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Aurelio Cafaro Stefania Bellino Fausto Titti Maria Teresa Maggiorella Leonardo Sernicola Roger W. Wiseman David Venzon Julie A. Karl David O'Connor Paolo Monini Marjorie Robert-Guroff Barbara Ensoli 《Journal of virology》2010,84(17):8953-8958
The effects of the challenge dose and major histocompatibility complex (MHC) class IB alleles were analyzed in 112 Mauritian cynomolgus monkeys vaccinated (n = 67) or not vaccinated (n = 45) with Tat and challenged with simian/human immunodeficiency virus (SHIV) 89.6Pcy243. In the controls, the challenge dose (10 to 20 50% monkey infectious doses [MID50]) or MHC did not affect susceptibility to infection, peak viral load, or acute CD4 T-cell loss, whereas in the chronic phase of infection, the H1 haplotype correlated with a high viral load (P = 0.0280) and CD4 loss (P = 0.0343). Vaccination reduced the rate of infection acquisition at 10 MID50 (P < 0.0001), and contained acute CD4 loss at 15 MID50 (P = 0.0099). Haplotypes H2 and H6 were correlated with increased susceptibility (P = 0.0199) and resistance (P = 0.0087) to infection, respectively. Vaccination also contained CD4 depletion (P = 0.0391) during chronic infection, independently of the challenge dose or haplotype.Advances in typing of the major histocompatibility complex (MHC) of Mauritian cynomolgus macaques (14, 20, 26) have provided the opportunity to address the influence of host factors on vaccine studies (13). Retrospective analysis of 22 macaques vaccinated with Tat or a Tat-expressing adenoviral vector revealed that monkeys with the H6 or H3 MHC class IB haplotype were overrepresented among aviremic or controller animals, whereas macaques with the H2 or H5 haplotype clustered in the noncontrollers (12). More recently, the H6 haplotype was reported to correlate with control of chronic infection with simian immunodeficiency virus (SIV) mac251, regardless of vaccination (18).Here, we performed a retrospective analysis of 112 Mauritian cynomolgus macaques, which included the 22 animals studied previously (12), to evaluate the impact of the challenge dose and class IB haplotype on the acquisition and severity of simian/human immunodeficiency virus (SHIV) 89.6Pcy243 infection in 45 control monkeys and 67 monkeys vaccinated with Tat from different protocols (Table (Table11).
Open in a separate windowaAll animals were inoculated with the indicated dose of Tat plasmid DNA (pCV-tat [8], adenovirus-tat [Ad-tat] [27]) or protein, Gag protein, or empty vectors (pCV-0, adenovirus [Ad]) by the indicated route. Doses are in micrograms unless indicated otherwise.bAlum, aluminum phosphate (4); RIBI oil-in-water emulsions containing squalene, bacterial monophosphoryl lipid A, and refined mycobacterial products (4); Iscom, immune-stimulating complex (4); H1D are biocompatible anionic polymeric microparticles used for vaccine delivery (10, 12, 25a).cs.c., subcutaneous; i.m., intramuscular; i.d., intradermal; i.n., intranasal; i.t., intratracheal.dAll animals were inoculated intravenously with the indicated dose of the same SHIV89.6.Pcy243 stock.eAccording to the virological outcome upon challenge, monkeys were grouped as aviremic (A), controllers (C), or viremic (V).fBecause of the short follow-up, controller status could not be determined and all infected monkeys of the ISS-TG protocol were therefore considered viremic.gNA, not applicable. 相似文献
TABLE 1.
Summary of treatment, challenge dose, and outcome of infection in cynomolgus monkeysProtocol code | No. of monkeys | Immunogen (dose)a | Adjuvantb | Schedule of immunization (wk) | Routec | Challenged (MID50) | Virological outcomee | Reference(s) or source | ||
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A | C | V | ||||||||
ISS-ST | 6 | Tat (10) | Alum or RIBI | 0, 2, 6, 12, 15, 21, 28, 32, 36 | s.c., i.m. | 10 | 4 | 1 | 1 | 4, 17 |
ISS-ST | 1 | Tat (6) | None | 0, 5, 12, 17, 22, 27, 32, 38, 42, 48 | i.d. | 10 | 1 | 0 | 0 | 4, 17 |
ISS-PCV | 3 | pCV-tat (1 mg) | Bupivacaine + methylparaben | 0, 2, 6, 11, 15, 21, 28, 32, 36 | i.m. | 10 | 3 | 0 | 0 | 6 |
ISS-ID | 3 | Tat (6) | none | 0, 4, 8, 12, 16, 20, 24, 28, 39, 43, 60 | i.d. | 10 | 1 | 1 | 1 | B. Ensoli, unpublished data |
ISS-TR | 6 | Tat (10) | Alum-Iscom | 0, 2, 6, 11, 16, 21, 28, 32, 36 | s.c., i.d., i.m. | 10 | 4 | 2 | 0 | Ensoli, unpublished |
ISS-TGf | 3 | Tat (10) | Alum | 0, 4, 12, 22 | s.c. | 15 | 0 | 3 | Ensoli, unpublished | |
ISS-TG | 3 | Tatcys22 (10) | Alum | 15 | 0 | 3 | Ensoli, unpublished | |||
ISS-TG | 4 | Tatcys22 (10) + Gag (60) | Alum | 15 | 0 | 4 | Ensoli, unpublished | |||
ISS-TG | 4 | Tat (10) + Gag (60) | Alum | 15 | 0 | 4 | Ensoli, unpublished | |||
ISS-MP | 3 | Tat (10) | H1D-Alum | 0, 4, 12, 18, 21, 38 | s.c., i.m. | 15 | 0 | 2 | 1 | Ensoli, unpublished |
ISS-MP | 3 | Tat (10) | Alum | s.c. | 15 | 0 | 0 | 3 | Ensoli, unpublished | |
ISS-GS | 6 | Tat (10) | H1D-Alum | 0, 4, 12, 18, 21, 36 | s.c., i.m. | 15 | 1 | 3 | 2 | Ensoli, unpublished |
NCI-Ad-tat/Tat | 7 | Ad-tat (5 × 108 PFU), Tat (10) | Alum | 0, 12, 24, 36 | i.n., i.t., s.c. | 15 | 2 | 3 | 2 | Ensoli, unpublished |
NCI-Tat | 9 | Tat (6 and 10) | Alum/Iscom | 0, 2, 6, 11, 15, 21, 28, 32, 36 | s.c., i.d., i.m. | 15 | 2 | 4 | 3 | 12 |
ISS-NPT | 3 | pCV-tat (1 mg) | Bupivacaine + methylparaben-Iscom | 0, 2, 8, 13, 17, 22, 28, 46, 71 | i.m. | 20 | 0 | 0 | 3 | Ensoli, unpublished |
ISS-NPT | 3 | pCV-tatcys22 (1 mg) | Bupivacaine + methylparaben-Iscom | 0, 2, 8, 13, 17, 22, 28, 46, 71 | i.m. | 20 | 1 | 1 | 1 | |
Total vaccinated | 67 | 19 | 17 | 31 | ||||||
Naive | 11 | None | None | NAg | NA | 10 or 15 | 1 | 3 | 7 | |
Control | 34 | None, Ad, or pCV-0 | Alum, RIBI, H1D, Iscom or bupivacaine + methylparaben-Iscom | s.c., i.d., i.n., i.t., i.m. | 10, 15, or 20 | 5 | 13 | 16 | ||
Total controls | 45 | 6 | 16 | 23 | ||||||
Total | 112 | 25 | 33 | 54 |
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Luís Pinto Hajer Radhouani Céline Coelho Paulo Martins da Costa Roméo Sim?es Ricardo M. L. Brand?o Carmen Torres Gilberto Igrejas Patrícia Poeta 《Applied and environmental microbiology》2010,76(12):4118-4120
Extended-spectrum β-lactamase-containing Escherichia coli isolates were detected in 32 of 119 fecal samples (26.9%) from birds of prey at Serra da Estrela, and these isolates contained the following β-lactamases: CTX-M-1 (n = 13), CTX-M-1 plus TEM-1 (n = 14), CTX-M-1 plus TEM-20 (n = 1), SHV-5 (n = 1), SHV-5 plus TEM-1 (n = 2), and TEM-20 (n = 1).The high and sometimes excessive use of antibiotics is directly related to the great spread and development of bacterial antibiotic resistance, a problem in public health nowadays. Thus, many studies have been done in order to understand the mechanisms of bacterial drug resistance, as many multidrug-resistant bacterial strains have been detected in domestic animals and humans (12). Recent studies from our research group have focused on wild animals, trying to understand how they acquire resistance to antibiotics without direct contact (11, 17, 20). One of the most clinically relevant antimicrobial resistance mechanisms is constituted by extended-spectrum β-lactamases (ESBLs) (3, 16) harbored by Enterobacteriaceae, such as Escherichia coli, commensals of the gastrointestinal tract of animals and humans, serving as a reservoir for resistances, namely, to β-lactam antibiotics (1, 15). Most ESBLs are derivatives of TEM or SHV enzymes, but a new family of plasmid-mediated ESBLs, CTX-M, has also been reported worldwide in E. coli and other Enterobacteriaceae. Different variants of the β-lactamase TEM, as in the case of TEM-1, are often reported in cases of plasmid-mediated β-lactamase resistance (2, 5, 8, 14, 21, 22). In this study, fecal samples from wild birds of prey at the Serra da Estrela Natural Reserve were analyzed in order to detect ESBL-containing E. coli isolates and characterize the type of ESBL-encoding genes and other associated resistance genes and the corresponding phylogenetic groups.One hundred nineteen fecal samples from birds of prey were recovered from April to July 2008 and studied for the presence of ESBL-containing E. coli strains (Table (Table1).1). All the fecal samples were collected individually from each animal and obtained in collaboration with CERVAS (Center of Ecology, Collecting, Welcome and Handling of Wild Animals). This Center receives injured animals found in its natural environments, is located at the Serra da Estrela Natural Reserve, and belongs to the Portuguese Institute of the Nature Management. None of the animals had been previously fed by humans or had received antibiotics. Each bird that arrives at CERVAS is placed in an individual cage to be treated and released back into the environment. Sampling was performed by recovering the fecal material in the cloaca by using a sterile swab. They were immediately transferred to peptone water and manipulated during the first 24 h of arrival to the laboratory. Samples were seeded in Levine agar supplemented with cefotaxime (2 mg/liter), and colonies with typical E. coli morphology were selected, identified by classical biochemical methods and by the API 20E system (bioMérieux, La Balme Les Grottes, France), and further studied. Susceptibility to 16 antibiotics (ampicillin, amoxicillin plus clavulanic acid [AMC], cefoxitin, cefotaxime [CTX], ceftazidime [CAZ], aztreonam [ATM], imipenem, gentamicin, amikacin, tobramycin, streptomycin, nalidixic acid, ciprofloxacin, sulfamethoxazole-trimethoprim, tetracycline, and chloramphenicol) was tested by the disc diffusion method (7) for all recovered E. coli isolates. E. coli ATCC 25922 was used as a quality control strain. The double-disc diffusion test (CTX, CAZ, and ATM in the presence or absence of AMC) was performed to detect ESBL production (7).
Open in a separate windowaNorthern goshawk (Accipiter gentilis; n = 5); European scops owl (Otus scops; n = 5); Eurasian sparrowhawk (Accipiter nisus; n = 4); common crane (Grus grus; n = 2); honey buzzard (Pernis apivorus; n = 2); carrion crow (Corvus corone; n = 2); yellow-legged gull (Larus cachinnans; n = 2); little owl (Athene noctua; n = 2); Eurasian marsh harrier (Circus aeruginosus; n = 1); Egyptian vulture (Neophron percnopterus; n = 1); common kestrel (Falco tinnunculus; n = 1); azure-winged magpie (Cyanopica cyanus; n = 1); European magpie (Pica pica; n = 1); common swift (Apus apus; n = 1); and red kite (Milvus milvus; n = 1).The presence of genes encoding TEM, SHV, OXA, CTX-M, and CMY type β-lactamases was studied by specific PCRs (17). All obtained amplicons were sequenced on both strands, and sequences were compared with those included in the GenBank database and at the Lahey Clinic website (http://www.lahey.org/Studies/) to identify the β-lactamase genes.Non-β-lactamase genes [tetA/tetB, aadA, aac(3)-II/aac(3)-IV, aac(6′), cmlA, and sul1/sul2/sul3, associated with tetracycline, streptomycin, gentamicin, amikacin, chloramphenicol, and trimethoprim-sulfamethoxazole resistance, respectively] were also studied by PCR (17). The presence of the genes intI1 and intI2, encoding class 1 and 2 integrases, respectively, was studied by PCR. Positive and negative controls from the bacterial collection of the University of Trás-os-Montes and Alto Douro were used in all assays. The ESBL-positive isolates were classified into one of the four main phylogenetic groups, A, B1, B2, and D, by PCR as described previously based on the presence or absence of the chuA, yjaA, or tspE4.C2 gene (6).E. coli isolates were detected in Levine-CTX plates for 32 of the 119 samples (26.9%) from wild birds of prey at the Serra da Estrela Natural Reserve (Table (Table1).1). All 32 isolates recovered in this survey resulted in a positive ESBL screening test, which also indicated a resistant phenotype to CTX and/or CAZ. The β-lactamase genes detected in these isolates were as follows: blaCTX-M-1 (n = 13), blaCTX-M-1 plus blaTEM-1b (n = 8), blaCTX-M-1 plus blaTEM-1d (n = 6), blaCTX-M-1 plus blaTEM-20 (n = 1), blaSHV-5 plus blaTEM-1b (n = 2), blaSHV-5 (n = 1), and blaTEM-20 (n = 1) (Table (Table2).2). The blaCTX-M-1 gene was found in most of our ESBL-positive E. coli isolates (n = 28); this ESBL was also reported in studies of healthy poultry (13) and by our research group in healthy pets (9) and wild animals (10, 17). It is interesting to underline that approximately half of the blaCTX-M-1-containing E. coli isolates also harbored a variant of a blaTEM gene, concretely blaTEM-1b, blaTEM-1d, or blaTEM-20. The blaTEM-1b gene is the most frequent variant found in β-lactam-resistant E. coli isolates of food, humans, and healthy animals or in wild boars in other studies (4, 18). The blaTEM-1d genetic variant is relatively infrequent, although it has been detected in some other studies (19). Two ESBL-containing E. coli isolates were positive for the presence of blaTEM-20, and another three isolates carried the blaSHV-5 gene. Most ESBL-positive E. coli isolates of this study were classified into the A or B1 phylogenetic group (72%), although nine isolates which harbored blaCTX-M-1 with or without blaTEM-1b were included in the B2 phylogenetic group. The predominance of CTX-M-1-producing isolates belonging to phylogenetic group B2 is of great concern, as, in fact, this phylogenetic group often carries virulence determinants that are less frequently present in other phylogenetic groups. Similar results were found in wild boars by our research group (18).
