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1.
Cj0859c variants fspA1 and fspA2 from 669 human, poultry, and bovine Campylobacter jejuni strains were associated with certain hosts and multilocus sequence typing (MLST) types. Among the human and poultry strains, fspA1 was significantly (P < 0.001) more common than fspA2. FspA2 amino acid sequences were the most diverse and were often truncated.Campylobacter jejuni is the leading cause of bacterial gastroenteritis worldwide and responsible for more than 90% of Campylobacter infections (7). Case-control studies have identified consumption or handling of raw and undercooked poultry meat, drinking unpasteurized milk, and swimming in natural water sources as risk factors for acquiring domestic campylobacteriosis in Finland (7, 9). Multilocus sequence typing (MLST) has been employed to study the molecular epidemiology of Campylobacter (4) and can contribute to virulotyping when combined with known virulence factors (5). FspA proteins are small, acidic, flagellum-secreted nonflagellar proteins of C. jejuni that are encoded by Cj0859c, which is expressed by a σ28 promoter (8). Both FspA1 and FspA2 were shown to be immunogenic in mice and protected against disease after challenge with a homologous strain (1). However, FspA1 also protected against illness after challenge with a heterologous strain, whereas FspA2 failed to do the same at a significant level. Neither FspA1 nor FspA2 protected against colonization (1). On the other hand, FspA2 has been shown to induce apoptosis in INT407 cells, a feature not exhibited by FspA1 (8). Therefore, our aim was to study the distributions of fspA1 and fspA2 among MLST types of Finnish human, chicken, and bovine strains.In total, 367 human isolates, 183 chicken isolates, and 119 bovine isolates (n = 669) were included in the analyses (3). PCR primers for Cj0859c were used as described previously (8). Primer pgo6.13 (5′-TTGTTGCAGTTCCAGCATCGGT-3′) was designed to sequence fspA1. Fisher''s exact test or a chi-square test was used to assess the associations between sequence types (STs) and Cj0859c. The SignalP 3.0 server was used for prediction of signal peptides (2).The fspA1 and fspA2 variants were found in 62.6% and 37.4% of the strains, respectively. In 0.3% of the strains, neither isoform was found. Among the human and chicken strains, fspA1 was significantly more common, whereas fspA2 was significantly more frequent among the bovine isolates (Table (Table1).1). Among the MLST clonal complexes (CCs), fspA1 was associated with the ST-22, ST-45, ST-283, and ST-677 CCs and fspA2 was associated with the ST-21, ST-52, ST-61, ST-206, ST-692, and ST-1332 CCs and ST-58, ST-475, and ST-4001. Although strong CC associations of fspA1 and fspA2 were found, the ST-48 complex showed a heterogeneous distribution of fspA1 and fspA2. Most isolates carried fspA2, and ST-475 was associated with fspA2. On the contrary, ST-48 commonly carried fspA1 (Table (Table1).1). In our previous studies, ST-48 was found in human isolates only (6), while ST-475 was found in both human and bovine isolates (3, 6). The strict host associations and striking difference between fspA variants in human ST-48 isolates and human/bovine ST-475 isolates suggest that fspA could be important in host adaptation.

TABLE 1.

