11H11++++stx1/stx2ββββ+/++++110, 128
        WH-01/27/009-8O26:H11H11++++stx1/stx2ββββ+/++110, 128
        WH-01/27/009-9O26:H11H11++++stx1/stx2ββββ+/++++110, 129
Day 309
    Animal 29 (farm A)
        WH-01/29/010-1O26:H11H11++stx1ββββ+/++++110, 35, 124
        WH-01/29/010-2O26:H11H11++stx1ββββ+/++130, 55, 358
        WH-01/29/010-3O26:H11H11++stx1ββββ+/++++130, 35, 128
Day 365
    Animal 8 (farm C)
        WH-04/08/008-6O26:H11H11++stx1ββββ+/++++110, 5512
Day 379
    Animal 9 (farm A)
        WH-01/09/016-2O26:H32H32++stx1/stx2−/−145, 130, 1.81
    Animal 27 (farm A)
        WH-01/27/014-3O26:H11H11++stx1ββββ+/++++110, 129
        WH-01/27/014-4O26:H11H11++stx1ββββ+/++++110, 129
        WH-01/27/014-5O26:H11H11++stx1ββββ+/++++110, 128
Day 407
    Animal 29 (farm A)
        WH-01/29/013-4O26:H11H11++stx1ββββ+/++++110, 12, 2.58
        WH-01/29/013-7O26:H11H11++stx1ββββ+/++++110, 12, 2.58
Day 478
    Animal 27 (farm A)
        WH-01/27/017-1O26:H11H11++++stx1/stx2ββββ+/++++110, 128
        WH-01/27/017-5O26:H11H11++++stx1/stx2ββββ+/++++110, 128
        WH-01/27/017-6O26:H11H11++++stx1/stx2ββββ+/++++1108
        WH-01/27/017-7O26:H11H11++++stx1/stx2ββββ+/++++1108
        WH-01/27/017-10O26:H11H11+++stx1ββββ+/++++130, 12, 2.58
Open in a separate windowastx1/stx2, gene stx1 or stx2.befa1 was detected by two hybridizations (with lifA1-lifA2 and lifA3-lifA4 probes). +/+, complete gene; +/− or −/+, incomplete gene; −/−, efa1 negative.The serotyping of the O26 isolates was confirmed by the results of the fliC PCR-restriction fragment length polymorphism (RFLP) analysis performed according to Fields et al. (25), with slight modifications described by Zhang et al. (62). All O26:H11 isolates showed the H11 pattern described by Zhang et al. (62). In contrast, the O26:H32 isolate demonstrated a different fliC RFLP pattern that was identical to the H32 pattern described by the same authors. It has been demonstrated that EHEC O26:H11 strains belong to at least four different sequence types (STs) in the common clone complex 29 (39). In the multilocus sequence typing analysis for E. coli (61), the tested five EHEC O26:H11 isolates (WH-01/02/003-1, WH-01/20/005-3, WH-01/27/009-9, WH-03/14/004-8, and WH-04/22/001-1) of different farms and clusters were characterized as two sequence types (ST 21 and ST 396). The isolates from farms A and C belong to ST 21, the most frequent ST of EHEC O26:H11 isolates found in humans and animals (39), but the single isolate from farm D was characterized as ST 396.Typing and subtyping of genes (stx1 and/or stx2, eae, tir, espA, espB, EHEC-hlyA, katP, and espP) associated with EHEC were performed with LightCycler fluorescence PCR (48) and different block-cycler PCRs. To identify the subtypes of the stx2 genes and of the locus of enterocyte effacement-encoding genes eae, tir, espA, and espB, the PCR products were digested by different restriction endonucleases (19, 26, 46). The complete pattern of virulence markers was detected in most bovine isolates examined in our study. An stx1 gene was present in all O26 isolates. In addition, an stx2 gene was found in nine O26:H11 isolates in farm A and in three isolates of the same type in farm C, as well as in the O26:H32 isolate. Both Stx1 and Stx2 were closely related to families of Stx1 and Stx2 variants or alleles. EHEC isolates with stx2 genes are significantly more often associated with HUS and other severe disease manifestations than isolates with an stx1 gene, which are more frequently associated with uncomplicated diarrhea and healthy individuals (13). In contrast to STEC strains harboring stx2 gene variants, however, STEC strains of the stx2 genotype were statistically significantly associated with HUS (26). The stx2 genotype was found in all O26 isolates with an stx2 gene, while the GK3/GK4 amplification products after digestion with HaeIII and FokI restriction enzymes showed the typical pattern for this genotype described by Friedrich et al. (26). The nucleotide sequences of the A and B subunits of the stx2 gene of the selected bovine O26:H11 isolate WH-01/27/017-1 (GenBank accession no. EU700491) were identical to the stx2 genes of different sorbitol-fermenting EHEC O157:H− strains associated with human HUS cases and other EHEC infections in Germany (10) and 99.3% identical in their DNA sequences to the stx2 gene of the EHEC type strain EDL933, a typical O157:H7 isolate from an HUS patient. A characteristic stx1 genotype was present in all O26 isolates. The nucleotide sequences of the A and B subunits of the stx1 gene of the tested bovine O26:H11 isolate WH-01/27/017-1 (GenBank accession no. EU700490) were nearly identical to those of the stx1 genes of the EHEC O26:H11 reference type strains H19 and DEC10B, which had been associated with human disease outbreaks in Canada and Australia. Nucleotide exchanges typical for stx1c and stx1d subtypes as described by Kuczius et al. (38) were not found. All bovine O26:H11 strains produced an Stx1 with high cytotoxicity for Vero cells tested by Stx enzyme-linked immunosorbent assay and Vero cell neutralization assay (53). The Stx2 cytotoxicity for Vero cells was also very high in the O26:H11 isolates.Not only factors influencing the basic and inducible Stx production are important in STEC pathogenesis. It has been suggested that the eae and EHEC-hlyA genes are likely contributors to STEC pathogenicity (2, 3, 13, 50). Ritchie et al. (50) found both genes in all analyzed HUS-associated STEC isolates. In all O26:H11 isolates we obtained, stx genes were present in combination with eae genes. Only the O26:H32 isolate lacked an eae gene. To date, 10 distinct variants of eae have been described (1, 19, 36, 45, 47). Some serotypes were closely associated with a particular intimin variant: the O157 serogroup was linked to γ-eae, the O26 serogroup to β-eae, and the O103 serogroup to ɛ-eae (4, 19, 20, 58). Our study confirms these associations. All bovine O26:H11 isolates were also typed as members of the β-eae subgroup. A translocated intimin receptor gene (tir gene) and the type III secreted proteins encoded by the espA and espB genes were found in all 56 O26:H11 isolates but not in the O26:H32 isolate. These other tested locus of enterocyte effacement-associated genes belonged to the β-subgroups. These results are in accord with the results of China et al. (19), who detected the pathotypes β-eae, β-tir, β-espA, and β-espB in all investigated human O26 strains. Like the eae gene, the EHEC-hlyA gene was found in association with severe clinical disease in humans (52). Aldick et al. (2) showed that EHEC hemolysin is toxic (cytolytic) to human microvascular endothelial cells and may thus contribute to the pathogenesis of HUS. In our study, the EHEC-hlyA gene was detected in 50 of the 56 bovine E. coli O26:H11 isolates which harbored virulence-associated plasmids of different sizes (Table (Table1).1). The presence of virulence-associated plasmids corresponded to the occurrence of additional virulence markers such as the espP and katP genes (17). The katP gene and the espP gene were detected in 49 and 50 of the 56 O26:H11 isolates, respectively. The espP gene was missing in six of the seven bovine O26:H11 isolates in which the katP genes were also absent. Both genes were not found in the O26:H32 isolate (Table (Table1).1). Although we found large plasmids of the same size in O26:H11 isolates, they lacked one or more of the plasmid-associated virulence factors (Table (Table1).1). Two DNA probes were used to detect the efa1 genes by colony hybridization. (DNA probes were labeled with digoxigenin [DIG] with lifA1-lifA2 and lifA3-lifA4 primers [14] using the PCR DIG probe synthesis kit [Roche Diagnostics, Mannheim, Germany]; DIG Easy Hyb solution [Roche] was used for prehybridization and hybridization.) Positive results with both DNA probes were obtained for 52 of 56 E. coli O26:H11 isolates. A positive signal was only found in three isolates with the lifA1-lifA2 DNA probe and in one isolate with the lifA3-lifA4 probe. An efa1 gene was not detected in the O26:H32 isolate (Table (Table11).We also analyzed the spatial and temporal behavior of the O26:H11/H32 isolates in the beef herds by cluster analysis (conducted in PAUP* for Windows version 4.0, 2008 [http://paup.csit.fsu.edu/about.html]). This was performed with distance matrices using the neighbor-joining algorithm, an agglomerative cluster method which generates a phylogenetic tree. The distance matrices were calculated by pairwise comparisons of the fragmentation patterns produced by genomic typing through pulsed-field gel electrophoresis analysis with four restriction endonucleases (XbaI, NotI, BlnI, and SpeI) and the presence or absence of potential virulence markers (Fig. (Fig.11 and Table Table1).1). To this end, the total character difference was used, which counts the pairwise differences between two given patterns. During a monitoring program of 3 years in four cattle farms (29), different O26:H11 cluster groups and one O26:H32 isolate were detected in three different farms. The genetic distance of the O26:H32 isolate was very high relative to the O26:H11 isolates. Therefore, the O26:H32 isolate was outgrouped. The O26:H11 isolates of each farm represented independent cluster groups. The single isolate from farm D fitted better to the isolates from farm C than to those from farm A. This finding is in accord with the geographical distance between the farms. The fact that the farms were located in neighboring villages may suggest that direct or indirect connections between the farms were possible (e.g., by person contacts or animal trade). However, the isolates from farm C and farm D belonged to different sequence types (ST 21 and ST 396), which may argue against a direct connection. Interestingly, O26:H11 isolates with and without stx2 genes were detected in the same clusters. This phenomenon was observed in both farm A and farm C. In farm A, the isolates with additional stx2 genes were found in animal 27 and were grouped in clusters 8 and 9 (day 218). An stx2 gene was repeatedly found (four isolates) in the same animal (animal 27). The isolates grouped in cluster 8 on a later day of sampling (day 478). All other O26:H11 isolates grouped in the same clusters and obtained from the same animals (27 and 29) on different sampling days lacked an stx2 gene. Also, the isolates obtained from animal 27 on previous sampling days, which grouped in clusters 3 and 5, exhibited no stx2 genes. In farm C, the three isolates with additional stx2 genes obtained from animal 7 grouped in clusters 11 and 12. An stx2 gene was absent from all other O26:H11 isolates grouped in the same cluster 12 on later sampling days, and no other isolates of cluster 11 were found later on. However, we detected members of many clusters over relatively long periods (clusters 5, 8, and 9 in farm A and cluster 12 in farm C), but members of other clusters were only found on single occasions. This patchy temporal pattern is apparently not a unique property of O26:H11, as we found similar results for cluster groups of other EHEC serotypes of bovine origin (28). The isolates grouped in the dominant cluster 8 were found on 5 of 9 sampling days over a period of 10 months. In contrast, we found the members of clusters 4, 5, 9, and 12 only on two nonconsecutive sampling days. The period during which isolates of these groups were not detected was particularly long for cluster 4 (231 days). We also observed the coexistence of different clusters over long periods in the same farm and in the same cattle (clusters 8 and 9), while one of the clusters dominated. Transmission of clusters between cattle was also observed. These results suggest that some of the EHEC O26:H11 strains had the potential for a longer persistence in the host population, while others had not. The reasons for this difference are not yet clear. Perhaps the incomplete efa1 gene found in isolates of clusters which were only detected once might explain why some strains disappeared rapidly. Efa1 has been discussed as a potential E. coli colonization factor for the bovine intestine used by non-O157 STEC, including O26 (54, 56). The O165:H25 cluster detected during a longer period in farm B may have disappeared after it had lost its efa1 gene (28). The precise biological activity of Efa1 in EHEC O26 is not yet known, but it has been demonstrated that the molecule is a non-Stx virulence determinant which can increase the virulence of EHEC O26 in humans (8).Open in a separate windowFIG. 1.Neighbor-joining tree of bovine E. coli O26:H11/H32 strains based on the restriction pattern obtained after digestion with XbaI, NotI, BlnI, and SpeI.We distinguished 12 different clusters, but complete genetic identity was only found in two isolates. The variations in the O26:H11 clusters may be due to increasing competition between the bacterial populations of the various subtypes in the bovine intestine or to potential interactions between EHEC O26:H11 and the host.The ephemeral occurrence of additional stx2 genes in different clusters and farms may be the result of recombination events due to horizontal gene transfer (16). The loss of stx genes may occur rapidly in the course of an infection, but the reincorporation by induction of an stx-carrying bacteriophage into the O26:H11 strains is possible at any time (9, 40). Nevertheless, an additional stx2 gene may increase the dangerousness of the respective EHEC O26:H11 strains. While all patients involved in an outbreak caused by an EHEC O26:H11 strain harboring the gene encoding Stx2 developed HUS (41), the persons affected by another outbreak caused by an EHEC O26:H11 strain that produced exclusively Stx1 had only uncomplicated diarrhea (60).In conclusion, our results showed that bovine O26:H11 isolates can carry virulence factors of EHEC that are strongly associated with EHEC-related disease in humans, particularly with severe clinical manifestations such as hemorrhagic colitis and HUS. Therefore, strains of bovine origin may represent a considerable risk for human infection. Moreover, some clusters of EHEC O26:H11 persisted in cattle and farms over longer periods, which may increase the risk of transmission to other animals and humans even further.  相似文献   