Open in a separate windowaAMP, ampicillin; AMC, amoxicillin-clavulanic acid; ATM, aztreonam.bGEN, gentamicin; TOB, tobramycin; STR, streptomycin; TET, tetracycline; SXT, sulfamethoxazole-trimethoprim; NAL, nalidixic acid; CIP, ciprofloxacin; CHL, chloramphenicol; KAN, kanamycin.The common phenotype of resistance to multiple antibiotics among our ESBL-producing isolates is probably due to the coexistence of blaCTX-M-1 with other antibiotic resistance genes in the same plasmid, contributing to maintain ESBL-producing populations under different antibiotic selective pressures. With this study, it was possible to detect and characterize ESBL-producing E. coli isolates from birds of prey at the Serra da Estrela Natural Reserve in Portugal, with two types of ESBLs detected as CTX-M-1 and SHV-5. However, we cannot exclude the possibility that these wild animals had been exposed to fecal material of farm animals or even that of humans. These facts might be involved in the acquisition and dissemination of antibiotic-resistant bacteria even in the absence of direct antibiotic pressure and might explain the presence of ESBL-positive E. coli isolates. However, more studies should be performed to better understand the role of these animals in the spread of this type of resistance. 相似文献
TABLE 1.
Animal species in which ESBL-positive E. coli isolates were recoveredAnimal species | No. of tested animals | No. of animals with ESBL-positive E. coli isolates |
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Common buzzard (Buteo buteo) | 23 | 11 |
Common barn owl (Tyto alba) | 8 | 4 |
Eurasian tawny owl (Strix aluco) | 10 | 2 |
Booted eagle (Hieraaetus pennatus) | 11 | 5 |
Montagu''s harrier (Circus pygargus) | 5 | 2 |
Black kite (Milvus migrans) | 16 | 2 |
Eurasian black vulture (Aegypius monachus) | 2 | 1 |
Bonelli''s eagle (Hieraaetus fasciatus) | 2 | 1 |
Eurasian eagle owl (Bubo bubo) | 8 | 2 |
Common raven (Corvus corax) | 3 | 2 |
Other speciesa | 31 | 0 |
TABLE 2.
Characteristics of the ESBL-positive fecal E. coli isolates recovered from birds of preyIsolate no. | Animal species | Resistance pattern to β-lactam antibioticsa | β-Lactamase-encoding genes detected | Resistance pattern to non-β-lactam antibioticsb | Resistance genes for non-β-lactam antibiotics | Integron-related genes | Phylogenetic group |
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B1 | Buteo buteo | AMP-CTX | blaCTX-M-1 plus blaTEM-20 | CIP-NAL-TET-STR-SXT | tetA, sul2 | intI1 | B1 |
B2 | Buteo buteo | AMP-CTX-ATM | blaCTX-M-1 plus blaTEM-1D | CIP-NAL-TET-SXT | tetA, sul2 | intI1 | B1 |
B3 | Buteo buteo | AMP-CTX | blaCTX-M-1 | NAL-SXT | sul2, sul3 | intI1 | B1 |
B9 | Buteo buteo | AMP-CTX-ATM | blaCTX-M-1 plus blaTEM-1b | CIP-NAL-TET-SXT | tetA, sul2 | intI1 | B1 |
B10 | Buteo buteo | AMP-CTX | blaCTX-M-1 plus blaTEM-1b | CIP-NAL-TET-STR-SXT | tetA, sul2 | intI1 | B1 |
B14 | Buteo buteo | AMP-CTX-ATM | blaCTX-M-1 plus blaTEM-1D | NAL-TET-STR-CHL-SXT | tetA, sul2 | intI1 | A |
B15 | Buteo buteo | AMP-CTX | blaCTX-M-1 plus blaTEM-1D | NAL-TET-STR-SXT | aadA, tetA, sul2 | intI1, intI2 | A |
B17 | Buteo buteo | AMP-CTX | blaCTX-M-1 | CIP-NAL-STR-SXT | sul2, sul3 | intI1 | B2 |
B18 | Buteo buteo | AMP-CTX-CAZ | blaTEM-20 | NAL-TET-STR-SXT-KAN | tetA, sul2 | intI1 | B1 |
B19 | Buteo buteo | AMP-CTX | blaCTX-M-1 | CIP-NAL-STR-SXT | sul2, sul3 | intI1 | B2 |
B24 | Buteo buteo | AMP-CTX | blaCTX-M-1 | CIP-NAL-TET-SXT | tetA, sul3 | B2 | |
H6 | Hieraaetus pennatus | AMP-CTX | blaCTX-M-1 | NAL-TET-SXT | sul2, sul3 | intI1 | B1 |
H7 | Hieraaetus pennatus | AMP-CTX | blaCTXM-1 | TET-SXT | sul2 | intI1 | A |
H8 | Hieraaetus pennatus | AMP-CTX | blaCTX-M-1 plus blaTEM-1b | CIP-NAL-TET-SXT | tetA, sul2 | intI1 | B1 |
H11 | Hieraaetus pennatus | AMP-CTX | blaCTX-M-1 plus blaTEM-1d | CIP- NAL-TET-SXT | tetA, sul2 | A | |
H12 | Hieraaetus pennatus | AMP-CTX | blaCTX-M-1 plus blaTEM-1d | CIP- NAL-TET-SXT | tetA, sul2 | A | |
T4 | Tyto alba | AMP-CTX-ATM | blaCTX-M-1 plus blaTEM-1d | NAL-TET-STR-SXT | aadA, tetA, sul2 | intI1, intI2 | A |
T28 | Tyto alba | AMP-CTX-AMC | blaCTX-M-1 plus blaTEM-1b | NAL-TET-STR-SXT | aadA, tetA, sul1 | intI1 | B2 |
T29 | Tyto alba | AMP-CTX-ATM-CAZ-AMC | blaSHV-5 | CIP-NAL-TET-STR-CHL- SXT-KAN-TOB-GEN | aadA, sul1 | intI1 | A |
T30 | Tyto alba | AMP-CTX-ATM | blaCTX-M-1 plus blaTEM-1b | TET-STR-CHL-SXT-KAN | aadA, sul1 | intI1, intI2 | B1 |
S5 | Strix aluco | AMP-CTX | blaCTX-M-1 | NAL-TET-SXT | sul2, sul3 | intI1 | B1 |
S20 | Strix aluco | AMP-CTX | blaCTX-M-1 | CIP-NAL-STR-SXT | sul2, sul3 | B2 | |
M13 | Milvus migrans | AMP-CTX | blaCTX-M-1 plus blaTEM-1b | CIP- NAL-TET-STR-SXT | sul1, sul2 | B1 | |
M21 | Milvus migrans | AMP-CTX-ATM | blaCTX-M-1 | NAL-SXT | sul3 | intI1 | B1 |
C22 | Circus pygargus | AMP-CTX | blaCTX-M-1 plus blaTEM-1b | NAL-TET-STR-SXT | tetA | B2 | |
C23 | Circus pygargus | AMP-CTX | blaCTX-M-1 | NAL-TET-SXT | sul3 | intI1 | A |
BU26 | Bubo bubo | AMP-CTX | blaCTX-M-1 | CIP- NAL-SXT | sul3 | intI1 | B2 |
BU27 | Bubo bubo | AMP-CTX | blaCTX-M-1 | CIP-NAL-SXT | sul3 | intI1 | B2 |
CO31 | Corvus corax | AMP-CTX-ATM | blaSHV-5 plus blaTEM-1b | TET-STR-CHL-SXT | cmlA, tetA, sul3 | intI1 | B1 |
CO32 | Corvus corax | AMP-CTX-ATM-CAZ | blaSHV-5 plus blaTEM-1b | TET- STR-CHL-SXT | cmlA, aadA, tetA, sul3 | intI1 | B1 |
A16 | Aegypius monachus | AMP-CTX | blaCTX-M-1 plus blaTEM-1b | CIP-NAL-STR-CHL-SXT | sul1, sul2 | intI1 | B1 |
HI25 | Hieraaetus fasciatus | AMP-CTX | blaCTX-M-1 | CIP-NAL-SXT | sul3 | intI1 | B2 |
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Chris A. Whitehouse Carson Baldwin Rangarajan Sampath Lawrence B. Blyn Rachael Melton Feng Li Thomas A. Hall Vanessa Harpin Heather Matthews Marina Tediashvili Ekaterina Jaiani Tamar Kokashvili Nino Janelidze Christopher Grim Rita R. Colwell Anwar Huq 《Applied and environmental microbiology》2010,76(6):1996-2001
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Camilla L. Nesb? Rajkumari Kumaraswamy Marlena Dlutek W. Ford Doolittle Julia Foght 《Applied and environmental microbiology》2010,76(14):4896-4900
All cultivated Thermotogales are thermophiles or hyperthermophiles. However, optimized 16S rRNA primers successfully amplified Thermotogales sequences from temperate hydrocarbon-impacted sites, mesothermic oil reservoirs, and enrichment cultures incubated at <46°C. We conclude that distinct Thermotogales lineages commonly inhabit low-temperature environments but may be underreported, likely due to “universal” 16S rRNA gene primer bias.Thermotogales, a bacterial group in which all cultivated members are anaerobic thermophiles or hyperthermophiles (5), are rarely detected in anoxic mesothermic environments, yet their presence in corresponding enrichment cultures, bioreactors, and fermentors has been observed using metagenomic methods and 16S rRNA gene amplification (6) (see Table S1 in the supplemental material). The most commonly detected lineage is informally designated here “mesotoga M1” (see Table S1 in the supplemental material). PCR experiments indicated that mesotoga M1 sequences amplified inconsistently using “universal” 16S rRNA gene primers, perhaps explaining their poor detection in DNA isolated from environmental samples (see text and Table S2 in the supplemental material). We therefore designed three 16S rRNA PCR primer sets (Table (Table1)1) targeting mesotoga M1 bacteria and their closest cultivated relative, Kosmotoga olearia. Primer set A was the most successful set, detecting a wider diversity of Thermotogales sequences than set B and being more Thermotogales-specific than primer set C (Table (Table22).
Open in a separate windowaHeterogeneity hot spots identified in reference 1.
Open in a separate windowaSee the supplemental material for site and methodological details. NA, not applicable; ND, not determined.bThe number of OTUs observed at a 0.01 distance cutoff is given for each primer set. The numbers of clones with Thermotogales sequences are in parentheses. —, PCR was attempted but no Thermotogales sequences were obtained or the PCR consistently failed.c+, sequence(s) detected; −, not detected. For more information on the enrichments, see the text and Table S3 in the supplemental material.dFrom April to May 2004, the temperature at the depth where the sample was taken was 12°C (7).eThere were no water samples from DWH and HSAT available for enrichment cultures, and no DNA was available from HWH.fThis reservoir has been treated with biocides; moreover, at this site, the water is filtered before being reinjected into the reservoir.gTemperatures of the oil pool where the water sample was obtained. The HSAT facility receives water from two oil pools, one at 41°C and one at 50°C.hWe screened DNA from samples taken in 2006 and 2008 but detected the same sequences in both, so sequences from the two samples were pooled.iThe mesotoga M1 and Kosmotoga sequences from DWH and DF were >99% similar and were assembled into one sequence in Fig. Fig.11.jThis reservoir has been injected with water from a neighboring oil reservoir.Since the putative mesophilic Thermotogales have been overwhelmingly associated with polluted and hydrocarbon-impacted environments and mesothermic oil reservoirs are the only natural environments where mesotoga M1 sequences previously were detected (see Table S1 in the supplemental material), we selected four oil reservoirs with in situ temperatures of 14°C to 53°C and two temperate, chronically hydrocarbon-impacted sites for analysis (Table (Table2).2). Total community DNA was extracted, the 16S rRNA genes were amplified, cloned, and sequenced as described in the supplemental material. 相似文献
TABLE 1.
Primers targeting mesotoga M1 bacteria constructed and used in this studyPrimer | Sequence (5′ to 3′) | Position in mesotoga 16S rRNA gene | No. of heterogeneity hot spotsa | Potential primer match in other Thermotogales lineages |
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Primer set A | 1 (helix 17) | |||
NMes16S.286F | CGGCCACAAGGAYACTGAGA | 286 | Perfect match in Kosmotoga olearia. The last 7 or 8 nucleotides at the 3′ end are conserved in other Thermotogales lineages. | |
NMes16S.786R | TGAACATCGTTTAGGGCCAG | 786 | One 5′ mismatch in Kosmotoga olearia and Petrotoga mobilis; 2-4 internal and 5′ mismatches in other lineages | |
Primer set B | None | |||
BaltD.42F | ATCACTGGGCGTAAAGGGAG | 540 | Perfect match in Kosmotoga olearia; one or two 3′ mismatches in most other Thermotogales lineages | |
BaltD.494R | GTGGTCGTTCCTCTTTCAAT | 992 | No match in other Thermotogaleslineages. The primer is located in heterogeneity hot spot helices 33 and 34. This primer also fails to amplify some mesotoga M1 sequences. | |
Primer set C | 9 (all 9 regions) | |||
TSSU-3F | TATGGAGGGTTTGATCCTGG | 3 | Perfect match in Thermotoga spp., Kosmotoga olearia, and Petrotoga mobilis; two or three 5′ mismatches in other Thermotogales lineages; one 5′ mismatch to mesotoga M1 16S rRNA genes | |
Mes16S.R | ACCAACTCGGGTGGCTTGAC | 1390 | One 5′ mismatch in Kosmotoga olearia; 1-3 internal or 5′ mismatches in other Thermotogales lineages |
TABLE 2.