Percent distributions of fspA1 and fspA2 variants among 669 human, poultry, and bovine Campylobacter jejuni strains and their associations with hosts, STs, and CCs
Host or ST complex/ST (no. of isolates)% of strains witha:
P valueb
fspA1fspA2
Host
    All (669)64.335.4
    Human (367)69.530.0<0.001
    Poultry (183)79.220.8<0.001
    Bovine (119)25.274.8<0.0001
ST complex and STs
    ST-21 complex (151)2.697.4<0.0001
        ST-50 (76)NF100<0.0001
        ST-53 (19)NF100<0.0001
        ST-451 (9)NF100<0.0001
        ST-883 (11)NF100<0.0001
    ST-22 complex (22)100NF<0.0001
        ST-22 (11)100NF<0.01
        ST-1947 (9)100NF0.03
    ST-45 complex (268)99.30.7<0.0001
        ST-11 (7)100NFNA
        ST-45 (173)99.40.6<0.0001
        ST-137 (22)95.54.50.001
        ST-230 (14)100NF<0.0001
    ST-48 complex (18)44.455.6NA
        ST-48 (7)100NFNA
        ST-475 (8)NF100<0.001
    ST-52 complex (5)NF100<0.01
        ST-52 (4)NF1000.02
    ST-61 complex (21)NF100<0.0001
        ST-61 (11)NF100<0.0001
        ST-618 (3)NF1000.04
    ST-206 complex (5)NF100<0.01
    ST-283 complex (24)100NF<0.0001
        ST-267 (23)100NF<0.0001
    ST-677 complex (59)100NF<0.0001
        ST-677 (48)100NF<0.0001
        ST-794 (11)100NF<0.001
    ST-692 complex (3)NF1000.04
    ST-1034 complex (5)NF80NA
        ST-4001 (3)NF1000.04
    ST-1287 complex/ST-945 (8)100NFNA
    ST-1332 complex/ST-1332 (4)NF1000.02
    Unassigned STs
        ST-58 (6)NF100<0.01
        ST-586 (6)100NFNA
Open in a separate windowaIn 0.3% of the strains, neither isoform was found. NF, not found.bNA, not associated.A total of 28 isolates (representing 6 CCs and 13 STs) were sequenced for fspA1 and compared to reference strains NCTC 11168 and 81-176. All isolates in the ST-22 CC showed the same one-nucleotide (nt) difference with both NCTC 11168 and 81-176 strains, resulting in a Thr→Ala substitution in the predicted protein sequence (represented by isolate FB7437, GenBank accession number HQ104931; Fig. Fig.1).1). Eight other isolates in different CCs showed a 2-nt difference (isolate 1970, GenBank accession number HQ104932; Fig. Fig.1)1) compared to strains NCTC 11168 and 81-176, although this did not result in amino acid substitutions. All 28 isolates were predicted to encode a full-length FspA1 protein.Open in a separate windowFIG. 1.Comparison of FspA1 and FspA2 isoforms. FspA1 is represented by 81-176, FB7437, and 1970. FspA2 is represented by C. jejuni strains 76763 to 1960 (GenBank accession numbers HQ104933 to HQ104946). Scale bar represents amino acid divergence.In total, 62 isolates (representing 7 CCs and 35 STs) were subjected to fspA2 sequence analysis. Although a 100% sequence similarity between different STs was found for isolates in the ST-21, ST-45, ST-48, ST-61, and ST-206 CCs, fspA2 was generally more heterogeneous than fspA1 and we found 13 predicted FspA2 amino acid sequence variants in total (Fig. (Fig.1).1). In several isolates with uncommon and often unassigned (UA) STs, the proteins were truncated (Fig. (Fig.1),1), with most mutations being ST specific. For example, all ST-58 isolates showed a 13-bp deletion (isolate 3074_2; Fig. Fig.1),1), resulting in a premature stop codon. Also, all ST-1332 CC isolates were predicted to have a premature stop codon by the addition of a nucleotide between nt 112 and nt 113 (isolate 1960; Fig. Fig.1),1), a feature shared with two isolates typed as ST-4002 (UA). A T68A substitution in ST-1960 (isolate T-73494) also resulted in a premature stop codon. Interestingly, ST-1959 and ST-4003 (represented by isolate 4129) both lacked one triplet (nt 235 to 237), resulting in a shorter FspA2 protein. SignalP analysis showed the probability of a signal peptide between nt 22 and 23 (ACA-AA [between the underlined nucleotides]). An A24C substitution in two other strains, represented by isolate 76580, of ST-693 and ST-993 could possibly result in a truncated FspA2 protein as well.In conclusion, our results showed that FspA1 and FspA2 showed host and MLST associations. The immunogenic FspA1 seems to be conserved among C. jejuni strains, in contrast to the heterogeneous apoptosis-inducing FspA2, of which many isoforms were truncated. FspA proteins could serve as virulence factors for C. jejuni, although their roles herein are not clear at this time.  相似文献   

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We analyzed the temporal and spatial diversity of the microbiota in a low-usage and a high-usage hospital tap. We identified a tap-specific colonization pattern, with potential human pathogens being overrepresented in the low-usage tap. We propose that founder effects and local adaptation caused the tap-specific colonization patterns. Our conclusion is that tap-specific colonization represents a potential challenge for water safety.Humans are exposed to and consume large amounts of tap water in their everyday life, with the tap water microbiota representing a potent reservoir for pathogens (8). Despite the potential impact, our knowledge about the ecological diversification processes of the tap water microbiota is limited (4, 11).The aim of the present work was to determine the temporal and spatial distribution patterns of the planktonic tap water microbiota. We compared the summer and winter microbiota from two hospital taps supplied from the same water source. We analyzed 16S rRNA gene clone libraries by using a novel alignment-independent approach for operational taxonomic unit (OTU) designation (6), while established OTU diversity and richness estimators were used for the ecological interpretations.Tap water samples (1 liter) from a high-usage kitchen and a low-usage toilet cold-water tap in Akershus University Hospital, Lørenskog, Norway, were collected in January and July 2006. The total DNA was isolated and the 16S rRNA gene PCR amplified and sequenced. Based on the sequences, we estimated the species richness and diversity, we calculated the distances between the communities, and trees were constructed to reflect the relatedness of the microbiota in the samples analyzed. Details about these analytical approaches are given in the materials and methods section in the supplemental material.Our initial analysis of species composition was done using the RDPII hierarchical classifier. We found that the majority of pathogen-related bacteria in our data set belonged to the class Gammaproteobacteria. The genera encompassed Legionella, Pseudomonas, and Vibrio (Table (Table1).1). We found a significant overrepresentation of pathogen-related bacteria in the toilet tap (P = 0.04), while there were no significant differences between summer and winter samples. Legionella showed the highest relative abundance for the pathogen-related bacteria. With respect to the total diversity, we found that Proteobacteria dominated the tap water microbiota (representing 86% of the taxa) (see Table S1 in the supplemental material). There was, however, a large portion (56%) of the taxa that could not be assigned to the genus level using this classifier.