12.
A 30-probe assay was developed for simultaneous classification of Listeria monocytogenes isolates by lineage (I to IV), major serogroup (4b, 1/2b, 1/2a, and 1/2c), and epidemic clone (EC) type (ECI, ECIa, ECII, and ECIII). The assay was designed to facilitate rapid strain characterization and the integration of subtype data into risk-based inspection programs.Listeria monocytogenes is a facultative intracellular pathogen that can cause serious invasive illness (listeriosis) in humans and other animals. L. monocytogenes is responsible for over 25% of food-borne-disease-related deaths attributable to known pathogens and is a leading cause of food recalls due to microbial adulteration (12, 21). However, not all L. monocytogenes subtypes contribute equally to human illness, and substantial differences in the ecologies and virulence attributes of different L. monocytogenes subtypes have been identified (9, 13, 14, 23, 24, 33, 35, 36). Among the four major evolutionary lineages of L. monocytogenes, only lineages I and II are commonly isolated from contaminated food and human listeriosis patients (19, 27, 29, 33). Lineage I strains are overrepresented among human listeriosis isolates, particularly those associated with epidemic outbreaks, whereas lineage II strains are overrepresented in foods and the environment (13, 14, 24). Lineage III strains account for approximately 1% of human listeriosis cases but are common among animal listeriosis isolates and appear to be a host-adapted group that is poorly adapted to food-processing environments (6, 34-36). The ecological and virulence attributes of lineage IV are poorly understood, as this lineage is rare and was only recently described based on a small number of strains (19, 26, 29, 33).L. monocytogenes is differentiated into 13 serotypes; however, four major serogroups (4b, 1/2b, 1/2a, and 1/2c) from within lineages I and II account for more than 98% of human and food isolates (16, 31). Serogroups refer to evolutionary complexes typified by a predominant serotype but which include very rare serotypes that represent minor evolutionary variants (7, 9, 33). Phylogenetic analyses have indicated that rare serotypes may have evolved recently, or even multiple times, from one of the major serotypes (9), and numerous molecular methods fail to discriminate minor serotypes as independent groups (1, 4, 7, 9, 18, 22, 33, 38, 39). Serotyping is one of the most common methods for L. monocytogenes subtyping, and serogroup classifications are a useful component of strain characterization because ecotype divisions appear largely congruent with serogroup distinctions (16, 34). Serogroup 4b strains are of particular public health concern because contamination with these strains appears to increase the probability that a ready-to-eat (RTE) food will be implicated in listeriosis (16, 28). Serogroup 4b strains account for approximately 40% of sporadic listeriosis and also are responsible for the majority of listeriosis outbreaks despite being relatively rare contaminants of food products (9, 13, 17, 30, 34). In addition, serogroup 4b strains are associated with more severe clinical presentations and higher mortality rates than other serogroups (11, 16, 20, 31, 34). Serogroups 1/2a and 1/2b are overrepresented among food isolates but also contribute significantly to human listeriosis, whereas serogroup 1/2c rarely causes human illness and may pose a lower risk of listeriosis for humans (16). Serogroup-specific differences in association with human listeriosis are consistent with the prevalence of virulence-attenuating mutations in inlA within these serogroups (32, 34); however, a number of additional factors likely contribute to these differences.Four previously described epidemic clones (ECs; ECI, ECIa, ECII, and ECIII) of L. monocytogenes have been implicated in numerous listeriosis outbreaks and have contributed significantly to sporadic illness (15, 34). ECI, ECIa, and ECII are distinct groups within serogroup 4b that were each responsible for repeated outbreaks of listeriosis in the United States and Europe. ECIII is a lineage II clone of serotype 1/2a that persisted in the same processing facility for more than a decade prior to causing a multistate outbreak linked to contaminated turkey (15, 25). While there has been speculation that epidemic clones possess unique adaptations that explain their frequent involvement in listeriosis outbreaks (9, 34, 37), it is not clear that epidemic clones are more virulent than other strains with the same serotype. However, contamination of RTE food with EC strains would be cause for increased concern due to the previous involvement of these clones in major outbreaks of listeriosis (16).As a result of the L. monocytogenes subtype-specific differences in ecology, virulence, and association with human illness, molecular subtyping technologies have the potential to inform assessments of relative risk and to improve risk-based inspection programs. The objective of the present study was to develop a single assay for rapid and accurate classification of L. monocytogenes isolates by lineage, major serogroup, and epidemic clone in order to facilitate strain characterization and the integration of subtype data into inspection programs that are based on assessment of relative risk.A database of more than 5.3 Mb of comparative DNA sequences from 238 L. monocytogenes isolates (9, 33-35) was scanned for single nucleotide polymorphisms that could be used to differentiate lineages, major serogroups, and epidemic clones via a targeted multilocus genotyping (TMLGT) approach. The acronym TMLGT is used to distinguish this approach from previously published multilocus genotyping (MLGT) assays that were lineage specific and designed for haplotype discrimination (9, 33). To provide for simultaneous interrogation of the selected polymorphisms via TMLGT, six genomic regions (Table (Table1)1) were coamplified in a multiplex PCR. While the previous MLGT assays were based on three lineage-specific multiplexes and required prior identification of lineage identity, TMLGT was designed to target variation across all of the lineages simultaneously and is based on a unique set of amplicons. PCR was performed in 50-μl volumes with 1× High Fidelity PCR buffer (Invitrogen Life Technologies), 2 mM MgSO4, 100 μM deoxynucleoside triphosphate (dNTP), 300 nM primer, 1.5 U Platinum Taq high-fidelity DNA polymerase (Invitrogen Life Technologies), and 100 ng of genomic DNA. PCR consisted of an initial denaturation of 90 s at 96°C, followed by 40 cycles of 30 s at 94°C, 30 s at 50°C, and 90 s at 68°C. Amplification products were purified using Montage PCR cleanup filter plates (Millipore) and served as a template for allele-specific primer extension (ASPE) reactions utilizing subtype-specific probes.

TABLE 1.

Primers used in multiplex amplification for the TMLGT assay
AmpliconPositionaGene(s)PrimerSequence (5′-3′)b
INLa455381-456505inlAinl2-a1GTCCTTGATAGTCTACTG
inl2-a2ACCAAATTAGTAATCTAGCAC
INLb457726-458752inlBinl-f1dGAATTRTTTAGYCAAGAATGT
inlb-rCTACCGGRACTTTATAGTAYG
LMO325116-326096lmo0298-lmo0300lmo-a1AAGGCTTACAAGATGGCT
lmo1a-1rAAATAATAYGTGATACCGAC
VGCa205366-206622plcA, hlyplca-fCTCATCGTATCRTGTGTACC
hly-rTCTGGAAGGTCKTGTAGGTTC
VGCb208447-209465mplra_mpl-fGTGGAYAGAACTCATAAAGG
ra_mpl-rACTCCCTCCTYGTGATASGCT
VGCc209728-211239actAvgc1a-2fTTCMATRCCAGCAGAACG
vgc1a-2rGCAGACCTAATAGCAATGTTG
Open in a separate windowaCorresponding nucleotide positions in the complete genome sequence of L. monocytogenes strain EGD-e (GenBank accession number NC_003210).bSee IUPAC codes for definition of degenerate bases.ASPE was performed in multiplex reactions including 30 probes, with each lineage (I to IV), major serogroup (4b, 1/2b, 1/2a, and 1/2c), and epidemic clone (ECI, ECIa, ECII, and ECIII) targeted by two different probes (Table (Table2).2). In addition, positive-control probes were included to confirm the presence of each amplicon in the multiplex PCR. As serogroups and epidemic clones are nested within a particular lineage, probes for these groups were designed to be specific within the appropriate lineage and values for these probes were evaluated only for isolates of the appropriate lineage. For example, serogroup 1/2a probes were evaluated only for isolates that were positive for lineage II probes. ASPE probes were designed with a unique 5′ sequence tag specific to individual sets of xMAP fluorescent polystyrene microspheres (Luminex Corporation) used to sort extension products. Extension and hybridization reactions were performed as described previously (9) except microspheres were twice pelleted by centrifugation (4 min at 2,250 × g) and resuspended in 75 μl 1× TM buffer prior to being pelleted and resuspended in 100 μl 1× TM buffer containing 2 μg/ml streptavidin-R-phycoerythrin (Invitrogen Life Technologies). Samples were incubated for 15 min at 37°C prior to detecting the microsphere complexes with a Luminex 100 flow cytometer (Luminex Corporation). The median fluorescence intensity (MFI) from biotinylated extension products attached to 100 microspheres was measured for each probe. The average MFI from three template-free control samples was also determined and subtracted from the raw MFI of each sample to account for background fluorescence. Probe performance was initially evaluated via the index of discrimination (ID) as described by Ducey et al. (9), and probes with ID values less than 2.0 were redesigned.

TABLE 2.