Mesotoga clade sequences detected in environmental samples and enrichment cultures screened in this studyaSite (abbreviation) | Temp in situ(°C) | Waterflooded | Environmental samplesb | Enrichment cultures | ||||||||
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Primer set A | Primer set B | Primer set C | Thermotogalesdetected by primer setc: | Lineage(s) detected | ||||||||
No. of OTUs (no. of clones) | Lineage | No. of OTUs (no. of clones) | Lineage | No. of OTUs (no. of clones) | Lineage | A | B | C | ||||
Sidney Tar Ponds sediment (TAR) | Temperate | NA | 1 (5) | M1 | 1 | M1 | — | — | + | + | + | M1, M2, M5 |
Oil sands settling basin tailings (05mlsb) | ∼12d | NA | — | — | 1 (6) | M1 | — | — | − | + | − | M1 |
Grosmont A produced water (GrosA) | 20 | No | 1 (15) | M1 | 1 (22) | M1 | 2 (14) | M1 | + | + | + | M1 |
Foster Creek produced water (FC) | 14 | No | 1 (21) | M1 | 1 (23) | M1 | 1 (1) | M1 | + | ND | − | M1 |
Oil field D wellhead water (DWH)e,f | 52-53g | Yes | 1 (14) | Kosmotogai | 1 (6) | M1i | 1 (1) | Kosmotogai | NA | NA | NA | NA |
Oil field D FWKO water (DF)f,h | 20-30 | Yes | 1 (45) | Kosmotogai | 1 (17) | M1i | — | — | + | + | − | M1, Kosmotoga, Petrotoga |
Oil field H FWKO water (HF)j | 30-32 | Yes | 7 (59) | M1, M2, M3, M4, Kosmotoga | 1 (29) | M1 | — | — | + | + | − | M1, Petrotoga |
Oil field H satellite water (HSAT)e,j | 41 and 50g | Yes | 1 (8) | M1 | — | — | 2 (16) | Kosmotoga, Thermotoga | NA | NA | NA | NA |
Oil field H wellhead water (HWH)e,j | 41 and 50g | Yes | NA | — | — | NA | NA | NA | + | + | + | M1, Petrotoga |
11.
Lisa Jacobsen Lisa Durso Tyrell Conway Kenneth W. Nickerson 《Applied and environmental microbiology》2009,75(13):4633-4635
Escherichia coli isolates (72 commensal and 10 O157:H7 isolates) were compared with regard to physiological and growth parameters related to their ability to survive and persist in the gastrointestinal tract and found to be similar. We propose that nonhuman hosts in E. coli O157:H7 strains function similarly to other E. coli strains in regard to attributes relevant to gastrointestinal colonization.Escherichia coli is well known for its ecological versatility (15). A life cycle which includes both gastrointestinal and environmental stages has been stressed by both Savageau (15) and Adamowicz et al. (1). The gastrointestinal stage would be subjected to acid and detergent stress. The environmental stage is implicit in E. coli having transport systems for fungal siderophores (4) as well as pyrroloquinoline quinone-dependent periplasmic glucose utilization (1) because their presence indicates evolution in a location containing fungal siderophores and pyrroloquinoline quinone (1).Since its recognition as a food-borne pathogen, there have been numerous outbreaks of food-borne infection due to E. coli O157:H7, in both ground beef and vegetable crops (6, 13). Cattle are widely considered to be the primary reservoir of E. coli O157:H7 (14), but E. coli O157:H7 does not appear to cause disease in cattle. To what extent is E. coli O157:H7 physiologically unique compared to the other naturally occurring E. coli strains? We feel that the uniqueness of E. coli O157:H7 should be evaluated against a backdrop of other wild-type E. coli strains, and in this regard, we chose the 72-strain ECOR reference collection originally described by Ochman and Selander (10). These strains were chosen from a collection of 2,600 E. coli isolates to provide diversity with regard to host species, geographical distribution, and electromorph profiles at 11 enzyme loci (10).In our study we compared the 72 strains of the ECOR collection against 10 strains of E. coli O157:H7 and six strains of E. coli which had been in laboratory use for many years (Table (Table1).1). The in vitro comparisons were made with regard to factors potentially relevant to the bacteria''s ability to colonize animal guts, i.e., acid tolerance, detergent tolerance, and the presence of the Entner-Doudoroff (ED) pathway (Table (Table2).2). Our longstanding interest in the ED pathway (11) derives in part from work by Paul Cohen''s group (16, 17) showing that the ED pathway is important for E. coli colonization of the mouse large intestine. Growth was assessed by replica plating 88 strains of E. coli under 40 conditions (Table (Table2).2). These included two LB controls (aerobic and anaerobic), 14 for detergent stress (sodium dodecyl sulfate [SDS], hexadecyltrimethylammonium bromide [CTAB], and benzalkonium chloride, both aerobic and anaerobic), 16 for acid stress (pH 6.5, 6.0, 5.0, 4.6, 4.3, 4.2, 4.1, and 4.0), four for the ability to grow in a defined minimal medium (M63 glucose salts with and without thiamine), and four for the presence or absence of a functional ED pathway (M63 with gluconate or glucuronate). All tests were done with duplicate plates in two or three separate trials. The data are available in Tables S1 to S14 in the supplemental material, and they are summarized in Table Table22.
Open in a separate window
Open in a separate windowaEight LB controls were run, two for each set of LB experiments: SDS, CTAB, benzalkonium chloride (BAC), and pH stress.bGrowth was measured as either +++, +, or 0 (good, poor, and none, respectively), with +++ being the growth achieved on the LB control plates. “Variable” means that two or three replicates did not agree. All experiments were done at 37°C.c“Anaerobic” refers to use of an Oxoid anaerobic chamber. Aerobic and anaerobic growth data are presented together when the results were identical and separately when the results were not the same or the anaerobic set had not been done. LB plates were measured after 1 (aerobic) or 2 (anaerobic) days, and the M63 plates were measured after 2 or 3 days.dCTAB used at 0.05, 0.2%, and 0.4%.eM63 defined medium (3) was supplemented with glucose, gluconate, or glucuronate, all at 0.2%.fIdentical results were obtained with and without 0.0001% thiamine.gND, not determined. 相似文献
TABLE 1.
E. coli strains used in this studyE. coli strain (n) | Source |
---|---|
ECOR strains (72) | Thomas Whittman |
Laboratory adapted (6) | |
K-12 Davis | Paul Blum |
CG5C 4401 | Paul Blum |
K-12 Stanford | Paul Blum |
W3110 | Paul Blum |
B | Tyler Kokjohn |
AB 1157 | Tyler Kokjohn |
O157:H7 (10) | |
FRIK 528 | Andrew Benson |
ATCC 43895 | Andrew Benson |
MC 1061 | Andrew Benson |
C536 | Tim Cebula |
C503 | Tim Cebula |
C535 | Tim Cebula |
ATCC 43889 | William Cray, Jr. |
ATCC 43890 | William Cray, Jr. |
ATCC 43888 | Willaim Cray, Jr. |
ATCC 43894 | William Cray, Jr. |
TABLE 2.
Physiological comparison of 88 strains of Escherichia coliGrowth medium or condition | Oxygenc | No. of strains with type of growthb
| |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
ECOR strains (n = 72)
| Laboratory strains (n = 6)
| O157:H7 strains (n = 10)
| |||||||||||
Good | Poor | None | Variable | Good | Poor | None | Variable | Good | Poor | None | Variable | ||
LB controla | Both | 72 | 0 | 0 | 0 | 6 | 0 | 0 | 0 | 10 | 0 | 0 | 0 |
1% SDS | Aerobic | 69 | 3 | 0 | 0 | 6 | 0 | 0 | 0 | 8 | 0 | 0 | 2 |
5% SDS | Aerobic | 68 | 4 | 0 | 0 | 6 | 0 | 0 | 0 | 8 | 2 | 0 | 0 |
1% SDS | Anaerobic | 53 | 15 | 4 | 0 | 2 | 3 | 1 | 0 | 1 | 7 | 0 | 2 |
5% SDS | Anaerobic | 0 | 68 | 4 | 0 | 0 | 4 | 2 | 0 | 0 | 7 | 0 | 4 |
CTABd (all) | Both | 0 | 0 | 72 | 0 | 0 | 0 | 6 | 0 | 0 | 0 | 10 | 0 |
0.05% BAC | Aerobic | 3 | 11 | 58 | 2 | 0 | 2 | 2 | 2 | 0 | 0 | 9 | 1 |
0.2% BAC | Aerobic | 0 | 1 | 71 | 0 | 1 | 0 | 5 | 0 | 0 | 0 | 10 | 0 |
0.05% BAC | Anaerobic | 2 | 3 | 67 | 0 | 0 | 1 | 5 | 0 | 0 | 0 | 9 | 1 |
0.2% BAC | Anaerobic | 0 | 0 | 72 | 0 | 0 | 0 | 6 | 0 | 0 | 0 | 10 | 0 |
pH 6.5 | Both | 72 | 0 | 0 | 0 | 6 | 0 | 0 | 0 | 10 | 0 | 0 | 0 |
pH 6 | Both | 72 | 0 | 0 | 0 | 6 | 0 | 0 | 0 | 10 | 0 | 0 | 0 |
pH 5 | Both | 70 | 2 | 0 | 0 | 6 | 0 | 0 | 0 | 9 | 0 | 0 | 1 |
pH 4.6 | Both | 70 | 2 | 0 | 0 | 6 | 0 | 0 | 0 | 10 | 0 | 0 | 0 |
pH 4.3 | Aerobic | 14 | 0 | 1 | 57 | 3 | 1 | 2 | 0 | 3 | 2 | 0 | 5 |
pH 4.3 | Anaerobic | 69 | 3 | 0 | 0 | 3 | 1 | 2 | 0 | 1 | 1 | 0 | 0 |
pH 4.1 or 4.2 | Aerobic | 0 | 0 | 72 | 0 | NDg | ND | ||||||
pH 4.0 | Both | 0 | 0 | 72 | 0 | 0 | 0 | 6 | 0 | 0 | 0 | 9 | 1 |
M63 with supplemente | |||||||||||||
Glucose | Aerobicf | 69 | 1 | 2 | 0 | 5 | 0 | 1 | 0 | 9 | 0 | 1 | 0 |
Glucose | Anaerobicf | 70 | 0 | 2 | 0 | 5 | 0 | 1 | 0 | 9 | 0 | 1 | 0 |
Gluconate | Both | 69 | 1 | 2 | 0 | 5 | 0 | 1 | 0 | 9 | 0 | 1 | 0 |
Glucuronate | Aerobic | 68 | 2 | 2 | 0 | 5 | 0 | 1 | 0 | 9 | 0 | 1 | 0 |
Glucuronate | Anaerobic | 69 | 1 | 2 | 0 | 5 | 0 | 1 | 0 | 9 | 0 | 1 | 0 |
12.
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).
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
TABLE 1.
Efficacies of simulated SODIS for 6 h alone and with 250 μM riboflavin (SODIS-R)Organism | Conditiona | Log10 reduction in viability at indicated h of exposureb | |||
---|---|---|---|---|---|
1 | 2 | 4 | 6 | ||
E. coli | SODIS | 0.0 ± 0.0 | 0.2 ± 0.1 | 5.7 ± 0.0 | 5.7 ± 0.0 |
SODIS-R | 1.1 ± 0.0 | 5.7 ± 0.0 | 5.7 ± 0.0 | 5.7 ± 0.0 | |
L. pneumophila | SODIS | 0.7 ± 0.2 | 1.3 ± 0.3 | 4.8 ± 0.2 | 4.8 ± 0.2 |
SODIS-R | 4.4 ± 0.0 | 4.4 ± 0.0 | 4.4 ± 0.0 | 4.4 ± 0.0 | |
P. aeruginosa | SODIS | 0.7 ± 0.0 | 1.8 ± 0.0 | 4.9 ± 0.0 | 4.9 ± 0.0 |
SODIS-R | 5.0 ± 0.0 | 5.0 ± 0.0 | 5.0 ± 0.0 | 5.0 ± 0.0 | |
S. aureus | SODIS | 0.0 ± 0.0 | 0.0 ± 0.0 | 6.2 ± 0.0 | 6.2 ± 0.0 |
SODIS-R | 0.2 ± 0.1 | 6.3 ± 0.0 | 6.3 ± 0.0 | 6.3 ± 0.0 | |
C. albicans | SODIS | 0.2 ± 0.0 | 0.4 ± 0.1 | 0.5 ± 0.1 | 1.0 ± 0.1 |
SODIS-R | 0.1 ± 0.0 | 0.7 ± 0.1 | 5.3 ± 0.0 | 5.3 ± 0.0 | |
F. solani conidia | SODIS | 0.2 ± 0.1 | 0.3 ± 0.0 | 0.2 ± 0.0 | 0.7 ± 0.1 |
SODIS-R | 0.3 ± 0.1 | 0.8 ± 0.1 | 1.3 ± 0.1 | 4.4 ± 0.0 | |
B. subtilis spores | SODIS | 0.3 ± 0.0 | 0.2 ± 0.0 | 0.0 ± 0.0 | 0.1 ± 0.0 |
SODIS-R | 0.1 ± 0.1 | 0.2 ± 0.1 | 0.3 ± 0.3 | 0.1 ± 0.0 | |
SODIS (250 W m−2) | 0.1 ± 0.0 | 0.1 ± 0.1 | 0.1 ± 0.1 | 0.0 ± 0.0 | |
SODIS-R (250 W m−2) | 0.0 ± 0.0 | 0.0 ± 0.0 | 0.2 ± 0.0 | 0.4 ± 0.0 | |
SODIS (320 W m−2) | 0.1 ± 0.1 | 0.1 ± 0.0 | 0.0 ± 0.1 | 4.3 ± 0.0 | |
SODIS-R (320 W m−2) | 0.1 ± 0.0 | 0.1 ± 0.1 | 0.9 ± 0.0 | 4.3 ± 0.0 | |
A. polyphaga trophozoites | SODIS | 0.4 ± 0.2 | 0.6 ± 0.1 | 0.6 ± 0.2 | 0.4 ± 0.1 |
SODIS-R | 0.3 ± 0.1 | 1.3 ± 0.1 | 2.3 ± 0.4 | 3.1 ± 0.2 | |
SODIS, naturalc | 0.3 ± 0.1 | 0.4 ± 0.1 | 0.5 ± 0.2 | 0.3 ± 0.2 | |
SODIS-R, naturalc | 0.2 ± 0.1 | 1.0 ± 0.2 | 2.2 ± 0.3 | 2.9 ± 0.3 | |
A. polyphaga cysts | SODIS | 0.4 ± 0.1 | 0.1 ± 0.3 | 0.3 ± 0.1 | 0.4 ± 0.2 |
SODIS-R | 0.4 ± 0.2 | 0.3 ± 0.2 | 0.5 ± 0.1 | 0.8 ± 0.3 | |
SODIS (250 W m−2) | 0.0 ± 0.1 | 0.2 ± 0.3 | 0.2 ± 0.1 | 0.1 ± 0.2 | |
SODIS-R (250 W m−2) | 0.4 ± 0.2 | 0.3 ± 0.2 | 0.8 ± 0.1 | 3.5 ± 0.3 | |
SODIS (250 W m−2), naturalc | 0.0 ± 0.3 | 0.2 ± 0.1 | 0.1 ± 0.1 | 0.2 ± 0.1 | |
SODIS-R (250 W m−2), naturalc | 0.1 ± 0.1 | 0.2 ± 0.2 | 0.6 ± 0.1 | 3.4 ± 0.2 |
13.
14.
Ana Roque Carmen Lopez-Joven Beatriz Lacuesta Laurence Elandaloussi Sariqa Wagley M. Dolores Furones Imanol Ruiz-Zarzuela Ignacio de Blas Rachel Rangdale Bruno Gomez-Gil 《Applied and environmental microbiology》2009,75(23):7574-7577
Presented here is the first report describing the detection of potentially diarrheal Vibrio parahaemolyticus strains isolated from cultured bivalves on the Mediterranean coast, providing data on the presence of both tdh- and trh-positive isolates. Potentially diarrheal V. parahaemolyticus strains were isolated from four species of bivalves collected from both bays of the Ebro delta, Spain.Gastroenteritis caused by Vibrio parahaemolyticus has been reported worldwide, though only sporadic cases have been reported in Europe (7, 14). The bacterium can be naturally present in seafood, but pathogenic isolates capable of inducing gastroenteritis in humans are rare in environmental samples (2 to 3%) (15) and are often not detected (10, 19, 20).The virulence of V. parahaemolyticus is based on the presence of a thermostable direct hemolysin (tdh) and/or the thermostable direct hemolysin-related gene (trh) (1, 5). Both are associated with gastrointestinal illnesses (2, 9).Spain is not only the second-largest producer in the world of live bivalve molluscs but also one of the largest consumers of bivalve molluscs, and Catalonia is the second-most important bivalve producer of the Spanish Autonomous Regions. Currently, the cultivation of bivalves in this area is concentrated in the delta region of the Ebro River. The risk of potentially pathogenic Vibrio spp. in products placed on the market is not assessed by existing legislative indices of food safety in the European Union, which emphasizes the need for a better knowledge of the prevalence of diarrheal vibrios in seafood products. The aim of this study was to investigate the distribution and pathogenic potential of V. parahaemolyticus in bivalve species exploited in the bays of the Ebro delta.Thirty animals of each species of Mytilus galloprovincialis, Crassostrea gigas, Ruditapes decussatus, and Ruditapes philippinarum were collected. They were sampled from six sites of the culture area, three in each bay of the Ebro River delta, at the beginning (40°37′112"N, 0°37′092"E [Alfacs]; 40°46′723"N, 0°43′943"E [Fangar]), middle (40°37′125"N, 0°38′570"E [Alfacs]; 40°46′666"N, 0°45′855"E [Fangar]), and end (40°37′309"N, 0°39′934"E [Alfacs]; 40°46′338"N, 0°44′941"E [Fangar]) of the culture polygon. Clams were sampled from only one site per bay as follows: in the Alfacs Bay from a natural bed of R. decussatus (40°37′44"N, 0°38′0"E) and in the Fangar Bay from an aquaculture bed of R. philippinarum (40°47′3"N, 0°43′8"E). In total, 367 samples were analyzed in 2006 (180 oysters, 127 mussels, 30 carpet shell clams, and 30 Manila clams) and 417 samples were analyzed in 2008 (178 oysters, 179 mussels, 30 carpet shell clams, and 30 Manila clams).All animals were individually processed and homogenized, and 1 ml of the homogenate was inoculated into 9 ml of alkaline peptone water (Scharlau, Spain). Following a 6-h incubation at 37°C, one loopful of the contents of each tube of alkaline peptone water was streaked onto CHROMagar vibrio plates (CHROMagar, France) and incubated for 18 h at 37°C. Mauve-purple colonies were purified, and each purified isolate was cryopreserved at −80°C (135 isolates in 2006 and 96 in 2008). From the initial homogenate portion, 100 μl was inoculated onto marine agar (Scharlau, Spain) and onto thiosulfate citrate-bile salts-sucrose agar (Scharlau, Spain) for total heterotrophic marine bacteria counts and total vibrio counts, respectively (Table (Table11).
Open in a separate windowaMg, Mytilus galloprovincialis; Cg, Crassostrea gigas; Rd, Ruditapes decussatus; Rp, R. phillipinarum; A, Alfacs; F, Fangar; ND, not determined; TCBS, thiosulfate citrate-bile salts-sucrose.Total DNA was extracted from each purified isolate using the Wizard genomic DNA purification kit (Promega), following the instructions of the manufacturer. A one-step PCR analysis was performed to identify/confirm which isolates were tl positive (species marker for V. parahaemolyticus). Further detection of the tdh or trh gene was carried out on all positive tl strains. All PCR analyses were carried out using the primers described by Bej et al. (2) with the following amplification conditions on the thermocycler (Eppendorf Mastercycler Personal): an initial denaturation at 95°C for 8 min, followed by 40 cycles of a 1-min denaturation at 94°C, annealing at 55°C for 1 min, elongation at 72° for 1 min, and a final extension of 10 min at 72°C. Positive and negative controls were included in all reaction mixtures: two positive controls, tl and tdh CAIM 1400 and trh CAIM 1772 (Collection of Aquatic Important Microorganisms [http://www.ciad.mx/caim/CAIM.html]), and negative control DNA-free molecular grade water (Sigma-Aldrich, Spain). Expected amplicons were visualized in 2% agarose gels stained with ethidium bromide.Fifty-eight isolates contained the gene tl in 2006 and 96 in 2008, which confirmed their identity as V. parahaemolyticus. In 2006, the distribution of the 58 isolates was as follows: 7 from 127 mussels, 34 from 180 oysters, and 17 from 30 R. decussatus clams. No tl-positive isolates were found in R. philippinarum. PCR analysis of the tl-positive isolates for the presence of the tdh or trh gene indicated that eight isolates contained the tdh gene and four contained the trh gene. In 2008, the source of the confirmed V. parahaemolyticus isolates was as follows: 31 from 88 oysters, 44 from 89 mussels, 9 from 30 R. decussatus clams, and 12 from 30 R. philippinarum clams. Of these, 17 were found to contain the tdh gene and 7 contained the trh gene. Two isolates (I806 and I1042) contained both toxigenic genes, tdh and trh.Putative tdh- and trh-positive PCR products were purified using the QIAquick PCR purification kit (Qiagen) following the manufacturer''s instructions and were sequenced bidirectionally by Macrogen Inc. Sequences were aligned using BioEdit (8) and analyzed using BLAST (National Center for Biotechnology Information). None of the toxigenic isolates was found positive by PCR analysis for the presence of open reading frame 8 of the phage 237 (16), a marker for the pandemic strain O3:K6.The isolates were fingerprinted by repetitive extragenic palindromic PCR (rep-PCR) as described previously (3), and the resulting electrophoretic band patterns were analyzed with the GelCompar II software (v4.5; Applied Maths). The similarity matrix was calculated with the Jaccard coefficient with a band position tolerance of 0.8%, and the dendrogram was constructed with the Ward algorithm. A high level of genomic diversity was found among the 32 toxigenic isolates characterized by rep-PCR. Three clonal groups were identified (those having identical rep-PCR band patterns) (Fig. 1a to c).Open in a separate windowFIG. 1.rep-PCR dendrogram of toxigenic isolates of V. parahaemolyticus isolated in the Ebro delta. Letters denote clonal groups of isolates.In vitro antibiotic susceptibility tests were performed using the diffusion disc test following a previously described protocol (18). The antibiotics used were gentamicin (10 μg), oxolinic acid (10 μg), amoxicillin (25 μg), polymyxin B (300 UI), vancomycin (30 μg), trimethoprim sulfamethoxazole (1.25/23.75 μg), nitrofurantoin (300 μg), doxycyclin (30 μg), ceftazidime (30 μg), streptomycin (10 μg), neomycin (30 UI), penicillin (6 μg), flumequine (30 μg), tetracycline (30 μg), ampicillin (10 μg), kanamycin (30 μg), ciprofloxacin (5 μg), and sulfonamide (300 μg). All tests were performed in duplicate. A Student t test for two samples with unequal variance was performed to compare the sensitivity of all 2006 isolates against the sensitivity of 2008 isolates for each antibiotic (Microsoft Office Excel 97-2003). Antibiogram results revealed a lower susceptibility in 2008 than in 2006, indicating a possible shift in overall susceptibility. Results from the t test indicated that significantly lower susceptibility in 2008 was detected (P ≤ 0.05; n = 36) for the following antibiotics: vancomycin, polymyxin B, ampicillin, amoxicillin, gentamicin, neomycin, trimethoprim sulfamethoxazole, nitrofurantoin, doxycyclin, ceftazidime, tetracycline, flumequine, and ciprofloxacin.The serological types for 27 strains were determined by the agglutination method using commercially available V. parahaemolyticus antisera (Denka Seiken Ltd.; Cosmos Biomedical Ltd, United Kingdom) following the manufacturer''s instructions. Potentially toxigenic V. parahaemolyticus isolates collected in 2006 were serologically heterogeneous (8 out of the 11 isolates) (Table (Table1).1). In isolates collected in 2008, results were more homogenous, with seven serotypes found among 19 isolates analyzed. The O3:K6 serotype was not detected in any of the strains analyzed, in agreement with the open reading frame 8 PCR results.The present study is the first to report the detection of potentially diarrheal V. parahaemolyticus strains isolated from cultured bivalves on Spanish Mediterranean coasts, providing data on the presence of both tdh- and trh-positive isolates. V. parahaemolyticus has previously been detected in several European countries (4, 13, 21, 22). A recent study carried out in Spain detected tdh-positive V. parahaemolyticus strains from patients who had consumed fresh oysters in a market in Galicia on the Atlantic coast of Spain (12) and potentially pathogenic V. parahaemolyticus strains have also been reported in France (17). These studies indicate that the risk of infections caused by V. parahaemolyticus in Europe is low compared to that in America or Asia (15). However, this risk could have been underestimated, since V. parahaemolyticus is not included in the current European surveillance programs, such as the European Network for Epidemiological Surveillance and Control of Communicable Diseases.Toxigenic V. parahaemolyticus strains detected in this study were genomically and serologically heterogeneous. The pandemic serotype O3:K6 was not detected, and although attempts to isolate O3:K6 from the environment and from seafood have not always been successful in previous studies reviewed by Nair and coauthors (15), this finding seems to be in agreement with the fact that no outbreak of diarrhea was observed in the area. Interestingly, isolates I806 and I1042 have been found positive for both tdh and trh in PCR tests. The coexistence of tdh and trh genes has already been reported in isolates from Japan, the United States, and Mexico (3, 6, 11, 19, 23). To our knowledge, no occurrence of an environmental isolate positive for both tdh and trh had previously been reported in Europe. All isolates tested were slightly different in their antibiotic resistance profiles. Typically, a high level of resistance could be determined. The detection of tdh- and/or trh-positive V. parahaemolyticus strains for the first time on the Mediterranean coast emphasizes the need to monitor for the presence of potentially diarrheal vibrios and bacterial gastroenteritis, and these data should be taken into consideration to revise the European legislation on the requirements for shellfish harvested for consumption in order to include the surveillance of these pathogens in Europe. 相似文献
TABLE 1.
Vibrio parahaemolyticus isolates, serotypes, and origins and total number of vibrios/heterotrophic bacteria contained in the bivalveaIsolate | Date of collection | Organism and site of origin | Temp (°C) | Salinity (‰) | Gene(s) | Serotype | Bacterial count using indicated medium (CFU ml−1) | |
---|---|---|---|---|---|---|---|---|
TCBS agar | Marine agar | |||||||
I745 | 8 August 2006 | Mg-F | 24.5 | 37 | tdh | ND | 1.5 × 104 | 1.2 × 104 |
I793 | 14 August 2006 | Cg-A | 25 | 35 | tdh | ND | 9.2 × 102 | 8.5 × 103 |
I805 | 14 August 2006 | Cg-A | 25 | 35 | tdh | O2:KUT | 7.2 × 102 | 9 × 103 |
I806 | 14 August 2006 | Cg-A | 25 | 35 | tdh and trh | O3:K33 | 1.9 × 103 | 4.6 × 103 |
I809 | 14 August 2006 | Cg-A | 25 | 35 | tdh | O2:K28 | 8 × 104 | 7.3 × 102 |
I678 | 4 July 2006 | Rd-A | 28.6 | 36 | tdh | O2:K28 | 3.1 × 105 | 2.5 × 105 |
I628 | 4 July 2006 | Rd-A | 28.6 | 36 | tdh | O4:KUT | 2.9 × 104 | 8.4 × 104 |
I775 | 8 August 2006 | Cg-A | 24.5 | 37 | tdh | ND | 4.21 × 103 | 1.1 × 104 |
I691 | 4 July 2006 | Rd-A | 28.6 | 36 | trh | O1:K32 | 2.2 × 105 | 2.6 × 105 |
I712 | 27 July 2006 | Mg-A | 29.4 | 35.5 | trh | O1:KUT | 8.6 × 103 | 8.4 × 103 |
I765 | 8 August 2006 | Cg-F | 24.5 | 37 | trh | O4:K34 | 1 × 104 | Uncountable |
I980 | 22 July 2008 | Cg-A | 26.7 | 33.5 | tdh | O1:K32 | 2.7 × 104 | 1.3 × 104 |
I981 | 22 July 2008 | Cg-A | 26.7 | 33.5 | trh | O1:KUT | 1 × 104 | 2.2 × 104 |
I993 | 22 July 2008 | Cg-A | 26.7 | 33.5 | tdh | O5:K17 | 3 × 103 | 1.1 × 104 |
I994 | 29 July 2008 | Mg-A | 27.7 | 37 | trh | O3:KUT | 3.4 × 103 | 7 × 103 |
I1031 | 5 August 2008 | Cg-F | 27.7 | 37 | tdh | O5:KUT | 5.5 × 104 | 3.3 × 104 |
I1034 | 5 August 2008 | Cg-F | 27.7 | 37 | tdh | O3:KUT | 8.7 × 104 | 4 × 104 |
I1040 | 5 August 2008 | Cg-F | 27.7 | 37 | tdh | O3:KUT | 1.6 × 104 | 3.2 × 104 |
I1042 | 5 August 2008 | Cg-F | 27.7 | 37 | tdh and trh | ND | 2.8 ×104 | 3 × 104 |
I1050 | 5 August 2008 | Cg-F | 27.7 | 37 | tdh | O1:KUT | 4.7 × 104 | 7.3 × 104 |
I1063 | 20 August 2008 | Mg-F | 25.9 | 36 | tdh | O3:KUT | 7.9 ×104 | 1.4 × 104 |
I1065 | 20 August 2008 | Mg-F | 25.9 | 36 | tdh | O2:KUT | 2.2 × 103 | 1.2 × 104 |
I1068 | 20 August 2008 | Mg-F | 25.9 | 36 | tdh | O5:KUT | 2.6 × 104 | 5.2 × 104 |
I1069 | 20 August 2008 | Mg-F | 25.9 | 36 | tdh | O3:KUT | 2.4 × 103 | 5.3 × 104 |
I1073 | 20 August 2008 | Mg-F | 25.9 | 36 | tdh | O5:KUT | 2.3 × 103 | 7.5 × 103 |
I1074 | 20 August 2008 | Mg-F | 25.9 | 36 | tdh | O3:KUT | 7.6 × 104 | 6.9 × 104 |
I1077 | 20 August 2008 | Mg-F | 25.9 | 36 | tdh | O4:KUT | 1.7 × 103 | 1.6 × 103 |
I1079 | 20 August 2008 | Mg-F | 25.9 | 36 | trh | O3:KUT | 2.5 × 103 | 1.1 × 104 |
I1092 | 20 August 2008 | Mg-F | 25.9 | 36 | tdh | ND | 1.7 × 103 | 1.6 × 103 |
I1130 | 25 August 2008 | Rd-A | 26.4 | 35 | tdh | ND | 1.7 × 104 | 3.8 × 104 |
I1143 | 25 August 2008 | Rd-A | 26.4 | 35 | tdh | ND | 1.1 × 104 | 1.9 × 104 |
I1165 | 25 August 2008 | Rd-A | 26.4 | 35 | trh | O2:KUT | 4.4 × 104 | 6.8 × 104 |
I1133 | 25 August 2008 | Rp-F | 25.5 | 36.5 | tdh | ND | 3.4 × 104 | 4 × 104 |
I1134 | 25 August 2008 | Rp-F | 25.5 | 36.5 | tdh | ND | 3.9 × 104 | 5.8 × 104 |
I1158 | 25 August 2008 | Rp-F | 25.5 | 36.5 | trh | O4:KUT | 6.6 × 104 | 4.7 × 104 |
I1161 | 25 August 2008 | Rp-F | 25.5 | 36.5 | trh | O3:KUT | 2.2 × 104 | 6.6 × 104 |
15.