TMLGT probes and probe performance data
ProbebTarget (n)cProbe sequencedIDeSensitivity (%)Specificity (%)
VGCb-21Lineage I (506)AATCCTTTCTTTAATCTCAAATCAgcggaagcttgggaagcggtc7.3100100
VGCa-94Lineage ICTTTCTATCTTTCTACTCAATAATcaacccgatgttcttcctgtc51.7100100
VGCc-8Lineage II (340)AATCCTTTTACATTCATTACTTACattagctgattcgctttcct14.1100100
INLb-51Lineage IITCATTTCAATCAATCATCAACAATagcgccaataaagctggc21.9100100
VGCb-19Lineage III (50)TCAATCAATTACTTACTCAAATACccgctattaaaatgtactcca31.0100100
VGCb-29Lineage IIIAATCTTACTACAAATCCTTTCTTTggtataccgctattaaaatgt45.1100100
LMO-17Lineage IV (10)CTTTAATCCTTTATCACTTTATCAgaaccaaacaatgttattggt11.8100100
VGCa-27Lineage IVCTTTTCAAATCAATACTCAACTTTttaacgacggtaacgtgccac58.3100100
INLb-84Serogroup 4b (213)TCAACTAACTAATCATCTATCAATggtaaaaatatgcgaatattg9.7100100
INLb-85Serogroup 4bATACTACATCATAATCAAACATCActcgtgaacaagctttcc5.5100100
INLb-16Serogroup 1/2b (293)AATCAATCTTCATTCAAATCATCAggtaaaaatatgcgtatctta11.7100100
INLb-100Serogroup 1/2bCTATCTTTAAACTACAAATCTAACgtgaataagctatcggtctat13.0100100
LMO-42Serogroup 1/2a (268)CTATCTTCATATTTCACTATAAACtggcgttgctgrctaagtttg6.6100100
VGCb-40Serogroup 1/2aCTTTCTACATTATTCACAACATTAaatcaagcsgctcatatgaag10.410098.6
LMO-9Serogroup 1/2c (72)TAATCTTCTATATCAACATCTTACtttactggtgaaatggcg13.5100100
VGCb-5Serogroup 1/2cCAATTCAAATCACAATAATCAATCaagattacgaatcgcttccac20.898.6100
LMO-10ECI (111)ATCATACATACATACAAATCTACAatgattaaaagtcagggaaag19.0100100
LMO-28ECICTACAAACAAACAAACATTATCAAaatcgaggcttacgaacgt23.7100100
VGCc-80ECIa (44)CTAACTAACAATAATCTAACTAACactacaacgaaaacagcgc10.7100100
VGCa-35ECIaCAATTTCATCATTCATTCATTTCAgttacttttatgtcgagt9.2100100
LMO-12ECII (35)TACACTTTCTTTCTTTCTTTCTTTataccgattatttggacggtt3.8100100
LMO-30ECIITTACCTTTATACCTTTCTTTTTACgacttgtagcagttgatttcaa7.5100100
VGCc-45ECIII (10)TCATTTCACAATTCAATTACTCAActcttatttgcttttgttggtc21.110099.4
INLa-3ECIIITACACTTTATCAAATCTTACAATCgagcttaatgaaaatcagcta17.010099.4
INLa-1INLa controlCTTTAATCTCAATCAATACAAATCagaagtggaagctgggaaNAaNANA
INLb-13INLb controlCAATAAACTATACTTCTTCACTAAtgcacctaaacctccgacNANANA
LMO-88LMO controlTTACTTCACTTTCTATTTACAATCccgtttccttatgccacaNANANA
VGCa-23VGCa controlTTCAATCATTCAAATCTCAACTTTcaagycctaagacgccaatcgNANANA
VGCb-25VGCb controlCTTTTCAATTACTTCAAATCTTCAgcatgcgttagttcatgrccaNANANA
VGCc-82VGCc controlTACATACACTAATAACATACTCATgactgcatgctagaatctaagNANANA
Open in a separate windowaNA, not applicable for positive amplicon control probes.bLuminex microsphere sets (Luminex Corporation) used for hybridization reactions are indicated following the hyphen.cn, number of isolates representing the target subtype among the 906 tested isolates.dThe 5′ sequence tag portions of extension probes are capitalized. See IUPAC codes for definitions of degenerate bases.eID, index of discrimination.Validation of the TMLGT assay was performed using 906 L. monocytogenes isolates for which the lineage, major serogroup, and epidemic clone type had been determined independently (see Table S1 in the supplemental material). A subset of 92 isolates, including at least five isolates from each lineage, serogroup, and epidemic clone type, was used to evaluate the discriminatory power of subtype-specific probes and the repeatability of the assay (see Table S1). Two independent runs of the 30-probe TMLGT assay produced identical results for these 92 isolates. In addition, genotypes matched expectations for all isolate/probe combinations, and the fluorescence intensities for positive genotypes (those targeted by a particular probe) were 3.8 to 58.3 (mean, 18.5) times as high as background values for isolates with negative genotypes (those not targeted by a particular probe) (Table (Table2).2). The performances of individual probes also were assessed in terms of sensitivity and specificity, where sensitivity is defined as the percentage of positive samples that produced positive results and specificity indicates the percentage of negative samples that produce negative results (5). Based on results from all 906 isolates analyzed by TMLGT, probe sensitivity was at least 98.6% and 23 of the 24 subtype-specific probes exhibited 100% sensitivity (Table (Table2).2). The specificities for all probes were also greater than 98.6%, and 21 of the 24 subtype-specific probes exhibited 100% specificity (Table (Table22).All but three of the 906 isolates in the validation panel were fully and accurately typed relative to lineage, serogroup, and epidemic clone by using the TMLGT assay (typeability, 99.9%; accuracy of isolate assignment, 99.8%). One of the lineage II isolates, NRRL B-33880, could not be assigned to a serogroup based on the TMLGT results because this isolate was positive for one of the serogroup 1/2a probes (VGCb-40) and one of the serogroup 1/2c probes (LMO-9). This isolate was previously identified as a member of serogroup 1/2c based on mapping lineage-specific MLGT data onto a multilocus phylogeny (34) but produced a serogroup 1/2a-specific banding pattern (data not shown) with the multiplex PCR assay described by Doumith et al. (7). Similar strains, including the common laboratory strain EGD-e, were found to have genomes that are more similar to serogroup 1/2c strains than to strains from the 1/2a serogroup (8, 33) and likely represent intermediates in the evolution of the 1/2c clade from 1/2a ancestors. There is a poor correlation between genomic and antigenic variation for such isolates (34), consistent with the ambiguous results produced by application of the TMLGT assay to NRRL B-33880. The two other problematic isolates, NRRL B-33555 and NRRL B-33559, were accurately identified based on TMLGT data as lineage II isolates from the 1/2a serogroup. However, these two isolates were positive for both ECIII-specific probes in the TMLGT assay but have lineage-specific MLGT haplotypes (Lm2.46), indicating that they are representatives of a sister group closely related to ECIII (33).In 2005, the Food Safety and Inspection Service (FSIS) implemented an approach to inspection that includes consideration of relative risk in order to determine L. monocytogenes sampling frequency among establishments that produce certain RTE products. This approach incorporates information on production volume, outgrowth potential in the product, steps taken to prevent postlethality contamination, and FSIS sampling history. However, L. monocytogenes subtype-specific variation in ecology and virulence indicates that information on the lineage, major serogroup, and epidemic clone identities of isolates could be used to inform assessments of relative risk and to improve inspection programs that are based on consideration of risk. Several PCR-based methods have been described for differentiation of various combinations of these subgroups (1-3, 5, 7, 10, 35, 37); however, these approaches have focused on a single subgroup or a smaller set of subgroups than is differentiated by TMLGT analysis. Although we previously developed a set of three MLGT assays that can be used to differentiate all of the major serogroups and epidemic clones of L. monocytogenes (9, 33, 34), those assays did not include probes for lineage discrimination and require identification of the lineage prior to application of one of three unique sets of probes. In addition, the MLGT assays were designed to maximize strain discrimination, as opposed to subgroup identification, and require the use of at least twice as many probes as is needed for TMLGT analysis. MLGT data analysis is also more complicated than analysis of TMLGT data, and serogroup or epidemic clone type identification via MLGT requires phylogenetic analyses to place novel haplotypes within an established phylogenetic framework.In the present study, we developed the first assay for simultaneous discrimination of the four lineages, the four major serogroups, and the four previously described epidemic clones of L. monocytogenes. The assay includes multiple markers for each of these subtype probes as well as control probes to ensure that negative probe data were not the result of amplification failure, providing a high degree of internal validation required for use in inspection programs that consider risk in making sampling decisions. In addition, the utility of the assay has been validated with a large and diverse panel of 906 isolates, including 567 isolates from FSIS surveillance of RTE products and processing facilities (see Table S1 in the supplemental material). Data produced by the TMLGT assay are amenable to high-throughput analysis, and a simple spreadsheet utility has been developed to semiautomate subtype identifications and to alert investigators to potentially conflicting probe data (available upon request). In addition to having a potential application in inspection programs, the TMLGT assay provides a rapid and accurate means of characterizing L. monocytogenes isolates from different environments, which would facilitate pathogen tracking and improve understanding of L. monocytogenes ecology.   相似文献   

13.
Simian immunodeficiency virus (SIV) infection of natural-host species, such as sooty mangabeys (SMs), is characterized by a high level of viral replication and a low level of generalized immune activation, despite evidence of an adaptive immune response. Here the ability of SIV-infected SMs to mount neutralizing antibodies (Nab) against autologous virus was compared to that of human immunodeficiency virus type 1 (HIV-1) subtype C-infected subjects. While high levels of Nab were observed in HIV-1 infection, samples obtained at comparable time points from SM exhibited relatively low titers of autologous Nab. Nevertheless, SM plasma with higher Nab titers also contained elevated peripheral CD4+ T-cell levels, suggesting a potential immunologic benefit for SMs. These data indicate that AIDS resistance in these primates is not due to high Nab titers and raise the possibility that low levels of Nab might be an inherent feature of natural-host SIV infections.More than 40 species of African nonhuman primates (NHPs) naturally harbor CD4+-tropic lentiviruses that are collectively known as simian immunodeficiency viruses (SIVs) and represent the ancestors of the human pathogens human immunodeficiency virus type 1 (HIV-1) and HIV-2. Interestingly, African NHPs infected with their cognate SIV generally do not progress to AIDS, despite high levels of sustained virus replication, with the only known exception being chimpanzee SIV (SIVcpz)-infected chimpanzees (16). Among the natural hosts for SIV infection, the sooty mangabey ([SM] Cercocebus atys) is of particular interest, because cross-species transmission of SM SIV (SIVsm) from this natural host into humans initiated the HIV-2 epidemic in West Africa (17). In addition, SIVsm (herein referred to as SIV) is the ancestor of the rhesus macaque SIV (SIVmac) viruses that are used in disease pathogenesis and vaccination studies in the rhesus macaque model (17). Both naturally infected and experimentally inoculated SMs remain healthy, maintain CD4+ T cells, and do not progress to AIDS-like disease, despite sustained high levels of virus replication (31).Nonpathogenic infection of SMs is characterized by low levels of immune activation during the chronic phase of infection, which are reached after a transient immune activation that occurs during primary infection (reviewed in reference 31). These findings have led to the hypothesis that the absence of generalized immune activation in SIV-infected SMs during the chronic phase of infection is an important feature that favors the preservation of CD4+ T-cell homeostasis, thereby avoiding disease progression (31). However, most of these earlier studies focused on T cells and innate immune cells, with a significant gap existing in our understanding of whether humoral immunity might also differ between pathogenic and nonpathogenic infections. In HIV-1-infected patients, B cells produce neutralizing antibodies against the infecting (autologous) virus, which drives viral escape, continuous de novo antibody production (26-28, 32), and B-cell dysfunction (24). The striking differences in both the clinical outcomes of infection and the levels of immune activation between SIV-infected SMs and HIV-1-infected humans prompted us to compare the neutralizing antibody (Nab) response against the autologous virus in these two populations. To this end, we utilized a pseudovirus assay that has been used extensively by our group and others to evaluate Nab against HIV-1 and SIV envelope (Env) glycoproteins (15, 19, 22, 26, 28, 32, 33; also unpublished data). All SMs were housed at the Yerkes National Primate Research Center (Atlanta, GA) and maintained in accordance with National Institutes of Health guidelines. The Emory University Animal Care and Use Committee approved these studies. Details of the Zambia Emory HIV Research Project (ZEHRP) have been described elsewhere (2, 10, 21). The Emory University Institutional Review Board and the University of Zambia School of Medicine Research Ethics Committee approved informed-consent and human subject protocols. None of the subjects received antiretroviral therapy during the evaluation period.In HIV-1 infection, autologous Nabs develop to relatively high titers against the newly transmitted virus within the first few months (15, 19, 26-28, 32). Here we sought to test whether a similar increase in Nab titer occurs during nonpathogenic SIV infection of SMs. Samples were obtained from five animals that were inoculated intravenously with plasma from a naturally infected SM as part of a previous study (30). Multiple, biologically functional Envs were cloned from plasma collected at day 14 postinoculation (Table (Table1),1), and Nab activity was evaluated in plasma collected at 6 months postinoculation. To facilitate comparison with early HIV-1 infection, Nab activity in plasma was also evaluated between 2 and 9 months against Envs that were cloned between 31 and 88 estimated days after infection from four subtype C HIV-1-infected seroconverters in Zambia (Table (Table1).1). Figure Figure1A1A demonstrates that Nab activity in plasma diluted 1:100 was readily detectable in all HIV-1-infected subjects at levels approaching 100% neutralization. However, Nab activity in the SM plasma was significantly lower than in the human subjects (median, 10% versus 93%, respectively; P = 0.02). Binding antibody was detected in all five SMs at titers greater than 1:51,200 by enzyme-linked immunosorbent assay (ELISA), demonstrating that all monkeys had seroconverted by 6 months and maintained high titers of binding antibody throughout the evaluation period (Fig. (Fig.1B).1B). Thus, the low level of Nab was not due to a diminished humoral immune response.Open in a separate windowFIG. 1.Autologous Nab activity and B-cell proliferation during experimental infection of SMs. (A) Neutralization activity levels in plasma from five SMs (filled black circles), which were experimentally inoculated with plasma from a naturally SIV-infected SM, and four HIV-1-infected Zambian subjects (half-filled squares), who were recently infected through heterosexual contact, are shown. The horizontal bars represent the median for each group. To assess neutralizing activity, pseudoviruses were created by expressing each cloned Env with an HIV-1 env-deficient backbone (ΔSG3). JC53-BL (Tzm-bl) cells were infected with each pseudovirus in the presence or absence of serially diluted autologous plasma. Each point represents the average level of neutralization at a 1:100 dilution of plasma for at least two Env clones (see Table Table11 for number of Envs tested). Each neutralization assay was performed twice independently, using duplicate wells. Statistical significance between the groups was determined by a Mann-Whitney test, using GraphPad Prism 5. Longitudinal measurements of endpoint antibody ELISA titers in plasma (filled green circles) (23) (B), autologous neutralization activity in plasma (filled blue diamonds) (C), percentages of Ki-67+ CD20+ cells in blood (filled black triangles) (D), and percentages of CD20+ cells in blood (filled red squares) (E) are shown for the five experimentally inoculated SMs combined. In panel C, each point represents average neutralization at a 1:100 dilution of plasma over time for at least two day 14 Env clones from each SM. For panels D and E, PBMCs were gated by forward and side scatter, and the CD3 CD20+ population was assessed for Ki-67 staining (D) by flow cytometry. SP34-2 was used to stain CD3, L27 was used for CD20, and B56 was used for Ki-67 (all from BD Biosciences). Error bars represent the standard errors of the means (SEMs). Plasma viral load peaked at day 14 (data not shown). Filled symbols in panels A through E indicate data generated from experimentally infected SMs.

TABLE 1.