Elian-Simplice Yaganza Russell J. Tweddell Joseph Arul 《Applied and environmental microbiology》2009,75(5):1465-1469
Twenty-one salts were tested for their effects on the growth of Pectobacterium carotovorum subsp. carotovorum and Pectobacterium atrosepticum. In liquid medium, 11 salts (0.2 M) exhibited strong inhibition of bacterial growth. The inhibitory action of salts relates to the water-ionizing capacity and the lipophilicity of their constituent ions.Different biochemical mechanisms have been put forth to explain the antimicrobial activity of organic and inorganic salts, including inhibition of several steps of the energy metabolism (benzoate, bicarbonate, propionate, sorbate, and sulfite salts) (2, 3, 11, 16, 17, 19, 25) and complexation to DNA and RNA (aluminum and sulfites) (12, 13, 15, 20, 27, 28). However, little is known about the physicochemical basis for the general antimicrobial action of salts. The objective of this work was to gain an understanding of the relationship between the inhibitory action of salts on bacterial growth and their physicochemical properties by using the bacteria Pectobacterium carotovorum subsp. carotovorum (formerly Erwinia carotovora subsp. carotovora) and Pectobacterium atrosepticum (formerly Erwinia carotovora subsp. atroseptica). These bacteria are responsible for soft rot, a disease of economic importance affecting numerous stored vegetable crops (14, 22).Pectobacterium carotovorum subsp. carotovorum (strain Ecc 1367) and P. atrosepticum (strain Eca 709), provided by the Laboratoire de Diagnostic en Phytoprotection (MAPAQ, Québec, Canada), were grown in 250-ml flasks containing 50 ml of 20% tryptic soy broth (Difco Laboratories, Becton Dickinson, Sparks, MD) amended with salts (200 mM) or unamended (control), by incubation at 24°C with agitation (150 rpm; Lab-Line Instruments Inc., Melrose Park, IL) for 24 h. The pHs of the media were not adjusted but varied with the type of salts, unless stated otherwise. Flasks were inoculated with 100 μl of each bacterial suspension (1 × 107 CFU/ml). Bacterial growth was determined by turbidimetry at 600 nm with a UV/visible spectrophotometer (Ultrospec 2000; Pharmacia Biotech Ltd, Cambridge, United Kingdom), using appropriate blanks. Results were expressed as the percentage of growth inhibition compared with the growth of the control. A completely randomized experimental design with three replicates was used, the experimental unit being a flask. Analysis of variance was carried out with the GLM (general linear model) procedure of SAS (SAS Institute, Cary, NC) software. When they were significant (P < 0.05), treatment means were compared using Fisher''s protected least-significant-difference test.Among the 21 salts tested, sodium carbonate, sodium metabisulfite, trisodium phosphate, aluminum lactate, aluminum chloride, sodium bicarbonate, sodium propionate, ammonium acetate, aluminum dihydroxy acetate, potassium sorbate, and sodium benzoate exhibited strong inhibition (≥97%) of the growth of both P. carotovorum subsp. carotovorum and P. atrosepticum (Table (Table1).1). Calcium chloride, sodium formate, sodium acetate, ammonium hydrogen phosphate, and sodium hydrogen phosphate exhibited a moderately inhibitory effect; sodium lactate and tartrate had no effect. On the other hand, ammonium chloride, potassium chloride, and sodium chloride stimulated the growth of P. atrosepticum.
Open in a separate windowaSalts were purchased from Sigma Chemical Co. (St. Louis, MO), except for ammonium acetate (BDH Inc., Toronto, Canada), sodium chloride (BDH), sodium bicarbonate (BDH), and aluminum lactate (Aldrich Chemical, Milwaukee, WI).bpH of the medium amended with each salt.cOsmotic pressure of the salt solution was calculated using van’t Hoff''s equation, Π = iRTc, where R is the gas constant, T is the absolute temperature (K), c is the concentration of the salt (mol/liter), and i is the number of ions into which the salt dissociates in solution.dPercentage of growth inhibition compared to growth of the control. Each value represents the mean of three replicates. Values in the same column followed by the same letter are not significantly different according to Fisher''s protected least-significant-difference test (P > 0.05). ND, not determined. Negative values signify bacterial growth stimulation.Several factors in the salt solutions can contribute to bacterial growth inhibition. Elevated osmolarity due to salt addition may trigger the osmoregulatory process, causing an increased maintenance metabolism and leading to reduction in bacterial growth. Thus, we calculated the osmotic pressure (Π) of salt solutions using van''t Hoff''s equation (26). As shown in Table Table1,1, salts with comparable osmolarities displayed complete or no bacterial growth inhibition, indicating that osmotic stress or reduction in water activity alone may not have brought about the inhibition of the bacterial growth. Therefore, other factors may play a role.The acidity or alkalinity of the medium resulting from the addition of some of the salts can have profoundly adverse effects on bacterial growth. Extreme pH conditions can lead to denaturation of proteins like enzymes present on the cell surface, depolarization of transport for essential ions and nutrients, modification of cytoplasmic pH, and DNA damage (12, 18). Table Table11 shows that the addition of aluminum lactate, aluminum chloride, and sodium metabisulfite, whose ΔpHs (ΔpH = |7.5 [the optimal pH for growth] − the pH of the salt-amended medium|) are ≥3, strongly acidified the medium, whereas the addition of sodium carbonate and trisodium phosphate strongly increased the pH (ΔpH ≥ 3.1). Except for ammonium acetate, sodium acetate, sodium bicarbonate, and the preservative salts (potassium sorbate, sodium benzoate, and sodium propionate), whose ΔpHs are <1, all the other salts generally display inhibitory effects when ΔpH values are ≥1 (Fig. (Fig.1).1). Based on this result, the effect of the highly acidic or alkaline salts (which strongly affected the pH of the medium) on the growth of P. atrosepticum was evaluated at pH 7.5. Sodium carbonate and sodium metabisulfite completely inhibited bacterial growth at pH 7.5, as they did at pHs 10.6 and 4.5, respectively; trisodium phosphate (pH 11.9) exhibited a slightly lower inhibitory effect (growth inhibition of 83.2%) at pH 7.5. These observations suggest that growth inhibition by sodium carbonate, sodium metabisulfite, and trisodium phosphate cannot be attributed solely to extreme pH and passive proton transfer (extreme pH) across the bacterial membrane. Since aluminum salts precipitate at pH 7.5 (due to formation of hydrated aluminum hydroxide), it was not possible to test their inhibitory effect at pH 7.5.Open in a separate windowFIG. 1.Relationship between ΔpH (|7.5 [the optimal pH for growth] − the pH of the salt-amended medium|) and growth inhibition of Pectobacterium atrosepticum. 1, Sodium chloride; 2, potassium chloride; 3, ammonium chloride; 4, sodium tartrate; 5, sodium lactate; 6, sodium formate; 7, ammonium hydrogen phosphate; 8, sodium acetate; 9, sodium hydrogen phosphate; 10, calcium chloride; 11, ammonium acetate; 12, sodium benzoate; 13, sodium propionate; 14, potassium sorbate; 15, sodium bicarbonate; 16, aluminum dihydroxy acetate; 17, sodium metabisulfite; 18, sodium carbonate; 19, aluminum lactate; 20, aluminum chloride; 21, trisodium phosphate.The dissociation of salts in aqueous medium generates ionic species which can participate in proton exchange reactions with water molecules. The capacity of an ion to dissociate water is an intrinsic characteristic, determined by its pK value (pKa for acidic species or pKb for basic ones) (4, 21, 24). For an ionic strength of >0.1 M, pKa and pKb values of the ions are more accurate when they are defined as apparent constants (pK′a or pK′b) in terms of the activities of hydronium and hydroxyl ions, ionic species concentrations and activity coefficients (6). Thus, for the acidic ions, we have the equation ), and for the basic anions, pK′b = pKb + log(γHB/γB−), where pK′a and pK′b are the apparent acidity constant and basicity constant, respectively; is the activity coefficient of the conjugate base (B−); and γHB is that of the acidic (HB) species. The activity coefficient (γ) of the species i can be expressed as a function of ionic strength (μ), using the Güntelberg approximation of the Debye-Hückel equation (21), as follows: −log γi=[(0.51Zi2 μ1/2)/(1 + μ1/2)], where Zi is the charge on the species i, and μ is the ionic strength. Thus, log(/γHB) = [(0.51μ1/2)/(1 + μ1/2)] (), and log(γHB/) = −[(0.51μ1/2)/(1 + μ1/2)] ().Polytropic acid-potentiating ions (bicarbonate, carbonate, monohydrogen phosphate, phosphate, sulfite, and tartrate) in an aqueous solution can exist as (n + 1) possible species for which the parent acid is HnA. These species may coexist in equilibrium under certain pH conditions. For these ions, pK′a or pK′b were expressed as the means of the coexisting species at a specified pH. Calculated values for pK′a of acidic anions and cations and calculated values for pK′b of basic anions are presented in Table Table2.2. Figure Figure2A2A shows a sigmoidal relationship between the inhibitory effect of salts on bacterial growth and the pK′b value of the basic ions (with a common cation, sodium or potassium, in the salt) and the pK′a value of the acidic ions (with a common anion, chloride, in the salt). The plot exhibits a sharp linear relationship in the pK′ range of 8.0 to 12.0. Below the pK′ value of 8.0, inhibition is maximal, whereas above the pK′ value of 11.0, ions appear to stimulate growth (growth was maximal above the pK′ value of 12). This result demonstrates that the capacity of the constitutive ions of the salts to either donate or subtract protons to water molecules, either in the growth environment (as reflected in the modification of the medium pH) or in the developing cells, generally plays a role in their inhibitory action. The consequent transmembrane pH gradient generated leads to a passive H+ transport across the microbial membrane and to acidification (in the case of ions with low pK′a) or alkalinization (in the case of ions with low pK′b) of the cytoplasm, once the capacity for proton-coupled active transport is outstripped. In both cases, proton exchange with outer membrane proteins will destabilize these proteins, their interaction with membrane lipids, and ultimately, their function in solute transport, leading to growth inhibition. The modification of cytoplasmic pH can also alter nucleic acid structures and functions and contribute to growth inhibition (18).Open in a separate windowFIG. 2.(A) Relationship between the growth inhibition of Pectobacterium atrosepticum and the apparent basicity constant (pK′b,•) of basic anions with common Na+ (or K+) cations in the salt, the apparent acidity constant (pK′a,○) of acidic bisulfite anion (HSO3−), and the cations with common Cl− ions in the salt. (B) Relationship between the growth inhibition of Pectobacterium atrosepticum and the addition parameter (pK′ + pPo/w) combining the partition coefficient (Po/w) and pK′b (•) of basic anions (common cation, Na+ or K+, in the salt) or pK′a (○) of cations (common anion, Cl−, in the salt) and the acidic bisulfite anion (HSO3−).
Open in a separate windowaCalculation of pK′ was performed according to Edsall and Wyman (6). pH values were measured at 0.2 M.bIncludes CO2·H2O and H2CO3.However, the water-ionizing capacity of the constituent ions of the salts and the consequent modification of the pH of the medium are not the sole factors accounting for growth inhibition, as suggested by the exceptional inhibitory actions of benzoate, propionate, and sorbate (Fig. (Fig.11 and and2A).2A). These ions provide a higher inhibition than is expected from their pK′ values (pK′b values of 10.0, 9.3, and 9.4, respectively), while the pH of their solution is optimal for bacterial growth (pHs of 7.4, 7.4, and 7.7, respectively). This suggests that they possess additional characteristics mediating their action, in addition to their water-ionization property. In fact, these preservative agents have been shown to be active either as undissociated acids (like other weak acids) or as anions (7, 8), due to their possibly hydrophobic nature which would allow them to interact with lipid constituents of the cell envelope of gram-negative bacteria such as Pectobacterium spp., and to modify their functionality (5), resulting in growth inhibition. They can also cross the cell envelope due to their lipophilicity, and their acidification inside the cell can cause additional adverse effects.Thus, we determined the octanol/water partition coefficient (Po/w), an indicator of the lipophilic character of a compound, for the effective salts with common sodium (or potassium) or chloride ions. The Po/w coefficients of the salts were determined in duplicate by using the general solvent-solvent separation procedure (9). Equal volumes (50 ml) of 1-octanol (Sigma Chemical Co., St. Louis, MO) and bidistilled water were poured into a separating flask and thoroughly shaken for 5 min. Four grams of each salt was then added, and the flask content was thoroughly mixed three times for 5 min each time, with a rest period of 5 min after each agitation. After complete separation (20 to 24 h at room temperature), the two phases were recovered separately in different flasks, and the concentration of the accompanying ion of the salt was measured in each phase by atomic absorption (model 3300 unit; Perkin-Elmer, Ueberlinger, Germany). The Po/w coefficient was calculated as the ratio of the concentration of ion in 1-octanol to the concentration of ion in the aqueous phase. Sodium benzoate was found to be the most lipophilic (Po/w = 1.41 × 10−2), followed by potassium sorbate (Po/w = 7.6 × 10−3) and sodium metabisulfite (Po/w = 2.0 × 10−4). Most other salts, sodium chloride (reference salt), sodium bicarbonate and carbonate, sodium propionate, sodium acetate, calcium chloride, and aluminum chloride mainly remained in the aqueous phase (Po/w = 2.0 × 10−5 to 5.0 × 10−5). This lipophilic characteristic of benzoate and sorbate ions would result from a reduced charge density in their molecules (due to the conjugated double bonds in their molecules). An addition parameter, pK′ + pPo/w, which combines the two properties of salts ions, i.e., the water-ionizing capacity (pK′) and the lipophilicity (pPo/w = −log Po/w), appears to provide a more general basis for the inhibitory effect of salts (Fig. (Fig.2B).2B). This suggests that while the dissociation constant of ions plays a major role in growth inhibition, as seen in Fig. Fig.2A,2A, the lipophilic character of the preservative-salt ions confers to them an added ability to penetrate the cell envelope and to inhibit bacterial growth (5, 10). The exclusion of ammonium (lower inhibition than expected from its pK′a value) and calcium (higher inhibition than expected from its pK′a value) ions from the sigmoidal pattern portrayed in Fig. Fig.2B2B would have resulted from their interactions with water and other molecules (NH4+) (1) or from cell membrane destabilization (Ca2+) (23).In conclusion, the study has shown that several salts (0.2 M concentration), including aluminum dihydroxy acetate, aluminum chloride, aluminum lactate, ammonium acetate, potassium sorbate, sodium benzoate, sodium metabisulfite, sodium bicarbonate, sodium carbonate, sodium propionate, and trisodium phosphate, strongly inhibited the growth of P. carotovorum subsp. carotovorum and P. atrosepticum. In addition, the study has established for the first time a basic sigmoidal relationship between the antimicrobial activity of the salts and the physicochemical characteristics of their constituent ions, namely their water-ionizing capacity and their lipophilicity. The constituent ions of the highly inhibiting salts generally displayed a high capacity to ionize water molecules (low pK′a or pK′b values) (Al3+, CO32−, PO43−, HCO3−, and HSO3−) or a high lipophilicity (benzoate− and sorbate−), and these two parameters in combination with known biochemical activities of salts ions would affect bacterial growth. 相似文献
TABLE 1.
Effect of salts on the growth of P. atrosepticum and P. carotovorum subsp. carotovorumSalt (0.2 M)a | pHb | Osmotic pressure (atm)c | Growth inhibition (%)d
| |
---|---|---|---|---|
P. atrosepticum | P. carotovorum subsp. carotovorum | |||
Aluminum dihydroxy acetate [Al(OH)2C2H3O2] | 4.9 | 9.79 | 100 a | 100 a |
Aluminum chloride (AlCl3·6H2O) | 2.5 | 19.57 | 100 a | 100 a |
Aluminum lactate [Al(C3H5O3)3] | 3.4 | 19.57 | 100 a | 100 a |
Ammonium acetate (NH4C2H3O2) | 7.2 | 9.79 | 100 a | 100 a |
Ammonium chloride (NH4Cl) | 7.0 | 9.79 | −18 d | ND |
Ammonium hydrogen phosphate [(NH4)2HPO4] | 8.3 | 14.68 | 43 b | 23 c |
Calcium chloride (CaCl2·2H2O) | 5.8 | 14.68 | 85 a | 70 b |
Potassium chloride (KCl) | 7.3 | 9.79 | −27 d | ND |
Potassium sorbate (KC6H7O2) | 7.7 | 9.79 | 100 a | 97 a |
Sodium acetate (NaC2H3O2·3H2O) | 7.4 | 9.79 | 63 b | ND |
Sodium benzoate (NaC7H5O2) | 7.4 | 9.79 | 100 a | 100 a |
Sodium bicarbonate (NaHCO3) | 8.1 | 9.79 | 100 a | 100 a |
Sodium carbonate (Na2CO3) | 10.6 | 14.68 | 100 a | 100 a |
Sodium chloride (NaCl) | 7.2 | 9.79 | −29 d | ND |
Sodium formate (NaCHO2) | 7.3 | 9.79 | 24 c | ND |
Sodium lactate (C3H5O3Na) | 7.3 | 9.79 | 3 c | ND |
Sodium metabisulfite (Na2S2O5) | 4.5 | 19.57 | 100 a | 100 a |
Sodium hydrogen phosphate (Na2HPO4) | 8.7 | 14.68 | 69 b | 61 b |
Sodium propionate (NaC3H5O2) | 7.4 | 9.79 | 100 a | 99 a |
Sodium tartrate (Na2C4H4O6·2H2O) | 7.3 | 14.68 | 2 c | ND |
Trisodium phosphate (Na3PO4·12H2O) | 11.9 | 19.57 | 100 a | 100 a |
TABLE 2.
Calculated apparent values for acidity, pK′a, and basicity, pK′baSalt | Basic anion
| Cation and acidic anion
| ||||
---|---|---|---|---|---|---|
pH | Ionic species or species in equilibrium | pK′b | pH | Ionic species or species in equilibrium | pK′a | |
Sodium acetate | 7.4 | Acetate− | 9.5 | |||
Sodium benzoate | 7.4 | Benzoate− | 10.0 | |||
Sodium bicarbonate | 8.1 | H2CO3/HCO3−b | 7.7 | |||
Sodium carbonate | 10.6 | HCO3−/CO32− | 6.1 | |||
Sodium formate | 7.3 | Formate− | 10.4 | |||
Sodium hydrogen phosphate | 8.7 | H2PO4−/HPO42− | 9.8 | |||
Sodium lactate | 7.3 | Lactate− | 11.1 | |||
Trisodium phosphate | 11.9 | HPO42−/PO43− | 5.3 | |||
Sodium propionate | 7.4 | Propionate− | 9.3 | |||
Potassium sorbate | 7.7 | Sorbate− | 9.4 | |||
Sodium tartrate | 7.3 | Tartrate2− | 10.6 | |||
Sodium chloride | 7.2 | Cl− | 17.2 | |||
Sodium metabisulfite | 4.5 | SO2·H2O/HSO3− | 4.0 | |||
Aluminum chloride | 2.5 | Al3+ | 6.2 | |||
Calcium chloride | 5.8 | Ca2+ | 13.4 | |||
Potassium chloride | 7.3 | K+ | 16.2 | |||
Sodium chloride | 7.2 | Na+ | 15.0 | |||
Ammonium chloride | 7.0 | NH4+ | 9.5 |
16.
One- and Two-Locus Population Models With Differential Viability Between Sexes: Parallels Between Haploid Parental Selection and Genomic Imprinting
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Alexey Yanchukov 《Genetics》2009,182(4):1117-1127
A model of genomic imprinting with complete inactivation of the imprinted allele is shown to be formally equivalent to the haploid model of parental selection. When single-locus dynamics are considered, an internal equilibrium is possible only if selection acts in the opposite directions in males and females. I study a two-locus version of the latter model, in which maternal and paternal effects are attributed to the single alleles at two different loci. A necessary condition for the allele frequency equilibria to remain on the linkage equilibrium surface is the multiplicative interaction between maternal and paternal fitness parameters. In this case the equilibrium dynamics are independent at both loci and results from the single-locus model apply. When fitness parameters are additive, analytic treatment was not possible but numerical simulations revealed that stable polymorphism characterized by association between loci is possible only in several special cases in which maternal and paternal fitness contributions are precisely balanced. As in the single-locus case, antagonistic selection in males and females is a necessary condition for the maintenance of polymorphism. I also show that the above two-locus results of the parental selection model are very sensitive to the inclusion of weak directional selection on the individual''s own genotypes.PARENTAL genetic effects refer to the influence of the mother''s and father''s genotypes on the phenotypes of their offspring, not attributable just to the transfer of genes. Examples have been documented across a wide range of areas of organism biology; see, for example, Wade (1998) and and22 in Rasanen and Kruuk (2007). Parental selection is a more formal concept used in theoretical modeling and concerns situations where the fitness of the offspring depends, besides other factors, on the genotypes of its parent(s) (generalizing from Kirkpatrick and Lande 1989).
Open in a separate windowParentheses in the first column indicate maternal genotype (parental selection model) or inactivation of the maternally derived allele (imprinting model). Whether selection occurs at the diploid (first column) or subsequent haploid (second column) stage does not change the resulting allele frequencies.
Open in a separate windowAnother well-known parent-of-origin phenomenon is genomic imprinting. Here, the level of expression of one of the alleles depends on which parent it is inherited from. Often it is difficult to tell apart the phenotypic patterns due to parental effects and genomic imprinting, and thus a problem arises in the process of identifying the candidate genes for such effects (Hager et al. 2008). Analytic methods (Weinberg et al. 1998; Santure and Spencer 2006; Hager et al. 2008) have been developed to quantify subtle differences between the two. In this article, I point out that a simple mathematical model, first suggested for genomic imprinting at a diploid locus, can be interpreted, without any formal changes, to describe parental selection on haploids.While there has been much progress in understanding the evolution of genomic imprinting (Hunter 2007), including advances in modeling (Spencer 2000, 2008), the population genetics theory of parental effects received less attention. Existing major-locus effect models of parental selection are single-locus, two-allele, and mostly concern uniparental (maternal) selection (Wright 1969; Spencer 2003; Gavrilets and Rice 2006; Santure and Spencer 2006), with only one specific case where the fitness effects of both parents interact studied by Gavrilets and Rice (2006). No attempt to extend this theory into multilocus systems has yet been made. Considering a two-locus model with both parents playing a role in selection on the offspring is called for by the observation that many maternal and paternal effects aim at the different traits or different life stages of their progeny. Among birds, for example, body condition soon after hatching is largely determined by the mother, while paternally transmitted sexual display traits develop much later in life (Price 1998). Such effects are therefore unlikely to be regulated within a single locus. Sometimes the effects are on the same trait, but still attributed to different loci: expression of gene Avy that causes the “agouti” phenotype (yellow fur coat and obesity) in mice is enhanced by maternal epigenetic modification (Rakyan et al. 2003), while paternal mutations at the other locus, MommeD4, contribute to a reverse phenotypic pattern in the offspring (Rakyan et al. 2003). The epigenetic state of the murine AxinFu allele is both maternally and paternally inherited (Rakyan et al. 2003).Focusing selection on haploids reduces the number of genotypes that need to be taken into account, while preserving the main properties of the multilocus system. Genes with haploid expression and a potential of parental effects can be found in two major taxonomic kingdoms. A notable candidate is Spam1 in mice, which is expressed during spermogenesis and encodes a factor that enables sperm to penetrate the egg cumulus (Zheng et al. 2001). This gene remains a target for effectively haploid selection, because its product is not shared via cytoplasm bridges between developing spermatides. Mutations at Spam1 alter performance of the male gametes that carry it and might indirectly, perhaps by altering the timing of fertilization, affect the fitness of the zygote. The highest estimated number of mouse genes expressed in the male gametes is currently 2375 (Joseph and Kirkpatrick 2004), and one might expect some of them to have similar paternal effects. Plants go through a profound haploid stage in their life cycles, and genes involved at this stage have an inevitable effect on the fitness of the future generations. In angiosperms, seed development is known to be controlled by both maternal (Chaudhury and Berger 2001; Yadegari and Drews 2004) and paternal (Nowack et al. 2006) effect genes, expressed, respectively, in female and male gametophytes.Under haploid selection, there can be no overdominance, and thus polymorphism is much more difficult to maintain than in diploid selection models (summarized in Feldman 1971). Nevertheless, differential or antagonistic selection between sexes can lead to a new class of stable internal equilibria in the diploid systems (Owen 1953; Bodmer 1965; Mandel 1971; Kidwell et al. 1977; Reed 2007), and I make use of this property in the haploid models developed below. In the experiment by Chippindale and colleagues (Chippindale et al. 2001), ∼75% of the total fitness variation in the adult stage of Drosophila melanogaster was negatively correlated between males and females, which suggests that a substantial portion of the fruit fly expressed genome is under sexually antagonistic selection. I assume that the effect of either parent on the fitness of the individual depends on the sex of the latter, which in respect to modeling is equivalent to the assumption of differential viability between the sexes in the progeny of the same parent(s). Biological systems that satisfy the latter assumptions can be found among colonial green algae: many members of the order Volvocales are haploid except for the short zygotic stage, and during sexual reproduction, they are also dioecious and anisogametic. I return to this example in the discussion. The possibility that genes expressed in animal gametes may be under antagonistic selection between sexes has been discussed (Bernasconi et al. 2004). For example, a (hypothetical) mutation increasing the ATP production in mitochondria would be beneficial in sperm, because of the increased mobility of the latter, but neutral or detrimental in the egg, due to a higher level of oxidative damage to DNA (Zeh and Zeh 2007).My main purpose was to derive conditions for existence and stability of the internal equilibria of the model(s). I begin with a simple one-locus case, which can be analyzed explicitly, and show how these one-locus results can be extended to the case of two recombining loci with multiplicative fitness. Then, I assume an additive relation between the maternal and paternal effect parameters and study the special cases where parental effects are symmetric. 相似文献
TABLE 1
Frequencies of genotypes and fitness parameterizations in model 1Gametes/haploids | Frequency before selection | Fitness
| ||
---|---|---|---|---|
Zygote | Male | Female | ||
(A)A | A | pfpm | 1 − α | 1 − δ |
(A)a | 1/2 A 1/2 a | pf(1 − pm) | 1 | 1 |
(a)A | 1/2 a 1/2 A | (1 − pf)pm | 1 − α | 1 − δ |
(a)a | A | (1 − pf)(1 − pm) | 1 | 1 |
TABLE 2
Offspring genotypic proportions from different mating types, sorted among four phenotypic groups/combinations of maternal and paternal effects: model 2Offspring genotypes/phenotypes
| |||||||||
---|---|---|---|---|---|---|---|---|---|
Parental genotypes
| Paternal (φ = 1)
| Joint (φ = 4)
| |||||||
Male | Female | AB | Ab | aB | Ab | AB | Ab | aB | ab |
AB | AB | 1 | |||||||
Ab | |||||||||
aB | |||||||||
ab | (1−r)/2 | r/2 | r/2 | (1−r)/2 | |||||
Ab | AB | ||||||||
Ab | 1 | ||||||||
aB | r/2 | (1−r)/2 | (1−r)/2 | r/2 | |||||
ab | |||||||||
Offspring genotypes/phenotypes
| |||||||||
Parental genotypes
| Maternal (φ = 2)
| None (φ = 3)
| |||||||
Male | Female | AB | Ab | aB | Ab | AB | Ab | aB | ab |
aB | AB | ||||||||
Ab | r/2 | (1 − r)/2 | (1 − r)/2 | r/2 | |||||
aB | 1 | ||||||||
ab | |||||||||
ab | AB | (1 − r)/2 | (1 − r)/2 | ||||||
Ab | |||||||||
aB | |||||||||
ab | 1 |
17.