Autologous Nab activity in experimentally SIV-infected SM and acutely HIV-1-infected humans
Subject IDaVirusNo. of mo postinfection Nab activity was evaluatedNo. of days postinfection Envs were cloned from plasmaNo. of Envs tested% neutralization at a 1:100 dilution of plasma
FuvSIVsm-Fuo614416.3
FSsSIVsm-Fuo614310.6
FWvSIVsm-Fuo614510.5
FFsSIVsm-Fuo614210.3
FRsSIVsm-Fuo61439.3
185FHIV-1533494.6
153MHIV-1988594.3
221MHIV-1631691.5
205FHIV-1248587.1
Open in a separate windowaID, identification.The low level of Nab activity observed in the five experimentally inoculated SMs persisted for 16 months and did not exceed 50% at a 1:100 dilution of plasma at any time point tested (Fig. (Fig.1C).1C). In contrast, the high levels of Nab activity in the HIV-1-infected subjects persisted for over 2 years, often exceeding 50% inhibitory titers of 1:3,000 against the early virus, as is characteristic of early subtype C HIV-1 infection (15, 19, 26, 28). Figure Figure1D1D demonstrates that a transient increase in proliferating B cells, as measured by positive Ki-67 staining (12), occurred in the SMs and peaked around day 30 postinfection and then declined to a level just above baseline by day 60. Analysis using a Wilcoxon signed-rank test for paired samples showed that the percentages of Ki-67-positive (Ki-67+) B cells were higher at days 21 and 30 than at day −5, reaching borderline significance at both time points (P = 0.06). In contrast, the percentages of Ki-67+ B cells on days 60 and 475 were not significantly different from that on day −5 (P = 0.8 and 0.3, respectively). An early but transient decrease in the percentage of circulating CD20+ B cells was also observed during the initial 20 days of infection (Fig. (Fig.1E).1E). Thus, the B-cell compartment within the SM underwent changes consistent with immune activation followed by resolution. Based on these results, it does not appear that a global defect in the B-cell response in the SM can account for the low-level Nab response elicited.To investigate Nab responses during established infection, we extended this analysis to a panel of 11 naturally SIV-infected SMs in the Yerkes colony and 5 chronically HIV-1-infected subjects in Zambia. Envs were cloned from these monkeys and human subjects using peripheral blood mononuclear cell (PBMC) DNA or plasma samples, and sensitivity to Nab was evaluated. Because Nab activity against contemporaneous Env is often low or undetectable in HIV-1 infection (1, 5, 14, 25, 27, 28, 32), we evaluated plasma collected between 6 and 55 months after the Envs were cloned from each individual. Table Table22 shows that the SM Envs reflected the four SIV subtypes that circulate in the Yerkes colony (3). Figure Figure2A2A demonstrates that Nab activity in the chronically HIV-1-infected subjects was high (median, 91%), whereas in the naturally SIV-infected SMs it was again significantly lower (median, 14%; P = 0.003). Nevertheless, Nab activity in the naturally infected SMs exhibited a considerable range, from undetectable to 84% neutralization (Fig. (Fig.2A).2A). This observation prompted us to investigate whether parameters associated with disease progression in HIV-1 infection were correlated with the level of Nab activity. Figure Figure2B2B demonstrates that the number of CD4+ T cells was positively correlated with the potency of neutralization (r = 0.69; P = 0.02), while the plasma viral load showed a trend toward an inverse correlation with neutralization (Fig. (Fig.2C)2C) (r = −0.54; P = 0.08). A correlation between plasma viral load and autologous Nab titer in established HIV-1 infection has not been observed (9).Open in a separate windowFIG. 2.Autologous Nab activity and its correlation with CD4+ count and plasma viral load during established natural infection of SMs. (A) Neutralization activity levels in plasma from 11 naturally SIV-infected SMs in the Yerkes colony (open circles) and 5 chronically HIV-1-infected human subjects from Zambia (half-filled squares) are shown. Statistical significance between groups was determined by a Mann-Whitney test using GraphPad Prism 5. Correlation between Nab activity and CD3+ CD4+ T cell counts or plasma viral load in naturally infected SMs (open circles) is shown in panels (B) and (C), respectively. The percent neutralization at a 1:100 dilution of plasma (shown in panel A) is plotted along the x axis. Each CD4+ T cell count and viral load value represents the average of three measurements from samples collected from the 11 SMs approximately 1 year apart. The significance of each correlation was determined using a nonparametric Spearman test. Open circles indicate data from naturally infected SMs.

TABLE 2.

Autologous Nab activity in naturally SIV-infected SMs and HIV-1-infected humans with established infections
Subject IDaVirusEnv subtypeNo. of mo between plasma collection and Env cloningNo. of Envs tested% neutralization at 1:100 dilution of plasma
FWkSIVsm228584.4
FNnSIVsm131463.5
FFvSIVsm150459.7
FFmSIVsm130442.0
FNgSIVsm548428.0
FBnSIVsm349213.9
FDoSIVsm36512.0
FZoSIVsm12838.8
FOhSIVsm1624.7
FPnSIVsm13250.6
FFjSIVsm15420.0
109MHIV-1C6591.4
55MHIV-1C15891.3
135FHIV-1C16497.4
106MHIV-1C17579.4
153FHIV-1C55599.0
Open in a separate windowaID, identification.This study is the first to directly compare the Nab response against the autologous virus in nonpathogenic SIV versus HIV-1 infection, including evaluation of both the early, developing Nab response in acute infection and the mature response in chronic infection. A significant difference in the magnitude of Nab activity was apparent during both early and later time points, with relatively strong but ultimately ineffective neutralization activity developing and persisting into chronic infection in humans but not in SMs. Although the SIV and HIV-1 samples were obtained during similar stages of infection, the disparity in the magnitude of autologous Nab activity during early infection could in part reflect differences such as the route of infection (intravenous versus mucosal) or the complexity of the founder virus (a single variant in HIV-1 versus multiple variants in SIV). In addition, the production of SIV Env pseudoviruses in human 293T cells could have altered the glycosylation pattern or the proteins that are embedded within the virion, decreasing the neutralization susceptibility of the SIV Env pseudoviruses. However, production of a subset of these pseudoviruses in an African green monkey-derived cell line (COS-1) did not alter their Nab sensitivity (data not shown).Despite the lack of potent autologous Nab, both naturally and experimentally SIV-infected SMs produce antibodies that bind Env in ELISAs or Western blotting (4, 6, 13, 18, 23). It is possible that the SIV Env glycoproteins elicit a different profile of Nab than does HIV-1 Env. The potential for structural and biological differences between SIV and HIV-1 Envs has not been thoroughly investigated, although they would not be unexpected due to the low level of amino acid sequence conservation between them. SIVsm/HIV-2 lineage-derived Envs (i.e., the SIVmac series) show a “wide evolutionary distance” and lack of cross-reactivity with SIVcpz/HIV-1-derived Envs, with an overall sequence identity in gp120 of ∼25% across HIV-1, HIV-2, and SIVsm (7, 8). Clear biological differences in immunogenicity have been described for HIV-1 group M subtypes, which all derive from a common SIV ancestor (reviewed in reference 20). Furthermore, SM IgG antibody molecules have less flexibility in the hinge region than human IgG, which could lead to a failure of the SM antibodies to recognize recessed neutralization targets such as the receptor binding domains (29). Thus, HIV-1 Env could elicit neutralizing antibodies that are qualitatively different from those induced by SIV Env.Early resolution of immune activation could be a key feature that distinguishes nonpathogenic from pathogenic infection (12, 31). The data presented here are consistent with that hypothesis, in that signs of early B-cell proliferation were present in the experimentally infected SMs but were resolved and did not result in potent neutralizing activity. However, later in infection, the naturally infected SMs did develop low-to-moderate levels of Nab activity, and these levels were positively correlated with the number of peripheral CD4+ T cells. This finding suggests that synergy between CD4+ T cells and B cells is maintained in this nonpathogenic setting. Other biologic factors could contribute to this correlation; however, differences in age and viral subtype in this cohort of SMs could not explain this finding (data not shown).Taken together, these results indicate that a low level of autologous Nab activity is a novel and previously unappreciated feature of nonpathogenic SIV infection of SMs. The fact that high-titer Nabs are not necessary to avoid disease progression during SIV infection of SMs is consistent with the notion that the apathogenicity of natural SIV infections is not the result of particularly effective adaptive immune responses against the virus (11). It is possible that this low level of autologous Nab activity in SMs stems in part from antibody recognition of targets that are poorly exposed on the native SIV Env glycoproteins. A low level of neutralizing activity in SM may therefore have a protective effect because it does not drive viral escape or induce chronic immune activation in the B-cell compartment. Moreover, a low level of immune activation in B cells and/or preservation of CD4+ T cells could enhance the quality of the neutralizing antibody response. It will be important, in future work, to assess how this low level of autologous Nab activity in SIV-infected SMs meshes with the lower levels of immune activation and dysregulation observed in these animals. Understanding the qualitative and quantitative differences in the Nab response during pathogenic versus nonpathogenic infection could provide critical information regarding protection from AIDS.  相似文献   

14.
A molecular diagnostic system using single nucleotide polymorphisms (SNPs) was developed to identify four Sclerotinia species: S. sclerotiorum (Lib.) de Bary, S. minor Jagger, S. trifoliorum Erikss., and the undescribed species Sclerotinia species 1. DNAs of samples are hybridized with each of five 15-bp oligonucleotide probes containing an SNP site midsequence unique to each species. For additional verification, hybridizations were performed using diagnostic single nucleotide substitutions at a 17-bp sequence of the calmodulin locus. The accuracy of these procedures was compared to that of a restriction fragment length polymorphism (RFLP) method based on Southern hybridizations of EcoRI-digested genomic DNA probed with the ribosomal DNA-containing plasmid probe pMF2, previously shown to differentiate S. sclerotiorum, S. minor, and S. trifoliorum. The efficiency of the SNP-based assay as a diagnostic test was evaluated in a blind screening of 48 Sclerotinia isolates from agricultural and wild hosts. One isolate of Botrytis cinerea was used as a negative control. The SNP-based assay accurately identified 96% of Sclerotinia isolates and could be performed faster than RFLP profiling using pMF2. This method shows promise for accurate, high-throughput species identification.Sclerotinia is distinguished morphologically from other genera in the Sclerotiniaceae (Ascomycota, Pezizomycotina, Leotiomycetes) by the production of tuberoid sclerotia that do not incorporate host tissue, by the production of microconidia that function as spermatia but not as a disseminative asexual state, and by the development of a layer of textura globulosa composing the outer tissue of apothecia (8). Two hundred forty-six species of Sclerotinia have been reported, most distinguished morphotaxonomically (Index Fungorum [www.indexfungorum.org]). These include the four species of agricultural importance now recognized plus many that are imperfectly known, seldom collected, or apparently endemic to relatively small geographic areas (2, 5, 6, 7, 8, 9, 17).The main species of phythopathological interest in the genus Sclerotinia are S. sclerotiorum (Lib.) de Bary, S. minor Jagger, S. trifoliorum Erikss., and the undescribed species Sclerotinia species 1. Sclerotinia species 1 is an important cause of disease in vegetables in Alaska (16) and has been found in association with wild Taraxacum sp., Caltha palustris, and Aconitum septentrionalis in Norway (7). It is morphologically indistinguishable from S. sclerotiorum, but it was shown to be a distinct species based on distinctive polymorphisms in sequences from internal transcribed spacer 2 (ITS2) of the nuclear ribosomal repeat (7). The other three species have been delimited using morphological, cytological, biochemical, and molecular characters (3, 8, 9, 10, 12, 15). Interestingly, given that the ITS is sufficiently polymorphic in many fungal genera to resolve species, in Sclerotinia, only species 1 and S. trifoliorum are distinguished by characteristic ITS sequence polymorphisms; S. sclerotiorum and S. minor cannot be distinguished based on ITS sequence (2, 7).Sclerotinia sclerotiorum is a necrotrophic pathogen with a broad host range (1). S. minor has a more restricted host range but causes disease in a variety of important crops such as lettuce, peanut, and sunflower crops (11). S. trifoliorum has a much narrower host range, limited to the Fabaceae (3, 8, 9). Sclerotial and ascospore characteristics also serve to differentiate among the three species. Sclerotinia minor has small sclerotia that develop throughout the colony in vitro and aggregate to form crusts on the host, while the sclerotia of S. sclerotiorum and S. trifoliorum are large and form at the colony periphery in vitro, remaining separate on the host (8, 9). The failure of an isolate to produce sclerotia or apothecia in vitro is not unusual, especially after serial cultivation (8). The presence of dimorphic, tetranucleate ascospores characterizes S. trifoliorum, while S. sclerotiorum and S. minor both have uniformly sized ascospores that are binucleate and tetranucleate, respectively (9, 14).With the apparent exception of Sclerotinia species 1, morphological characteristics are sufficient to delimit Sclerotinia species given that workers have all manifestations of the life cycle in hand. In cultures freshly isolated from infected plants, investigators usually have mycelia and sclerotia but not apothecia. Restriction fragment length polymorphisms (RFLPs) in ribosomal DNA (rDNA) are diagnostic for Sclerotinia species (3, 10), but the assay requires cloned probes (usually accessed from other laboratories) hybridized to Southern blots from vertical gels, an impractical procedure for large samples. We have analyzed sequence data from previous phylogenetic studies (2) and have identified diagnostic variation for the rapid identification of the four Sclerotinia species. The single nucleotide polymorphism (SNP) assay that we report here is amenable to a high throughput of samples and requires only PCR amplification with a standard set of primers and oligonucleotide hybridizations to Southern blots in a dot format.The SNP assay was performed using two independent sets of species-specific oligonucleotide probes, all with SNP sites shown to differentiate the four Sclerotinia species (Fig. (Fig.1).1). A panel of 49 anonymously coded isolates (Table (Table1)1) was screened using these species-specific SNP probes, as outlined in Fig. Fig.1.1. The assay was validated by comparison to Southern hybridizations of EcoRI-digested genomic DNA hybridized with pMF2, a plasmid probe containing the portion of the rDNA repeat with the 18S, 5.8S, and 26S rRNA cistrons of Neurospora crassa (4, 10).Open in a separate windowFIG. 1.Protocol for the SNP-based identification of Sclerotinia species, with diagnostic SNP sites underlined and in boldface type for each hybridization probe.

TABLE 1.

Isolates and hybridization results for all SNP-based oligonucleotide probesf
Collector''s isolateAnonymous codePrescreened presumed species identityOriginHostSpecies-specific SNP
IGS50CAL448 S.trifolCAL124CAL448 S.minorRAS148CAL446 S.sp1CAL19ACAL19BCAL448 S.sclero
LMK1849Botrytis cinereaOntario, CanadaAllium cepa
FA2-13Sclerotinia minorNorth CarolinaArachis hypogaea++
W15Sclerotinia minorNorth CarolinaCyperus esculentus++
W1030Sclerotinia minorNorth CarolinaOenothra laciniata++
PF1-138Sclerotinia minorNorth CarolinaArachis hypogaea++
PF18-49714Sclerotinia minorOklahomaArachis hypogaea++
PF17-48246Sclerotinia minorOklahomaArachis hypogaea++
PF19-51948Sclerotinia minorOklahomaArachis hypogaea++
LF-2720Sclerotinia minorUnited StatesLactuca sativa++
AR12811Sclerotinia sclerotiorumArgentinaArachis hypogaea++
AR128216Sclerotinia sclerotiorumArgentinaArachis hypogaea++
LMK2116Sclerotinia sclerotiorumCanadaBrassica napus++
LMK5725Sclerotinia sclerotiorumNorwayRanunculus ficaria++
LMK75415Sclerotinia sclerotiorumNorwayRanunculus ficaria++
UR1939Sclerotinia sclerotiorumUruguayLactuca sativa++
UR4789Sclerotinia sclerotiorumUruguayLactuca sativa++
CA90132Sclerotinia sclerotiorumCaliforniaLactuca sativa++
CA99540Sclerotinia sclerotiorumCaliforniaLactuca sativa++
CA104441Sclerotinia sclerotiorumCaliforniaLactuca sativa++
1980a34Sclerotinia sclerotiorumNebraskaPhaseolus vulgaris++
Ss00113Sclerotinia sclerotiorumNew YorkbGlycine max++
Ssp00531Sclerotinia sclerotiorumNew YorkGlycine max++
H02-V2833Sclerotinia species 1AlaskacUnknown vegetable crop++
H01-V1426Sclerotinia species 1AlaskaUnknown vegetable crop++
LMK74521Sclerotinia species 1NorwayTaraxacum sp.++
02-2611Sclerotinia trifoliorumFinlanddTrifolium pratense+
06-1429Sclerotinia trifoliorumFinlandTrifolium pratense++
2022Sclerotinia trifoliorumFinlandTrifolium pratense++
2-L945Sclerotinia trifoliorumFinlandTrifolium pratense++
3-A524Sclerotinia trifoliorumFinlandTrifolium pratense
5-L912Sclerotinia trifoliorumFinlandTrifolium pratense++
K14Sclerotinia trifoliorumFinlandTrifolium pratense++
K237Sclerotinia trifoliorumFinlandTrifolium pratense++
L-11223Sclerotinia trifoliorumFinlandTrifolium pratense++
L-11944Sclerotinia trifoliorumFinlandTrifolium pratense++
LMK3619Sclerotinia trifoliorumTasmaniaTrifolium repens++
Ssp00118Sclerotinia trifoliorumNew YorkLotus corniculatus++
Ssp00210Sclerotinia trifoliorumNew YorkLotus corniculatus++
Ssp00328Sclerotinia trifoliorumNew YorkLotus corniculatus++
Ssp00436Sclerotinia trifoliorumNew YorkLotus corniculatus++
LMK4743Sclerotinia trifoliorumVirginiaMedicago sativa++
MBRS-127UnknownAustraliaeBrassica spp.++
MBRS-27UnknownAustraliaBrassica spp.++
MBRS-342UnknownAustraliaBrassica spp.++
MBRS-522UnknownAustraliaBrassica spp.++
WW-135UnknownAustraliaBrassica spp.++
WW-28UnknownAustraliaBrassica spp.++
WW-317UnknownAustraliaBrassica spp.++
WW-447UnknownAustraliaBrassica spp.++
Open in a separate windowaThe annotated genome for S. sclerotiorum strain 1980 (ATCC 18683) is publicly available through the Broad Institute, Cambridge, MA (http://www.broad.mit.edu/annotation/genome/sclerotinia_sclerotiorum/Home.html).bAll isolates from New York were provided by Gary C. Bergstrom, Cornell University, Ithaca, NY. Isolates Ss001 and Ssp005 were submitted as S. sclerotiorum, and Ssp001 through Ssp004 were submitted as S. trifoliorum.cAll isolates from Alaska, submitted as Sclerotinia species 1, were provided by Lori Winton, USDA-ARS Subarctic Agricultural Research Unit, University of Alaska, Fairbanks.dAll isolates from Finland, submitted as S. trifoliorum, were provided by Tapani Yli-Mattila, University of Turku, Turku, Finland.eAll isolates from Australia, presumed to be S. sclerotiorum but requiring species confirmation, were provided by Martin Barbetti, DAF Plant Protection Branch, South Perth, Australia.fThe probes that are diagnostic for S. minor, S. sclerotiorum, S. trifoliorum, and Sclerotinia species 1 are listed, with a “+” indicating a positive hybridization for the probe and a “−” indicating no hybridization of the probe.  相似文献   

15.
Arthrobacter sp. strain JBH1 was isolated from nitroglycerin-contaminated soil by selective enrichment. Detection of transient intermediates and simultaneous adaptation studies with potential intermediates indicated that the degradation pathway involves the conversion of nitroglycerin to glycerol via 1,2-dinitroglycerin and 1-mononitroglycerin, with concomitant release of nitrite. Glycerol then serves as the source of carbon and energy.Nitroglycerin (NG) is manufactured widely for use as an explosive and a pharmaceutical vasodilator. It has been found as a contaminant in soil and groundwater (7, 9). Due to NG''s health effects as well as its highly explosive nature, NG contamination in soils and groundwater poses a concern that requires remedial action (3). Natural attenuation and in situ bioremediation have been used for remediation in soils contaminated with certain other explosives (16), but the mineralization of NG in soil and groundwater has not been reported.To date, no pure cultures able to grow on NG as the sole carbon, energy, and nitrogen source have been isolated. Accashian et al. (1) observed growth associated with the degradation of NG under aerobic conditions by a mixed culture originating from activated sludge. The use of NG as a source of nitrogen has been studied in mixed and pure cultures during growth on alternative sources of carbon and energy (3, 9, 11, 20). Under such conditions, NG undergoes a sequential denitration pathway in which NG is transformed to 1,2-dinitroglycerin (1,2DNG) or 1,3DNG followed by 1-mononitroglycerin (1MNG) or 2MNG and then glycerol, under both aerobic and anaerobic conditions (3, 6, 9, 11, 20), and the enzymes involved in denitration have been characterized in some detail (4, 8, 15, 21). Pure cultures capable of completely denitrating NG as a source of nitrogen when provided additional sources of carbon include Bacillus thuringiensis/cereus and Enterobacter agglomerans (11) and a Rhodococcus species (8, 9). Cultures capable of incomplete denitration to MNG in the presence of additional carbon sources were identified as Pseudomonas putida, Pseudomonas fluorescens (4), an Arthobacter species, a Klebsiella species (8, 9), and Agrobacterium radiobacter (20).Here we describe the isolation of bacteria able to degrade NG as the sole source of carbon, nitrogen, and energy. The inoculum for selective enrichment was soil historically contaminated with NG obtained at a facility that formerly manufactured explosives located in the northeastern United States. The enrichment medium consisted of minimal medium prepared as previously described (17) supplemented with NG (0.26 mM), which was synthesized as previously described (18). During enrichment, samples of the inoculum (optical density at 600 nm [OD600] ∼ 0.03) were diluted 1/16 in fresh enrichment medium every 2 to 3 weeks. Isolates were obtained by dilution to extinction in NG-supplemented minimal medium. Cultures were grown under aerobic conditions in minimal medium at pH 7.2 and 23°C or in tryptic soy agar (TSA; 1/4 strength).Early stages of enrichment cultures required extended incubation with lag phases of over 200 h and exhibited slow degradation of NG (less than 1 μmol substrate/mg protein/h). After a number of transfers over 8 months, the degradation rates increased substantially (2.2 μmol substrate/mg protein/h). A pure culture capable of growth on NG was identified based on 16S rRNA gene analysis (504 bp) as an Arthrobacter species with 99.5% similarity to Arthrobacter pascens (GenBank accession no. GU246730). Purity of the cultures was confirmed microscopically and by formation of a single colony type on TSA plates. 16S gene sequencing and identification were done by MIDI Labs (Newark, DE) and SeqWright DNA Technology Services (Houston, TX). The Arthrobacter cells stained primarily as Gram-negative rods with a small number of Gram-positive cocci (data not shown); Gram variability is also a characteristic of the closely related Arthrobacter globiformis (2, 19). The optimum growth temperature is 30°C, and the optimum pH is 7.2. Higher pH values were not investigated because NG begins to undergo hydrolysis above pH 7.5 (data not shown). The isolated culture can grow on glycerol, acetate, succinate, citrate, and lactate, with nitrite as the nitrogen source. Previous authors described an Arthrobacter species able to use NG as a nitrogen source in the presence of additional sources of carbon. However, only dinitroesters were formed, and complete mineralization was not achieved (9).To determine the degradation pathway, cultures of the isolated strain (5 ml of inoculum grown on NG to an OD600 of 0.3) were grown in minimal medium (100 ml) supplemented with NG at a final concentration of 0.27 mM. Inoculated bottles and abiotic controls were continuously mixed, and NG, 1,2DNG, 1,3DNG, 1MNG, 2MNG, nitrite, nitrate, CO2, total protein, and optical density were measured at appropriate intervals. Nitroesters were analyzed with an Agilent high-performance liquid chromatograph (HPLC) equipped with an LC-18 column (250 by 4.6 mm, 5 μm; Supelco) and a UV detector at a wavelength of 214 nm (13). Methanol-water (50%, vol/vol) was used as the mobile phase at a flow rate of 1 ml/min. Nitrite and nitrate were analyzed with an ion chromatograph (IC) equipped with an IonPac AS14A anion-exchange column (Dionex, CA) at a flow rate of 1 ml/min. Carbon dioxide production was measured with a Micro Oxymax respirometer (Columbus Instruments, OH), and total protein was quantified using the Micro BCA protein assay kit (Pierce Biotechnology, IL) according to manufacturer''s instructions. During the degradation of NG the 1,2DNG concentration was relatively high at 46 and 72 h (Fig. (Fig.1).1). 1,3DNG, detected only at time zero, resulted from trace impurities in the NG stock solution. Trace amounts of 1MNG appeared transiently, and trace amounts of 2MNG accumulated and did not disappear. Traces of nitrite at time zero were from the inoculum. The concentration of NG in the abiotic control did not change during the experiment (data not shown).Open in a separate windowFIG. 1.Growth of strain JBH1 on NG. ×, NG; ▵, 1,2DNG; ⋄, 1MNG; □, 2MNG; ○, protein.Results from the experiment described above were used to calculate nitrogen and carbon mass balances (Tables (Tables11 and and2).2). Nitrogen content in protein was approximated using the formula C5H7O2N (14). Because all nitrogen was accounted for throughout, we conclude that the only nitrogen-containing intermediate compounds are 1,2DNG and 1MNG, which is consistent with previous studies (6, 9, 20). The fact that most of the nitrogen was released as nitrite is consistent with previous reports of denitration catalyzed by reductase enzymes (4, 8, 21). The minor amounts of nitrate observed could be from abiotic hydrolysis (5, 12) or from oxidation of nitrite. Cultures supplemented with glycerol or other carbon sources assimilated all of the nitrite (data not shown).

TABLE 1.

Nitrogen mass balance
Time (h)% of total initial nitrogen by mass recovered ina:
Total recovery (%)
1MNG2MNG1,2DNG1,3DNGNGProteinNitriteNitrate
0NDbND0.9 ± 0.70.8 ± 0.682 ± 5.20.8 ± 0.214 ± 0.70.8 ± 0.3100 ± 5.3
460.1 ± 0.00.8 ± 0.27.9 ± 0.4ND35 ± 3.62.0 ± 0.549 ± 1.11.7 ± 0.096 ± 4.2
720.1 ± 0.00.9 ± 0.24.3 ± 4.2ND5.0 ± 0.43.3 ± 0.281 ± 4.23.9 ± 1.998 ± 6.8
94ND0.6 ± 0.4NDND0.6 ± 0.43.2 ± 0.095 ± 102.6 ± 1.6102 ± 10
Open in a separate windowaData represent averages of four replicates ± standard deviations.bND, not detected.

TABLE 2.