Banumathi Sankaran Shilah A. Bonnett Kinjal Shah Scott Gabriel Robert Reddy Paul Schimmel Dmitry A. Rodionov Valérie de Crécy-Lagard John D. Helmann Dirk Iwata-Reuyl Manal A. Swairjo 《Journal of bacteriology》2009,191(22):6936-6949
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 bacteriaaOpen 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18.
Vertebrate genomic assemblies were analyzed for endogenous sequences related to any known viruses with single-stranded DNA genomes. Numerous high-confidence examples related to the Circoviridae and two genera in the family Parvoviridae, the parvoviruses and dependoviruses, were found and were broadly distributed among 31 of the 49 vertebrate species tested. Our analyses indicate that the ages of both virus families may exceed 40 to 50 million years. Shared features of the replication strategies of these viruses may explain the high incidence of the integrations.It has long been appreciated that retroviruses can contribute significantly to the genetic makeup of host organisms. Genes related to certain other viruses with single-stranded RNA genomes, formerly considered to be most unlikely candidates for such contribution, have recently been detected throughout the vertebrate phylogenetic tree (1, 6, 13). Here, we report that viruses with single-stranded DNA (ssDNA) genomes have also contributed to the genetic makeup of many organisms, stretching back as far as the Paleocene period and possibly the late Cretaceous period of evolution.Determining the evolutionary ages of viruses can be problematic, as their mutation rates may be high and their replication may be rapid but also sporadic. To establish a lower age limit for currently circulating ssDNA viruses, we analyzed 49 published vertebrate genomic assemblies for the presence of sequences derived from the NCBI RefSeq database of 2,382 proteins from known viruses in this category, representing a total of 23 classified genera from 7 virus families. Our survey uncovered numerous high-confidence examples of endogenous sequences related to the Circoviridae and to two genera in the family Parvoviridae: the parvoviruses and dependoviruses (Fig. (Fig.11).Open in a separate windowFIG. 1.Phylogenetic tree of vertebrate organisms and history of ssDNA virus integrations. Times of integration of ancestral dependoviruses (yellow icosahedrons), parvoviruses (blue icosahedrons), and circoviruses (triangles) are approximate.The Dependovirus and Parvovirus genomes are typically 4 to 6 kb in length, include 2 major open reading frames (encoding replicase proteins [Rep and NS1, respectively] and capsid proteins [Cap and VP1, respectively]), and have characteristic hairpin structures at both ends (Fig. (Fig.2).2). For replication, these viruses depend on host enzymes that are recruited by the viral replicase proteins to the hairpin regions, where self-primed viral DNA synthesis is initiated (2). Circovirus genomes are typically ∼2-kb circles. DNA of the type species, porcine circovirus 1 (PCV-1), contains a stem-loop structure within the origin of replication (Fig. (Fig.2),2), and the largest open reading frame includes sequences that are homologous to the Parvovirus replicase open reading frame (9, 11). The circoviruses also depend on host enzymes for replication, and DNA synthesis is self-primed from a 3′-OH end formed by endonucleolytic cleavage of the stem-loop structure (4). The frequency of Dependovirus infection is estimated to be as high as 90% within an individual''s lifetime. None of the dependoviruses have been associated with human disease, but related viruses in the family Parvoviridae (e.g., erythrovirus B19 and possibly human bocavirus) are pathogenic for humans, and members of both the Parvoviridae and the Circoviridae can cause a variety of animal diseases (2, 4).Open in a separate windowFIG. 2.Schematics illustrating the structure and organization of Parvoviridae and Circoviridae genomes and origins of several of the longest-integrated ancestral viral sequences found in vertebrates. Integrations were aligned to the Dependovirus adeno-associated virus 2 (AAV2), the Parvovirus minute virus of mice (MVM), and the Circovirus porcine circovirus 1 (PCV-1). The inverted terminal repeat (ITR) sequences in the Dependovirus and Parvovirus genomes are depicted on an expanded scale. A linear representation of the circular genome of PCV-1 is shown with the 10-bp stem-loop structure on an expanded scale. Horizontal lines beneath the maps indicate the lengths of similar sequences that could be identified by BLAST. The numbers indicate the locations of amino acids in the viral proteins where the sequence similarities in the endogenous insertions start and end. The actual ancestral virus-derived integrated sequences may extend beyond the indicated regions.With some ancestral endogenous sequences that we identified, phylogenetic comparisons can be used to estimate age. For example, as a Dependovirus-like sequence is present at the same location in the genomes of mice and rats, the ancestral virus must have existed before their divergence, more than 20 million years ago. Some Circovirus- and Dependovirus-related integrations also predate the split between dog and panda, about 42 million years ago. However, in most other cases, we rely on an indirect method for estimating age (1). As genomic sequences evolve, they accumulate new stop codons and insertion/deletion-induced frameshifts. The rates of these events can be tied directly to the rates of neutral sequence drift and, therefore, the time of evolution. To apply this method, we first performed a BLAST search of vertebrate genomes for all known ssDNA virus proteins (BLAST options, -p tblastn -M BLOSUM62 -e 1e−4). Candidate sequences were then recorded, along with 5 kb of flanking regions, and then again aligned against the database of ssDNA viruses to find the most complete alignment (BLAST options, -t blastx -F F -w 15 -t 1500 -Z 150 -G 13 -E 1 -e 1e−2). Detected alignments were then compared with a neutral model of genome evolution, as described in the supplemental material, and the numbers of stop codons and frameshifts were converted into the expected genomic drift undergone by the sequences. The age of integration was then estimated from the known phylogeny of vertebrates (7, 10). Using these methods, we discovered that as many as 110 ssDNA virus-related sequences have been integrated into the 49 vertebrate genomes considered, during a time period ranging from the present to over 40 to 60 million years ago (Table (Table1;1; see also Tables S1 to S3 in the supplemental material).
Open in a separate windowaSome ambiguity in choosing the most similar virus is possible. We generally used the alignment with the lowest E value in the BLAST results. However, one or two points in the exponent of an E value were sometimes sacrificed to achieve a longer sequence alignment.baa, amino acids.cThese sequences have long insertions compared to the present-day viruses. In all cases tested, these insertions originated from short interspersed elements (SINEs). These insertions were excluded from the counts of stop codons and frameshifts and the estimation of integration age.dChr, chromosome.It is important to recognize that there is an intrinsic limit on how far back in time we can reach to identify ancient endogenous viral sequences. First, the sequences must be identified with confidence by BLAST or similar programs. This requirement places a lower limit on sequence identity at about 20 to 30% of amino acids, or about 75% of nucleotides (nucleotides evolve nearly 2.5 times slower than the amino acid sequence they encode). Second, the related, present-day virus must have evolved at a rate that is not much higher than that of the endogenous sequences. The viruses for which ancestral endogenous sequences were identified in this study exhibit sequence drift similar to that associated with mammalian genomes. Setting this rate at 0.14% per million years of evolution (8), we arrive at 90 million years as the theoretical limit for the oldest sequences that can be identified using our methods. This limit drops to less than 35 million years for endogenous viral sequences in rodents and even lower for sequences related to viruses that evolve faster than mammalian genomes.The most widespread integrations found in our survey are derived from the dependoviruses. These include nearly complete genomes related to adeno-associated virus (AAV) in microbat, wallaby, dolphin, rabbit, mouse, and baboon (Fig. (Fig.2).2). We did not detect inverted terminal repeats in several integrations tested, even though repeats are common in the present-day dependoviruses. This result could be explained by sequence decay or the absence of such structures in the ancestral viruses. However, we do see sequences that resemble degraded hairpin structures to which Dependovirus Rep proteins bind, with an example from microbat integration mlEDLG-1 shown in Fig. Fig.3.3. The second most widespread endogenous sequences are related to the parvoviruses. They are found in 6 of 49 vertebrate species considered, with nearly complete genomes in rat, opossum, wallaby, and guinea pig (Fig. (Fig.22).Open in a separate windowFIG. 3.Hairpin structure of the inverted terminal repeat of adeno-associated virus 2 (left) and a candidate degraded hairpin structure located close to the 5′ end of the mlEDLG-1 integration in microbats (right). Structures and mountain plots were generated using default parameters of the RNAfold program (5), with nucleotide coloring representing base-pairing probabilities: blue is below average, green is average, and red is above average. Mountain plots represent hairpin structures based on minimum free energy (mfe) calculations and partition function (pf) calculations, as well as the centroid structure (5). Height is expressed in numbers of nucleotides; position represents nucleotide.The Dependovirus AAV2 has strong bias for integration into human chromosome 19 during infection, driven by a host sequence that is recognized by the viral Rep protein(s). Rep mediates the formation of a synapse between viral and cellular sequences, and the cellular sequences are nicked to serve as an origin of viral replication (14). The related integrations in mice and rats, located in the same chromosomal locations, might be explained by such a mechanism. However, the extent of endogenous sequence decay and the frequency of stop codons indicate that these integrations occurred some 30 to 35 million years ago, implying that they are derived from a single event in a rodent ancestor rather than two independent integration events at the same location. Similarly, integrations EDLG-1 in dog and panda lie in chromosomal regions that can be readily aligned (based on University of California—Santa Cruz [UCSC] genome assemblies) and show sequence decay consistent with the age of the common ancestor, about 42 million years. Endogenous sequences related to the family Parvoviridae can thus be traced to over 40 million years back in time, and viral proteins related to this family have remained over 40% conserved.Sequences related to circoviruses were detected in five vertebrate species (Table (Table11 and Table S1 in the supplemental material). At least one of these sequences, the endogenous sequence in opossum, likely represents a recent integration. Several integrations in dog, cat, and panda, on the other hand, appear to date from at least 42 million years ago, which is the last time when pandas and dogs shared a common ancestor. We see evidence for this age in data from sequence degradation (Table (Table1),1), phylogenetic analyses of endogenous Circovirus-like genomes (see Fig. S2 in the supplemental material), and genomic synteny where integration ECLG-3 is surrounded by genes MTA3 and ARID5A in both dog and panda and integration ECLG-2 lies 35 to 43 kb downstream of gene UPF3A. In fact, Circovirus integrations may even precede the split between dogs and cats, about 55 million years ago, although the preliminary assembly and short genomic contigs for cats make synteny analysis impossible.The most common Circovirus-related sequences detected in vertebrate genomes are derived from the rep gene. We speculate that, like those of the Parvoviridae, the ancestral Circoviridae sequences might have been copied using a primer sequence in the host DNA that resembled the viral origin and was therefore recognized by the virus Rep protein. Higher incidence of rep gene identifications may represent higher conservation of this gene with time, or alternatively, possession of these sequences may impart some selective advantage to the host species. The largest Circovirus-related integration detected, in the opossum, comprises a short fragment of what may have been the cap gene immediately adjacent to and in the opposite orientation from the rep gene. This organization is similar to that of the present day Circovirus genome in which these genes share a promoter in the hairpin regions but are translated in opposite directions (Fig. (Fig.22).In summary, our results indicate that sequences derived from ancestral members of the families Parvoviridae and Circoviridae were integrated into their host''s genomes over the past 50 million years of evolution. Features of their replication strategies suggest mechanisms by which such integrations may have occurred. It is possible that some of the endogenous viral sequences could offer a selective advantage to the virus or the host. We note that rep open reading frame-derived proteins from some members of these families kill tumor cells selectively (3, 12). The genomic “fossils” we have discovered provide a unique glimpse into virus evolution but can give us only a lower estimate of the actual ages of these families. However, numerous recent integrations suggest that their germ line transfer has been continuing into present times. 相似文献
TABLE 1.