Carbon mass balance
Time (h)% of total initial carbon by mass recovered in:
Total recovery (%)
1MNGa2MNGa1,2DNGa1,3DNGaNGaProteinaCO2b
0NDcND1.6 ± 1.21.9 ± 0.492 ± 5.84.4 ± 0.9100 ± 8.4
460.5 ± 0.22.6 ± 0.613 ± 0.7ND39 ± 3.913 ± 3.028 ± 5.796 ± 14.1
720.4 ± 0.02.9 ± 0.77.3 ± 7.0ND5.6 ± 0.422 ± 1.259 ± 8.397 ± 17.6
94ND2.8 ± 0.3NDND0.8 ± 0.518 ± 0.371 ± 4.593 ± 5.6
Open in a separate windowaData represent averages of four replicates ± standard deviations.bData represent averages of duplicates ± standard deviations.cND, not detected.In a separate experiment cells grown on NG were added to minimal media containing 1,3DNG, 1,2DNG, 1MNG, or 2MNG and degradation over time was measured. 1,2DNG, 1,3DNG, and 1MNG were degraded at rates of 6.5, 3.8, and 8 μmol substrate/mg protein/hour. No degradation of 2MNG was detected (after 250 h), which indicates that 2MNG is not an intermediate in a productive degradation pathway. Because 1,3DNG was not observed at any point during the degradation of NG and its degradation rate is approximately one-half the degradation rate of 1,2DNG, it also seems not to be part of the main NG degradation pathway used by Arthrobacter sp. strain JBH1. The above observations indicate that the degradation pathway involves a sequential denitration of NG to 1,2DNG, 1MNG, and then glycerol, which serves as the source of carbon and energy (Fig. (Fig.2).2). The productive degradation pathway differs from that observed by previous authors using both mixed (1, 3, 6) and pure cultures (4, 9, 11, 20), in which both 1,3- and 1,2DNG were intermediates during NG transformation. Additionally, in previous studies both MNG isomers were produced regardless of the ratio of 1,2DNG to 1,3DNG (3, 4, 6, 9, 20). Our results indicate that the enzymes involved in denitration of NG in strain JBH1 are highly specific and catalyze sequential denitrations that do not involve 1,3DNG or 2MNG. Determination of how the specificity avoids misrouting of intermediates will require purification and characterization of the enzyme(s) involved.Open in a separate windowFIG. 2.Proposed NG degradation pathway.Mass balances of carbon and nitrogen were used to determine the following stoichiometric equation that describes NG mineralization by Arthrobacter sp. strain JBH1: 0.26C3H5(ONO2)3 + 0.33O2 → 0.03C5H7O2N + 0.63CO2 + 0.75NO2 + 0.75H+ + 0.17H2O. The result indicates that most of the NG molecule is being used for energy. The biomass yield is relatively low (0.057 mg protein/mg NG), with an fs (fraction of reducing equivalents of electron donor used for protein synthesis) of 0.36 (10), which is low compared to the aerobic degradation of other compounds by pure cultures, for which fs ranges between 0.4 and 0.6 (10, 14). The results are consistent with the requirement for relatively large amounts of energy during the initiation of the degradation mechanism (each denitration probably requires 1 mole of NADH or NADPH [21]).Although NG degradation rates were optimal at pH 7.2, they were still substantial at values as low as 5.1. The results suggest that NG degradation is possible even at low pH values typical of the subsurface at sites where explosives were formerly manufactured or sites where nitrite production lowers the pH.NG concentrations above 0.5 mM are inhibitory, but degradation was still observed at 1.2 mM (data not shown). The finding that NG can be inhibitory to bacteria at concentrations that are well below the solubility of the compound is consistent with those of Accashian et al. (1) for a mixed culture.The ability of Arthrobacter sp. strain JBH1 to grow on NG as the carbon and nitrogen source provides the basis for a shift in potential strategies for natural attenuation and bioremediation of NG at contaminated sites. The apparent specificity of the denitration steps raises interesting questions about the evolution of the pathway.  相似文献   

16.
Resistance of greenhouse-selected strains of the cabbage looper, Trichoplusia ni, to Bacillus thuringiensis subsp. kurstaki was countered by a hybrid strain of B. thuringiensis and genetically modified toxins Cry1AbMod and Cry1AcMod, which lack helix α-1. Resistance to Cry1AbMod and Cry1AcMod was >100-fold less than resistance to native toxins Cry1Ab and Cry1Ac.Insecticidal proteins from Bacillus thuringiensis are used widely for pest control, but evolution of resistance by pests can reduce their efficacy (3, 4, 6, 14). Resistance to B. thuringiensis toxins has been reported in field populations of four species of Lepidoptera, one species in response to sprays (3, 14) and three species in response to transgenic crops (10, 15, 16). Here, we focus on understanding and countering resistance to sprays of Bacillus thuringiensis subsp. kurstaki that evolved in commercial greenhouse populations of the cabbage looper, Trichoplusia ni (7, 17).We compared responses to single toxins and formulations of B. thuringiensis by two resistant strains (GipBtR and GlenBtR) and two related susceptible strains (GipS and GlenS) of T. ni. All four strains were started by the collection of larvae in 2001 from commercial greenhouses near Vancouver in British Columbia, Canada (7). Resistance evolved in the greenhouses in response to repeated sprays of DiPel (7), a formulation of B. thuringiensis subsp. kurstaki strain HD1 containing Cry1Aa, Cry1Ab, Cry1Ac, and Cry2Aa (9). Previously reported concentrations required to kill 50% of larvae (LC50s) indicated that, relative to a susceptible laboratory strain, initial resistance to DiPel was 113-fold in the Gip population (labeled T2c in reference 7) and 24-fold in the Glen population (labeled P5 in reference 7).We reared larvae on a wheat germ diet (5) at 26°C on a light-to-dark schedule of 16 h:8 h. GipS and GlenS were reared on diet without B. thuringiensis toxins, which allowed resistance to decline (7). To maintain resistance, GipBtR and GlenBtR were reared each generation on a diet treated with 5 or 10 mg of DiPel WP (Abbott Laboratories, Ontario, Canada) per milliliter of diet (7). In bioassays, groups of five third-instar larvae were put in 60-ml plastic cups containing diet, and mortality was assessed after 3 days by gently probing larvae for movement.We used diet overlay bioassays to evaluate the toxicity to GipBtR and GipS of the protoxin forms of Cry1Ab, Cry1Ac, Cry1AbMod, and Cry1AcMod produced in B. thuringiensis strains (12). Cry1AbMod and Cry1AcMod are genetically engineered variants of Cry1Ab and Cry1Ac, respectively, each lacking 56 amino acids from the amino-terminal region, including helix α-1 (12). An 80-μl aliquot containing distilled water and toxin was dispensed evenly over the surfaces of 2 ml of diet (a mean surface area of 7.1 cm2) and allowed to dry. Fifty to 200 larvae from each strain were tested at five to eight concentrations of each toxin.We used diet incorporation bioassays (7) to evaluate the toxicities of DiPel and Agree WG (Certis, Columbia, MD) to GipS, GipBtR, GlenS, and GlenBtR. Agree is a formulation of hybrid strain GC91, which was created from the conjugation-like transfer of a plasmid from B. thuringiensis subsp. kurstaki strain HD191 into B. thuringiensis subsp. aizawai strain HD135, and it contains Cry1Ac, Cry1C, and Cry1D (1, 8). DiPel and Agree were diluted in distilled water and mixed into diet (7). Twenty-five to 50 larvae from each strain were tested at six to seven concentrations of DiPel and Agree.We used probit analysis (13) to estimate the LC50s and their 95% fiducial limits (FL), as well as the slopes of concentration-mortality lines and their standard errors. The mortality of larvae fed treated diet was not adjusted for the mortality of control larvae on untreated diet, because the control mortality was low (mean, 3.6%; range, 0 to 16%). LC50s with nonoverlapping 95% FL are significantly different. Resistance ratios were calculated as the LC50 of a resistant strain (GipBtR or GlenBtR) divided by the LC50 of its susceptible counterpart (GipS or GlenS).The genetically modified toxins Cry1AbMod and Cry1AcMod were much more effective than the native toxins Cry1Ab and Cry1Ac against larvae of T. ni from the resistant GipBtR strain (Table (Table1).1). Resistance ratios of GipBtR were 580 for Cry1Ab and 1,400 for Cry1Ac but only 5.5 for Cry1AbMod and 9.3 for Cry1AcMod (Table (Table1).1). Against GipBtR, the LC50 was 53-fold higher for Cry1Ab than for Cry1AbMod and 11-fold higher for Cry1Ac than for Cry1AcMod (Table (Table1).1). Against GipS, however, the LC50 was 2-fold higher for Cry1AbMod than for Cry1Ab and 14-fold higher for Cry1AcMod than for Cry1Ac (Table (Table11).

TABLE 1.

Responses of resistant (GipBtR and GlenBtR) and susceptible (GipS and GlenS) strains of T. ni to native toxins (Cry1Ab and Cry1Ac), modified toxins (Cry1AbMod and Cry1AcMod), and formulations (DiPel and Agree)
Toxin or formulationStrainNo. of larvaeLC50 (95% FL)aSlope ± SEResistance ratiob
Cry1AbGipBtR400180 (59-2,900)c0.41 ± 0.09580
GipS3760.30 (0.21-0.41)0.56 ± 0.06
Cry1AbModGipBtR4003.4 (2.6-4.6)0.52 ± 0.055.5
GipS3750.62 (0.51-0.75)0.99 ± 0.09
Cry1AcGipBtR60054 (35-110)d0.50 ± 0.071,400
GipS1,4500.038 (0.031-0.046)0.44 ± 0.02
Cry1AcModGipBtR6005.1 (4.4-5.8)0.85 ± 0.069.3
GipS1,1450.55 (0.47-0.64)0.60 ± 0.03
DiPelGipBtR12566 (21-420,000)e0.43 ± 0.17370
GipS1250.18 (0.08-0.27)0.73 ± 0.16
AgreeGipBtR3004.9 (3.6-7.7)0.81 ± 0.129.9
GipS3000.49 (0.42-0.57)1.4 ± 0.14
DiPelGlenBtR1503.2 (2.7-3.9)1.9 ± 0.2726
GlenS1250.13 (0.05-0.17)1.5 ± 0.44
AgreeGlenBtR3002.0 (1.7-2.4)1.2 ± 0.125.9
GlenS2950.34 (0.29-0.39)1.4 ± 0.17
Open in a separate windowaConcentration that killed 50% and its 95% FL in mg protoxin per cm2 diet for toxins and mg formulation per ml of diet for DiPel and Agree.bLC50 of the resistant strain divided by the LC50 of the related susceptible strain for each toxin or formulation.cTotal of 17% mortality at the highest toxin concentration tested (17 mg protoxin/cm2 diet).dTotal of 35% mortality at the highest toxin concentration tested (23 mg protoxin/cm2 diet).eTotal of 24% mortality at the highest toxin concentration tested (15 mg DiPel/ml diet).Agree was more effective than DiPel against the two resistant strains GipBtR and GlenBtR (Table (Table1).1). Resistance ratios for DiPel were 370 for GipBtR and 26 for GlenBtR compared to resistance ratios for Agree, which were 9.9 for GipBtR and 5.9 for GlenBtR (Table (Table1).1). For the two resistant strains, LC50s were higher for DiPel than for Agree (13-fold higher against GipBtR and 1.6-fold higher against GlenBtR) (Table (Table1).1). Conversely, against the two susceptible strains, the LC50s were higher for Agree than for DiPel (2.7-fold higher against GipBtR and 2.6-fold higher against GlenBtR).The resistant GipBtR strain examined here (Table (Table1)1) and the resistant GLEN-Cry1Ac-BCS strain of T. ni studied by Wang et al. (17) had >500-fold resistance to Cry1Ab and Cry1Ac. Both GipBtR and GLEN-Cry1Ac-BCS were derived from greenhouse populations of T. ni that had been sprayed repeatedly with DiPel (7, 17), which contains Cry1Ab and Cry1Ac but not Cry1C or Cry1D (9). The GLEN-Cry1Ac-BCS strain had cross-resistance of only 2.5-fold to Cry1C and 2.4-fold to Cry1D (17). Agree contains Cry1C and Cry1D (8), which probably boosted its efficacy against GipBtR and GlenBtR (Table (Table11).The results here with Cry1AbMod and Cry1AcMod extend those of previous work indicating that modified toxins killed larvae of Manduca sexta in which susceptibility to Cry1Ab was decreased via RNA interference and also killed larvae of Pectinophora gossypiella that had laboratory-selected, genetically based resistance to Cry1Ab and Cry1Ac (12). The efficacy of Cry1AbMod and Cry1AcMod against greenhouse-selected T. ni suggests that the modified toxins may be useful against resistance that evolves in commercial agricultural settings. The results here also increase the number of lepidopteran species against which the modified toxins were effective to three, with each species representing a different family (Sphingidae, Gelechiidae, and Noctuidae). In the two other species, decreased susceptibility to native Cry1A toxins was mediated by alterations in a cadherin protein that binds Cry1Ac (2, 11, 12), whereas the role of cadherin in T. ni resistance has not been demonstrated or excluded.Similar to patterns observed with P. gossypiella (12), modified toxins were more effective than native toxins against resistant T. ni larvae, but native toxins were more effective than modified toxins against susceptible T. ni larvae (Table (Table1).1). This raises the intriguing possibility that combinations of native and modified toxins might be especially effective against populations with a mixture of susceptible and resistant individuals. In any case, the Cry1AMod toxins and hybrid B. thuringiensis products applied either jointly or separately may be useful for countering or delaying evolution of resistance in T. ni. However, further work is needed to determine how native and modified toxins interact when used in combination and how modified toxins perform in the greenhouse and field.  相似文献   

17.
18.
Riboflavin significantly enhanced the efficacy of simulated solar disinfection (SODIS) at 150 watts per square meter (W m−2) against a variety of microorganisms, including Escherichia coli, Fusarium solani, Candida albicans, and Acanthamoeba polyphaga trophozoites (>3 to 4 log10 after 2 to 6 h; P < 0.001). With A. polyphaga cysts, the kill (3.5 log10 after 6 h) was obtained only in the presence of riboflavin and 250 W m−2 irradiance.Solar disinfection (SODIS) is an established and proven technique for the generation of safer drinking water (11). Water is collected into transparent plastic polyethylene terephthalate (PET) bottles and placed in direct sunlight for 6 to 8 h prior to consumption (14). The application of SODIS has been shown to be a simple and cost-effective method for reducing the incidence of gastrointestinal infection in communities where potable water is not available (2-4). Under laboratory conditions using simulated sunlight, SODIS has been shown to inactivate pathogenic bacteria, fungi, viruses, and protozoa (6, 12, 15). Although SODIS is not fully understood, it is believed to achieve microbial killing through a combination of DNA-damaging effects of ultraviolet (UV) radiation and thermal inactivation from solar heating (21).The combination of UVA radiation and riboflavin (vitamin B2) has recently been reported to have therapeutic application in the treatment of bacterial and fungal ocular pathogens (13, 17) and has also been proposed as a method for decontaminating donor blood products prior to transfusion (1). In the present study, we report that the addition of riboflavin significantly enhances the disinfectant efficacy of simulated SODIS against bacterial, fungal, and protozoan pathogens.Chemicals and media were obtained from Sigma (Dorset, United Kingdom), Oxoid (Basingstoke, United Kingdom), and BD (Oxford, United Kingdom). Pseudomonas aeruginosa (ATCC 9027), Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 6633), Candida albicans (ATCC 10231), and Fusarium solani (ATCC 36031) were obtained from ATCC (through LGC Standards, United Kingdom). Escherichia coli (JM101) was obtained in house, and the Legionella pneumophila strain used was a recent environmental isolate.B. subtilis spores were produced from culture on a previously published defined sporulation medium (19). L. pneumophila was grown on buffered charcoal-yeast extract agar (5). All other bacteria were cultured on tryptone soy agar, and C. albicans was cultured on Sabouraud dextrose agar as described previously (9). Fusarium solani was cultured on potato dextrose agar, and conidia were prepared as reported previously (7). Acanthamoeba polyphaga (Ros) was isolated from an unpublished keratitis case at Moorfields Eye Hospital, London, United Kingdom, in 1991. Trophozoites were maintained and cysts prepared as described previously (8, 18).Assays were conducted in transparent 12-well tissue culture microtiter plates with UV-transparent lids (Helena Biosciences, United Kingdom). Test organisms (1 × 106/ml) were suspended in 3 ml of one-quarter-strength Ringer''s solution or natural freshwater (as pretreated water from a reservoir in United Kingdom) with or without riboflavin (250 μM). The plates were exposed to simulated sunlight at an optical output irradiance of 150 watts per square meter (W m−2) delivered from an HPR125 W quartz mercury arc lamp (Philips, Guildford, United Kingdom). Optical irradiances were measured using a calibrated broadband optical power meter (Melles Griot, Netherlands). Test plates were maintained at 30°C by partial submersion in a water bath.At timed intervals for bacteria and fungi, the aliquots were plated out by using a WASP spiral plater and colonies subsequently counted by using a ProtoCOL automated colony counter (Don Whitley, West Yorkshire, United Kingdom). Acanthamoeba trophozoite and cyst viabilities were determined as described previously (6). Statistical analysis was performed using a one-way analysis of variance (ANOVA) of data from triplicate experiments via the InStat statistical software package (GraphPad, La Jolla, CA).The efficacies of simulated sunlight at an optical output irradiance of 150 W m−2 alone (SODIS) and in the presence of 250 μM riboflavin (SODIS-R) against the test organisms are shown in Table Table1.1. With the exception of B. subtilis spores and A. polyphaga cysts, SODIS-R resulted in a significant increase in microbial killing compared to SODIS alone (P < 0.001). In most instances, SODIS-R achieved total inactivation by 2 h, compared to 6 h for SODIS alone (Table (Table1).1). For F. solani, C. albicans, ands A. polyphaga trophozoites, only SODIS-R achieved a complete organism kill after 4 to 6 h (P < 0.001). All control experiments in which the experiments were protected from the light source showed no reduction in organism viability over the time course (results not shown).

TABLE 1.

Efficacies of simulated SODIS for 6 h alone and with 250 μM riboflavin (SODIS-R)
OrganismConditionaLog10 reduction in viability at indicated h of exposureb
1246
E. coliSODIS0.0 ± 0.00.2 ± 0.15.7 ± 0.05.7 ± 0.0
SODIS-R1.1 ± 0.05.7 ± 0.05.7 ± 0.05.7 ± 0.0
L. pneumophilaSODIS0.7 ± 0.21.3 ± 0.34.8 ± 0.24.8 ± 0.2
SODIS-R4.4 ± 0.04.4 ± 0.04.4 ± 0.04.4 ± 0.0
P. aeruginosaSODIS0.7 ± 0.01.8 ± 0.04.9 ± 0.04.9 ± 0.0
SODIS-R5.0 ± 0.05.0 ± 0.05.0 ± 0.05.0 ± 0.0
S. aureusSODIS0.0 ± 0.00.0 ± 0.06.2 ± 0.06.2 ± 0.0
SODIS-R0.2 ± 0.16.3 ± 0.06.3 ± 0.06.3 ± 0.0
C. albicansSODIS0.2 ± 0.00.4 ± 0.10.5 ± 0.11.0 ± 0.1
SODIS-R0.1 ± 0.00.7 ± 0.15.3 ± 0.05.3 ± 0.0
F. solani conidiaSODIS0.2 ± 0.10.3 ± 0.00.2 ± 0.00.7 ± 0.1
SODIS-R0.3 ± 0.10.8 ± 0.11.3 ± 0.14.4 ± 0.0
B. subtilis sporesSODIS0.3 ± 0.00.2 ± 0.00.0 ± 0.00.1 ± 0.0
SODIS-R0.1 ± 0.10.2 ± 0.10.3 ± 0.30.1 ± 0.0
SODIS (250 W m−2)0.1 ± 0.00.1 ± 0.10.1 ± 0.10.0 ± 0.0
SODIS-R (250 W m−2)0.0 ± 0.00.0 ± 0.00.2 ± 0.00.4 ± 0.0
SODIS (320 W m−2)0.1 ± 0.10.1 ± 0.00.0 ± 0.14.3 ± 0.0
SODIS-R (320 W m−2)0.1 ± 0.00.1 ± 0.10.9 ± 0.04.3 ± 0.0
A. polyphaga trophozoitesSODIS0.4 ± 0.20.6 ± 0.10.6 ± 0.20.4 ± 0.1
SODIS-R0.3 ± 0.11.3 ± 0.12.3 ± 0.43.1 ± 0.2
SODIS, naturalc0.3 ± 0.10.4 ± 0.10.5 ± 0.20.3 ± 0.2
SODIS-R, naturalc0.2 ± 0.11.0 ± 0.22.2 ± 0.32.9 ± 0.3
A. polyphaga cystsSODIS0.4 ± 0.10.1 ± 0.30.3 ± 0.10.4 ± 0.2
SODIS-R0.4 ± 0.20.3 ± 0.20.5 ± 0.10.8 ± 0.3
SODIS (250 W m−2)0.0 ± 0.10.2 ± 0.30.2 ± 0.10.1 ± 0.2
SODIS-R (250 W m−2)0.4 ± 0.20.3 ± 0.20.8 ± 0.13.5 ± 0.3
SODIS (250 W m−2), naturalc0.0 ± 0.30.2 ± 0.10.1 ± 0.10.2 ± 0.1
SODIS-R (250 W m−2), naturalc0.1 ± 0.10.2 ± 0.20.6 ± 0.13.4 ± 0.2
Open in a separate windowaConditions are at an intensity of 150 W m−2 unless otherwise indicated.bThe values reported are means ± standard errors of the means from triplicate experiments.cAdditional experiments for this condition were performed using natural freshwater.The highly resistant A. polyphaga cysts and B. subtilis spores were unaffected by SODIS or SODIS-R at an optical irradiance of 150 W m−2. However, a significant reduction in cyst viability was observed at 6 h when the optical irradiance was increased to 250 W m−2 for SODIS-R only (P < 0.001; Table Table1).1). For spores, a kill was obtained only at 320 W m−2 after 6-h exposure, and no difference between SODIS and SODIS-R was observed (Table (Table1).1). Previously, we reported a >2-log kill at 6 h for Acanthamoeba cysts by using SODIS at the higher optical irradiance of 850 W m−2, compared to the 0.1-log10 kill observed here using the lower intensity of 250 W m−2 or the 3.5-log10 kill with SODIS-R.Inactivation experiments performed with Acanthamoeba cysts and trophozoites suspended in natural freshwater gave results comparable to those obtained with Ringer''s solution (P > 0.05; Table Table1).1). However, it is acknowledged that the findings of this study are based on laboratory-grade water and freshwater and that differences in water quality through changes in turbidity, pH, and mineral composition may significantly affect the performance of SODIS (20). Accordingly, further studies are indicated to evaluate the enhanced efficacy of SODIS-R by using natural waters of varying composition in the areas where SODIS is to be employed.Previous studies with SODIS under laboratory conditions have employed lamps delivering an optical irradiance of 850 W m−2 to reflect typical natural sunlight conditions (6, 11, 12, 15, 16). Here, we used an optical irradiance of 150 to 320 W m−2 to obtain slower organism inactivation and, hence, determine the potential enhancing effect of riboflavin on SODIS.In conclusion, this study has shown that the addition of riboflavin significantly enhances the efficacy of simulated SODIS against a range of microorganisms. The precise mechanism by which photoactivated riboflavin enhances antimicrobial activity is unknown, but studies have indicated that the process may be due, in part, to the generation of singlet oxygen, H2O2, superoxide, and hydroxyl free radicals (10). Further studies are warranted to assess the potential benefits from riboflavin-enhanced SODIS in reducing the incidence of gastrointestinal infection in communities where potable water is not available.  相似文献   

19.
This work demonstrates that Vibrio vulnificus biotype 2, serovar E, an eel pathogen able to infect humans, can become resistant to quinolone by specific mutations in gyrA (substitution of isoleucine for serine at position 83) and to some fluoroquinolones by additional mutations in parC (substitution of lysine for serine at position 85). Thus, to avoid the selection of resistant strains that are potentially pathogenic for humans, antibiotics other than quinolones must be used to treat vibriosis on farms.Vibrio vulnificus is an aquatic bacterium from warm and tropical ecosystems that causes vibriosis in humans and fish (http://www.cdc.gov/nczved/dfbmd/disease_listing/vibriov_gi.html) (33). The species is heterogeneous and has been subdivided into three biotypes and more than eight serovars (6, 15, 33; our unpublished results). While biotypes 1 and 3 are innocuous for fish, biotype 2 can infect nonimmune fish, mainly eels, by colonizing the gills, invading the bloodstream, and causing death by septicemia (23). The disease is rapidly transmitted through water and can result in significant economic losses to fish farmers. Surviving eels are immune to the disease and can act as carriers, transmitting vibriosis between farms. Interestingly, biotype 2 isolates belonging to serovar E have been isolated from human infections, suggesting that serovar E is zoonotic (2). This serovar is also the most virulent for fish and has been responsible for the closure of several farms due to massive losses of fish. A vaccine, named Vulnivaccine, has been developed from serovar E isolates and has been successfully tested in the field (14). Although the vaccine provides fish with long-term protection from vibriosis, at present its use is restricted to Spain. For this reason, in many fish farms around the world, vibriosis is treated with antibiotics, which are usually added to the food or water.Quinolones are considered the most effective antibiotics against human and fish vibriosis (19, 21, 31). These antibiotics can persist for a long time in the environment (20), which could favor the emergence of resistant strains under selective pressure. In fact, spontaneous resistances to quinolones by chromosomal mutations have been described for some gram-negative bacteria (10, 11, 17, 24, 25, 26). Therefore, improper antibiotic treatment of eel vibriosis or inadequate residue elimination at farms could favor the emergence of human-pathogenic serovar E strains resistant to quinolones by spontaneous mutations. Thus, the main objective of the present work was to find out if the zoonotic serovar of biotype 2 can become quinolone resistant under selective pressure and determine the molecular basis of this resistance.Very few reports on resistance to antibiotics in V. vulnificus have been published; most of them have been performed with biotype 1 isolates. For this reason, the first task of this study was to determine the antibiotic resistance patterns in a wide collection of V. vulnificus strains belonging to the three biotypes that had been isolated worldwide from different sources (see Table S1 in the supplemental material). Isolates were screened for antimicrobial susceptibility to the antibiotics listed in Table S1 in the supplemental material by the agar diffusion disk procedure of Bauer et al. (5), according to the standard guideline (9). The resistance pattern found for each isolate is shown in Table S1 in the supplemental material. Less than 14% of isolates were sensitive to all the antibiotics tested, and more than 65% were resistant to more than one antibiotic, irrespective of their biotypes or serovars. The most frequent resistances were to ampicillin-sulbactam (SAM; 65.6% of the strains) and nitrofurantoin (F; 60.8% of the strains), and the least frequent were to tetracycline (12%) and oxytetracycline (8%). In addition, 15% of the strains were resistant to nalidixic acid (NAL) and oxolinic acid (OA), and 75% of these strains came from fish farms (see Table S1 in the supplemental material). Thus, high percentages of strains of the three biotypes were shown to be resistant to one or more antibiotics, with percentages similar to those found in nonbiotyped environmental V. vulnificus isolates from Asia and North America (4, 27, 34). In those studies, resistance to antibiotics could not be related to human contamination. However, the percentage of quinolone-resistant strains found in our study is higher than that reported in other ones, probably due to the inclusion of fish farm isolates, where the majority of quinolone-resistant strains were concentrated. This fact suggests that quinolone resistance could be related to human contamination due to the improper use of these drugs in therapy against fish diseases, as has been previously suggested (18, 20). Although no specific resistance pattern was associated with particular biotypes or serovars, we found certain differences in resistance distribution, as shown in Table Table1.1. In this respect, biotype 3 displayed the narrowest spectrum of resistances and biotype 1 the widest. The latter biotype encompassed the highest number of strains with multiresistance (see Table S1 in the supplemental material). Within biotype 2, there were differences among serovars, with quinolone resistance being restricted to the zoonotic serovar (Table (Table11).