Selected endogenous sequences in vertebrate genomes related to single-stranded DNA virusesVirus group and vertebrate species | Initial genomic search using TBLASTN | Best sequence homology identified using BLASTX | Predicted nucleotide drift (%) | Integration label | Age (million yr) or timing of integration based on sequence aging | |||||
---|---|---|---|---|---|---|---|---|---|---|
Chromosomal or scaffold location | Protein | BLAST E value/% sequence identity | Most similar virusa | Protein | Coordinates | No. of stop codons/frameshifts | ||||
Circoviruses | ||||||||||
Cat | Scaffold_62068 | Rep | 6E−05/37 | Canary circovirus | Rep | 4-283 | 3/7 in 268 aab | 14.2 | fcECLG-1 | 82 |
Scaffold_24038 | Rep | 6E−06/51 | Columbid circovirus | Rep | 44-317 | 4/5 in 231 aac | 15.2 | fcECLG-2 | 87 | |
Dog | Chr5d | Rep | 7E−16/46 | Raven circovirus | Rep | 16-263 | 6/5 in 250 aa | 17.6 | cfECLG-1 | 98 |
Chr22 | Rep | 1E−14/43 | Beak and feather disease virus | Rep | 7-264 | 2/1 in 261 aac | 4.5 | cfECLG-2 | 54 | |
Opossum | Chr3 | Rep | 4E−46/44 | Finch circovirus | Rep | 2-291 | 0/2 in 282 aa | 2.3 | mdECLG | 12 |
Cap | 6-36 | 0/0 in 30 aa | ||||||||
Dependoviruses | ||||||||||
Dog | ChrX | Rep | 6E−05/55 | AAV5 | Rep | 239-445 | 3/4 in 200 aa | 14.0 | cfEDLG-1 | 78 |
Dolphin | GeneScaffold1475 | Rep | 8E−39/39 | Avian AAV DA1 | Rep | 79-486 | 3/4 in 379 aac | 6.6 | ttEDLG-2 | 55 |
Cap | 4E−61/47 | Cap | 1-738 | 4/7 in 678 aac | ||||||
Elephant | Scaffold_4 | Rep | 0/55 | AAV5 | Rep | 3-589 | 0/0 in 579 aa | 0.0 | laEDLG | Recent |
Hyrax | GeneScaffold5020 | Cap | 3E−34/53 | AAV3 | Cap | 485-735 | 0/5 in 256 aa | 7.0 | pcEDLG-1 | 29 |
Scaffold_19252 | Rep | 9E−72/47 | Bovine AAV | Rep | 2-348 | 8/4 in 348 aa | 14.3 | pcEDLG-2 | 60 | |
Megabat | Scaffold_5601 | Rep | 2E−13/31 | AAV2 | Rep | 315-479 | 1/5 in 175 aa | 13.1 | pvEDLG-3 | 76 |
Microbat | GeneScaffold2026 | Rep | 1E−117/50 | AAV2 | Rep | 1-617 | 2/5 in 612 aa | 5.8 | mlEDLG-1 | 27 |
Cap | 9E−33/51 | Cap | 1-731 | 2/9 in 509 aac | ||||||
Scaffold_146492 | Cap | 6E−32/42 | AAV2 | Cap | 479-732 | 0/3 in 252 aa | 4.2 | mlEDLG-2 | 19 | |
Mouse | Chr1 | Rep | 2E−06/34 | AAV2 | Rep | 4-206 | 3/5 in 191 aa | 17.1 | mmEDLG-1 | 39 |
Chr3 | Rep | 2E−24/31 | AAV5 | Rep | 71-478 | 12/7 in 389 aa | 16.5 | mmEDLG-2 | 37 | |
Cap | 2E−22/45 | Cap | 22-724 | 12/10 in 649aac | ||||||
Chr8 | Rep | 1E−08/46 | AAV2 | Rep | 314-473 | 3/3 in 147 aa | 13.8 | mmEDLG-3 | 31 | |
Cap | 1-137 | 1/2 in 114 aa | ||||||||
Panda | Scaffold2359 | Rep | 2E−06/37 | Bovine AAV | Rep | 238-426 | 2/3 in 186 aa | 10.4 | amEDLG-1 | 59 |
Pika | Scaffold_9941 | Rep | 4E−14/28 | AAV5 | Rep | 126-415 | 2/2 in 282 aa | 5.4 | opEDLG | 14 |
Platypus | Chr2 | Rep | 9E−10/35 | Bovine AAV | Rep | 297-437 | 4/3 in 138 aa | 17.1 | oaEDLG-1 | 79 |
Cap | 272-419 | 1/2 in 150 aac | ||||||||
Contig12430 | Rep | 2E−09/47 | Bovine AAV | Rep | 353-450 | 3/1 in 123 aa | 12.0 | oaEDLG-2 | 55 | |
Cap | 2E−05/32 | Cap | 253-367 | 2/1 in 116 aa | ||||||
Rabbit | Chr10 | Rep | 3E−97/39 | AAV2 | Rep | 1-619 | 3/9 in 613 aa | 9.3 | ocEDLG | 43 |
Cap | 5E−50/45 | Cap | 1-723 | 10/9 in 675 aa | ||||||
Rat | Chr13 | Rep | 2E−09/33 | AAV2 | Rep | 4-175 | 2/4 in 177 aa | 13.3 | rnEDLG-1 | 28 |
Chr2 | Rep | 4E−18/40 | AAV5 | Rep | 1-461 | 12/12 in 454 aa | 22.7 | rnEDLG-2 | 51 | |
Chr19 | Rep | 2E−07/33 | AAV5 | Rep | 329-464 | 2/4 in 136 aa | 16.1 | rnEDLG-3 | 35 | |
Cap | 31-133 | 2/1 in 93 aa | ||||||||
Tarsier | Scaffold_178326 | Rep | 4E−14/23 | AAV5 | Rep | 96-465 | 2/3 in 356 aa | 5.3 | tsEDLG | 23 |
Parvoviruses | ||||||||||
Guinea pig | Scaffold_188 | Rep | 3E−24/46 | Porcine parvovirus | Rep | 313-567 | 5/3 in 250 aa | 12.3 | cpEPLG-1 | 40 |
Cap | 1E−16/36 | Cap | 10-689 | 11/12 in 672 aa | ||||||
Scaffold_27 | Rep | 1E−50/39 | Canine parvovirus | Rep | 11-640 | 1/4 in 616 aa | 5.3 | cpEPLG-2 | 17 | |
Cap | 1E−38/39 | Porcine parvovirus | Cap | 3-719 | 2/14 in 700 aa | |||||
Tenrec | Scaffold_260946 | Rep | 2E−20/38 | LuIII virus | Rep | 406-598 | 4/4 in 190 aa | 19.0 | etEPLG-2 | 60 |
Cap | 11-639 | 16/15 in 595 aa | ||||||||
Rat | Chr5 | Rep | 6E−10/56 | Canine parvovirus | Rep | 1-282 | 0/0 in 312 aa | 0.6 | rnEPLG | Recent |
Cap | 0/62 | Cap | 637-667 | 0/2 in 760 aa | ||||||
Rep | 0/63 | 1-751 | ||||||||
Opossum | Chr3 | Rep | 2E−39/33 | LuIII virus | Rep | 7-570 | 11/3 in 502 aa | 10.9 | mdEPLG-2 | 56 |
Cap | 7E−8/33 | Cap | 11-729 | 14/7 in 704 aa | ||||||
Chr6 | Rep | 6E−58/44 | Porcine parvovirus | Rep | 16-563 | 3/7 in 534 aac | 4.6 | mdEPLG-3 | 24 | |
Cap | 6E−60/38 | Cap | 10-715 | 2/5 in 707 aac | ||||||
Wallaby | Scaffold_108040 | Rep | 4E−74/62 | Canine parvovirus | Rep | 341-645 | 0/0 in 287 aa | 1.3 | meEPLG-3 | 7 |
Cap | 8E−37/32 | Cap | 35-738 | 0/4 in 687 aa | ||||||
Scaffold_72496 | Rep | 2E−61/42 | Porcine parvovirus | Rep | 23-567 | 4/3 in 531 aa | 5.7 | meEPLG-6 | 30 | |
Cap | 2E−31/38 | Cap | 10-532 | 6/4 in 514 aa | ||||||
Scaffold_88340 | Rep | 7E−37/55 | Mouse parvovirus 1 | Rep | 344-566 | 0/3 in 223 aa | 6.7 | meEPLG-16 | 36 | |
Cap | 7E−22/33 | Cap | 11-713 | 6/9 in 700 aa |
19.
Flavia Pichiorri Hiroshi Okumura Tatsuya Nakamura Preston N. Garrison Pierluigi Gasparini Sung-Suk Suh Teresa Druck Kelly A. McCorkell Larry D. Barnes Carlo M. Croce Kay Huebner 《The Journal of biological chemistry》2009,284(2):1040-1049
We have previously shown that Fhit tumor suppressor protein interacts with
Hsp60 chaperone machinery and ferredoxin reductase (Fdxr) protein.
Fhit-effector interactions are associated with a Fhit-dependent increase in
Fdxr stability, followed by generation of reactive oxygen species and
apoptosis induction under conditions of oxidative stress. To define Fhit
structural features that affect interactions, downstream signaling, and
biological outcomes, we used cancer cells expressing Fhit mutants with amino
acid substitutions that alter enzymatic activity, enzyme substrate binding, or
phosphorylation at tyrosine 114. Gastric cancer cell clones stably expressing
mutants that do not bind substrate or cannot be phosphorylated showed
decreased binding to Hsp60 and Fdxr and reduced mitochondrial localization.
Expression of Fhit or mutants that bind interactor proteins results in
oxidative damage and accumulation of cells in G2/M or
sub-G1 fractions after peroxide treatment; noninteracting mutants
are defective in these biological effects. Gastric cancer clones expressing
noncomplexing Fhit mutants show reduction of Fhit tumor suppressor activity,
confirming that substrate binding, interaction with heat shock proteins,
mitochondrial localization, and interaction with Fdxr are important for Fhit
tumor suppressor function.Fhit protein is a powerful tumor suppressor that is frequently lost or
reduced in cancer cells because of rearrangement of the exquisitely DNA
damage-sensitive fragile FHIT gene. Restoration of Fhit expression
suppresses tumorigenicity of cancer cells of various types, and the ability to
induce apoptosis in cancer cells in vitro is reduced by specific Fhit
mutations (1,
2).Through studies of signal pathways affected by Fhit expression, by searches
for Fhit protein effectors, and by in vitro analyses of Fhit
activity, we and others have defined Fhit enzymatic activity in vitro
(3), apoptotic activity in
cells and tumors
(4–6),
and most recently identification of a Fhit protein complex that affects Fhit
stability, mitochondrial localization, and interaction with ferredoxin
reductase (Fdxr)5
(7). The complex includes Hsp60
and Hsp10 that mediate Fhit stability and may affect import into mitochondria,
where Fhit interacts with Fdxr, which is responsible for transferring
electrons from NADPH to cytochrome P450 via ferredoxin. Virally mediated Fhit
restoration in Fhit-deficient cancer cells increases production of
intracellular reactive oxygen species (ROS), followed by increased apoptosis
of cancer cells under oxidative stress conditions; conversely, Fhit-negative
cells escape apoptosis, likely carrying oxidative DNA damage that contributes
to accumulation of mutations.The Fhit protein sequence, showing high homology to the histidine triad
(HIT) family of proteins, suggested that the protein product would hydrolyze
diadenosine tetraphosphate or diadenosine triphosphate (Ap3A)
(8), and in vitro
studies showed that Ap3A was cleaved into ADP and AMP by Fhit. The
catalytic histidine triad within Fhit was essential for catalytic activity
(3), and a Fhit mutant that
substituted Asn for His at the central histidine (H96N mutant) was
catalytically inactive, although it bound substrate well
(3). Early tumor suppression
studies showed that cancer cells stably transfected with wild type (WT) or
H96N mutant Fhit were suppressed for tumor growth in nude mice. This suggested
the hypothesis that the Fhit-substrate complex sends the tumor suppression
signal (9,
10). To test this hypothesis,
a series of FHIT alleles was designed to reduce substrate-binding
and/or hydrolytic rates and was characterized by quantitative cell-death
assays on cancer cells virally infected with each allele. The allele series
covered defects as great as 100,000-fold in kcat and
increases as large as 30-fold in Km. Mutants with
2–7-fold increases in Km had significantly reduced
apoptotic indices and the mutant with a 30-fold increase in
Km retained little apoptotic function. Thus, the
proapoptotic function of Fhit, which is likely associated with tumor
suppressor function, is limited by substrate binding and is unrelated to
substrate hydrolysis (11).Fhit, a homodimeric protein of 147 amino acids, is a target of tyrosine
phosphorylation by the Src family protein kinases, which can phosphorylate
Tyr-114 of Fhit in vitro and in vivo
(12). After co-expression of
Fhit with the Elk tyrosine kinase in Escherichia coli to generate
phosphorylated forms of Fhit, unphosphorylated, mono-, and diphosphorylated
Fhit were purified, and enzyme kinetics studies showed that monophosphorylated
Fhit exhibited monophasic kinetics with Km and
kcat values ∼2- and ∼7-fold lower, respectively,
than for unphosphorylated Fhit. Diphosphorylated Fhit exhibited biphasic
kinetics; one site had Km and kcat
values ∼2- and ∼140-fold lower, respectively, than for
unphosphorylated Fhit; the second site had a Km
∼60-fold higher and a kcat ∼6-fold lower than for
unphosphorylated Fhit (13).
Thus, it was possible that the alterations in Km and
kcat values for phosphorylated forms of Fhit might favor
formation and lifetime of the Fhit-Ap3A complex and enhance tumor
suppressor activity (see Fhit forms
Kinetic parameters
% Sub-G1
Direct binding
Subcellular location
Co-IP in vivo
8-OHdG
Apoptosis
Tumor suppressor
Km (mm) kcat (s–1) A549 MKN74 Hsp60 Fdxr Hsp60 Fdxr
Fhit WT
1.6 +/– 0.19
2.7 +/– 0.95
43
24
Yes
Yes
Cyt & mito
Yes
Yes
Yes
Yes
Yes
Catalyt mutants H96D
Up 2-fold
Down >2 × 104 29
NT
NT
NT
Cyt & mito
Yes
Yes
NT
Yes
NT
H96N
Up 2-fold
Down >5 × 105
31
14.4
NT
NT
Cyt & mito
Yes
Yes
Yes
Yes
Yes
Loop mutants Y114A
Up 23-fold
Down 2-fold
3.7
NT
NT
NT
Cyt
+/–
+/–
+/–
No
No
Y114D
NT
NT
2.9
6
NT
NT
Cyt
+/–
+/–
–
No
–/+
Y114E
NT
NT
NT
NT
NT
NT
Cyt & mito
–/+
–/+
–
No
NT
Y114F
Up 5-fold
Up 1.1-fold
11.5
3
NT
NT
Cyt & mito
–/+
–/+
–
No
No
Y114W
Up 5-fold
Up 1.4-fold
NT
NT
NT
NT
Cyt & mito
–/+
–
–
NT
NT
del113–117
Up 10-fold
Down 38-fold
5
NT
NT
NT
NT
NT
NT
–
No
NT
Other mutants L25W
Up 7-fold
Down 4-fold
15
NT
NT
NT
Cyt
–
–
–
NT
–/+
I10W,L25W
Up 32-fold
Down 6-fold
11
NT
NT
NT
NT
NT
NT
NT
NT
NT
F5W
Up 3.3 fold
NT
NT
5
NT
NT
NT
NT
NT
+/–
No
NT
Purified pFhit pFhit
Down 0.4-fold
Down 7-fold
NA
NA
–/+
Yes
NA
NA
NA
NA
NA
NA
ppFhit
Down 0.4-fold
Down > 100-fold
NA
NA
–/+
Yes
NA
NA
NA
NA
NA
NA
Up 60-fold
Down 6-fold