TABLE 1.

Percentage of resistant strains distributed by biotypes and serovars
V. vulnificusNo. of isolatesResistance distribution (%) for indicated antibiotica
SAMCTXENALFOTOASXT-TMPTE
Biotype 14975.524.514.330.683.78.230.628.68.2
Biotype 2 (whole)7258.313.912.54.247.29.74.24.213.9
Biotype 2
    Serovar E3630.312.139.127.315.29.1321.2
    Serovar A231009.118.2077.3009.14.6
    Nontypeable82914.325057.114.30014.3
    Serovar I5100202002020000
Biotype 3510002008000020
Open in a separate windowaCTX, cefotaxime; E, erythromycin; OT, oxytetracycline; SXT-TMP, sulfamethoxazole-trimethoprim; TE, tetracycline.The origin of resistance to quinolones in the zoonotic serovar was further investigated. To this end, spontaneous mutants of sensitive strains were selected from colonies growing within the inhibition halo around OA or NAL disks. Two strains (strain CG100 of biotype 1 and strain CECT 4604 of biotype 2, serovar E) developed isolated colonies within the inhibition zone. These colonies were purified, and maintenance of resistance was confirmed by serial incubations on medium without antibiotics. Using the disk diffusion method, CG100 was shown to be resistant to SAM and F and CECT 4604 to F (see Table S1 in the supplemental material). The MICs for OA, NAL, flumequine (UB), and ciprofloxacin (CIP) were determined by using the microplate assay according to the recommendations of the Clinical and Laboratory Standards Institute and the European Committee for Antimicrobial Susceptibility Testing of the European Society of Clinical Microbiology and Infectious Diseases (8, 12) and interpreted according to the European Committee for Antimicrobial Susceptibility Testing of the European Society of Clinical Microbiology and Infectious Diseases (13). The MICs for OA and NAL and for the fluoroquinolones UB and CIP exhibited by the mutants and their counterparts are shown in Table Table2.2. The inhibition zone diameters correlated well with MICs (data not shown). Mutants FR1, FR2, FR3, and FR4 were resistant to NAL and sensitive to the remaining quinolones, although they showed higher resistances than their parental strains (Table (Table2).2). Thus, these four mutants showed increases of 32- to 128-fold for NAL MICs, 4- to 8-fold for UB MICs, and 16-fold for CIP MICs (Table (Table2).2). The fifth mutant, FR5, was resistant to the two tested quinolones and to UB, a narrow-spectrum fluoroquinolone. This mutant, although sensitive to CIP, multiplied its MIC for this drug by 128 with respect to the parental strain (Table (Table22).

TABLE 2.

MICs for quinolones and fluoroquinolones and mutations in gyrA, gyrB, and parC detected in naturally and artificially induced resistant strains
Strain(s)MIC (μg ml−1) for indicated antibioticb
Gene mutationa
gyrA
gyrB
parC
Position
Codon changeaa changePosition
Codon changeaa changePosition
Codon changeaa change
NALOAUBCIPntaantaantaa
CG1000.5 (S)0.125 (S)0.0625 (S)0.0078 (S)
FR116 (R)1 (S)0.25 (S)0.125 (S)24883AGT→ATTS→INCNCNCNCNCNCNCNC
FR216 (R)1 (S)0.25 (S)0.125 (S)24883AGT→ATTS→INCNCNCNCNCNCNCNC
CECT 46040.25 (S)0.0625 (S)0.0625 (S)0.0078 (S)
FR332 (R)2 (S)0.5 (S)0.125 (S)24883AGT→ATTS→INCNCNCNCNCNCNCNC
FR432 (R)2 (S)0.5 (S)0.125 (S)24883AGT→ATTS→INCNCNCNCNCNCNCNC
FR5256 (R)16 (R)16 (R)1 (S)24883AGT→ATTS→I1156386GCA→ACAA→T25485TCA→TTAS→L
1236412CAG→CACQ→H
CECT 4602128 (R)8 (R)64 (R)1 (S)24883AGT→ATTS→INCNCNCNC25485TCA→TTAS→L
CECT 4603, CECT 4606, CECT 4608, PD-5, PD-12, JE32 (R)2 (S)<1 (S)<1 (S)24883AGT→ATTS→INCNCNCNCNCNCNCNC
CECT 486264 (R)2 (S)2 (S)<1 (S)24983AGT→AGAS→RNCNCNCNCNCNCNCNC
A2, A4, A5, A6, A7, PD-1, PD-364-128 (R)2 (S)4 (S)<1 (S)24883AGT→ATTS→INCNCNCNC338113GCA→GTAA→V
V1128 (R)4 (S)4 (S)<1 (S)24883AGT→ATTS→I1274425GAG→GGGE→GNCNCNCNC
1314438AAC→AAAN→K
Open in a separate windowaMutations in a nucleotide (nt) that gave rise to a codon change and to a change in amino acids (aa) are indicated. NC, no change detected.bThe resistance (R) or sensitivity (S) against the antibiotic determined according to the Clinical and Laboratory Standards Institute and the European Committee for Antimicrobial Susceptibility Testing of the European Society of Clinical Microbiology and Infectious Diseases (9, 13) is indicated in parentheses.For other gram-negative pathogens, quinolone resistance relies on spontaneous mutations in the gyrA, gyrB, parC, and parE genes that occur in a specific region of the protein known as the quinolone resistance-determining region (QRDR) (1, 11, 17, 24, 25, 26, 28). To test the hypothesis that mutations in these genes could also produce quinolone resistance in V. vulnificus, the QRDRs of these genes were sequenced in the naturally resistant strains and in the two sensitive strains that had developed resistances by selective pressure in vitro. The genomic DNA was extracted (3), and the QRDRs of gyrA, gyrB, parE, and parC were amplified using the primers shown in Table Table3,3, which were designed from the published genomes of biotype 1 strains YJ016 and CMCP6 (7, 22). PCR products of the predicted size were sequenced in an ABI 3730 sequencer (Applied Biosystems). Analysis of the QRDR sequences for gyrA, gyrB, parC, and parE of the mutants and the naturally resistant strains revealed that all naturally resistant strains, except one, shared a specific mutation at nucleotide position 248 with the laboratory-induced mutants (Table (Table2).2). This mutation gave rise to a change from serine to isoleucine at amino acid position 83. The exception was a mutation in the adjacent nucleotide that gave rise to a substitution of arginine for serine at the same amino acid position (Table (Table2).2). All the isolates that were resistant to the quinolone NAL had a unique mutation in the gyrA gene, irrespective of whether resistance was acquired naturally or in the laboratory (Table (Table2).2). This result strongly suggests that a point mutation in gyrA that gives rise to a change in nucleotide position 83 can confer resistance to NAL in V. vulnificus biotypes 1 and 2 and that this mutation could be produced by selective pressure under natural conditions. gyrA mutations consisting of a change from serine 83 to isoleucine have also been described in isolates of Aeromonas from water (17) and in diseased fish isolates of Vibrio anguillarum (26). Similarly, replacement of serine by arginine at amino acid position 83 in diseased fish isolates of Yersinia ruckeri (16) suggests that this mechanism of quinolone resistance is widespread among gram-negative pathogens. In all cases, these single mutations were also related to increased resistance to other quinolones (OA) and fluoroquinolones (UB and CIP) (Table (Table2),2), although the mutants remained sensitive according to the standards of the Clinical and Laboratory Standards Institute and the European Committee for Antimicrobial Susceptibility Testing of the European Society of Clinical Microbiology and Infectious Diseases (9, 13). A total of 50% of the naturally resistant strains, all of them of biotype 1, showed additional mutations that affected parC (a change in amino acid position 113) or gyrB (changes in amino acids at positions 425 and 438) (Table (Table2).2). These strains exhibited higher MICs for OA and fluoroquinolones (Table (Table2),2), although they were still sensitive to these drugs (9, 13). Finally, one isolate of biotype 2, serovar E, which was naturally resistant to quinolones and UB, showed a mutation in parC that gave rise to a substitution of leucine for serine at amino acid position 85 (Table (Table2).2). This mutation was shared only with the laboratory-induced mutant, also a biotype 2, serovar E mutant, which was resistant to the fluoroquinolone UB. The same mutation in parC had been previously described in diseased fish isolates of V. anguillarum that were highly resistant to quinolones (28), but this had not been related to fluoroquinolone resistance in Vibrio spp. nor in other gram-negative bacteria. These results strongly suggest that resistance to fluoroquinolones in V. vulnificus is related to specific mutations in gyrA and parC and that mutations in different positions for parC or in gyrB could contribute to increased resistance to quinolones and fluoroquinolones. Our results also agree with previous studies confirming that the acquisition of higher quinolone resistance is more probable when arising from a gyrA parC double mutation than from a gyrA gyrB double mutation (29).

TABLE 3.

Oligonucleotides used in this study
PrimerSequenceAnnealing temp (°C)Size (bp)
GyrAFGGCAACGACTGGAATAAACC55.8416
GyrARCAGCCATCAATCACTTCCGTC
ParCFCGCAAGTTCACCGAAGATGC56.6411
ParCRGGCATCCGCAACTTCACG
GyrBFCGACTTCTGGTGACGATGCG57.4642
GyrBRGACCGATACCACAACCTAGTG
ParEFGCCAGGTAAGTTGACCGATTG56.8512
ParERCACCCAGACCTTTGAATCGTTG
Open in a separate windowFinally, the evolutionary history for each protein was inferred from previously published DNA sequences of the whole genes from different Vibrio species after multiple sequence alignment with MEGA4 software (32) by applying the neighbor-joining method (30) with the Poisson correction (35). The distance tree for each whole protein showed a topology similar to the phylogenetic tree based on 16S rRNA analysis, with the two isolates of V. vulnificus forming a single group, closely related to Vibrio parahaemolyticus, Vibrio cholerae, V. anguillarum, and Vibrio harveyi (see Fig. S1A in the supplemental material). A second analysis was performed with the QRDR sequences of the different mutants and isolates of V. vulnificus (GenBank accession numbers FJ379836 to FJ379927) to infer the intraspecies relationships (see Fig. S1B in the supplemental material). This analysis showed that QRDRs of gyrA, gyrB, parC, and parE were highly homogeneous within V. vulnificus.In summary, the zoonotic serovar of V. vulnificus can mutate spontaneously to gain quinolone resistance, under selective pressure in vitro, due to specific mutations in gyrA that involve a substitution of isoleucine for serine at amino acid position 83. This mutation appears in biotype 2, serovar E diseased-fish isolates and biotype 1 strains, mostly recovered from fish farms. An additional mutation in parC, resulting in a substitution of lysine for serine at amino acid position 85, seems to endow partial fluoroquinolone resistance on biotype 2, serovar E strains. This kind of double mutation is present in diseased-fish isolates of the zoonotic serovar but not in resistant biotype 1 isolates, which show different mutations in gyrB or in parC that increase their resistance levels but do not make the strains resistant to fluoroquinolones. Thus, antibiotics other than quinolones should be used at fish farms to prevent the emergence and spread of quinolone resistances, especially to CIP, a drug widely recommended for human vibriosis treatment.  相似文献   

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