TABLE 1.

Cloned sequences related to human pathogensa
Sampling placeSampling timePathogenNCBI accession no.Identity (%)
ToiletSummerEscherichia coliEF41861499
ToiletSummerEscherichia sp.EF07430799
ToiletSummerLegionella sp.AY92415595
ToiletSummerLegionella sp.AY92415395
ToiletSummerLegionella sp.AY92415396
ToiletWinterLegionella sp.AY92406196
ToiletWinterLegionella sp.AY92415897
ToiletWinterLegionella sp.AY92415897
KitchenWinterLegionella sp.AY92399697
ToiletSummerPseudomonas fluorescensEF41307398
ToiletSummerPseudomonas fluorescensEF41307398
KitchenSummerPseudomonas fluorescensDQ20773199
ToiletWinterVibrio sp.DQ40838898
ToiletWinterVibrio sp.AB27476098
KitchenWinterVibrio sp.DQ40838898
KitchenWinterVibrio lentusAY29293699
KitchenWinterVibrio sp.AM18376597
ToiletWinterStenotrophomonas maltophiliaAY83773099
KitchenWinterStenotrophomonas maltophiliaDQ42487098
ToiletWinterStreptococcus suisAF28457898
ToiletWinterStreptococcus suisAF28457898
Open in a separate windowaThe relatedness between the cloned sequences and potential pathogens was determined by BLAST searches of the NCBI database, carried out using default settings.To obtain a better resolution of the uncharacterized microbiota, we analyzed the data using a clustering approach that is not dependent on a predefined bacterial group (see the materials and methods section in the supplemental material for details). These analyses showed that there were three relatively tightly clustered groups in our data set (Fig. (Fig.1A).1A). The largest group (n = 590) was only distantly related to characterized betaproteobacteria within the order Rhodocyclales. We also identified another large betaproteocaterial group (n = 320) related to Polynucleobacter. Finally, a tight group (n = 145) related to the alphaproteobacterium Sphingomonas was identified.Open in a separate windowFIG. 1.Tap water microbiota diversity, determined by use of a principal component analysis coordinate system. (A) Each bacterium is classified by coordinates, with the following color code: brown squares, kitchen summer; red diamonds, toilet summer; green triangles, kitchen winter; and green circles, toilet winter. (B and C) Each square represents a 1 × 1 (B) or 5 × 5 (C) OTU. PC1, first principal component; PC2, second principal component.The tap-specific distributions of the bacterial groups were investigated using density distribution analyses. A dominant population related to Polynucleobacter was identified for the toilet summer samples, while for the winter samples there was a dominance of the Rhodocyclales-related bacteria. The kitchen summer samples revealed a dominance of Sphingomonas. The corresponding winter samples did not reveal distinct high-density bacterial populations (see Table S2 in the supplemental material).Hierarchical clustering for the 1 × 1 OTU density distribution confirmed the relatively low overlap for the microbiota in the samples analyzed (Fig. (Fig.2).2). We found that the microbiota clustered according to tap and not season.Open in a separate windowFIG. 2.Hierarchical clustering for the density distribution of the tap water microbiota. The density of 1 × 1 OTUs was used as a pseudospecies for hierarchical clustering. The tree for the Cord distance matrix is presented, while the distances calculated using the three distance matrices Cord, Brad Curtis, and Sneath Sokal, respectively, are shown for each branch.We have described the species diversity and richness of the microbiota in Table S3 in the supplemental material. For the low taxonomic level, these analyses showed that the diversity and species richness were greater for the winter samples than for the summer samples. Comparing the two taps, the diversity and richness were greater in the kitchen tap than in the toilet tap. In particular, the winter sample from the kitchen showed great richness and diversity. The high taxonomic level, however, did not reveal the same clear differences as did the low level, and the distributions were more even. Rarefaction analyses for the low taxonomic level confirmed the richness and diversity estimates (see Fig. S1 in the supplemental material).Our final analyses sought to fit the species rank distributions to common rank abundance curves. Generally, the rank abundance curves were best fitted to log series or truncated log normal distributions (see Table S4 in the supplemental material). The log series distribution could be fit to all of the samples except the kitchen summer samples at the low taxonomic level, while the truncated log normal distribution could not be fit to the kitchen samples at the high taxonomic level. Interestingly, however, the kitchen winter sample was best fit to a geometric curve at both the high and the low taxonomic level.Diversifying, adaptive biofilm barriers have been documented for tap water bacteria (7), and it is known that planktonic bacteria can interact with biofilms in an adaptive manner (3). On the other hand, tap usage leads to water flowthrough and replacement of the global with the local water population by stochastic founder effects (1).Therefore, we propose that parts of the local diversity observed can be explained by local adaptation (10) and parts by founder effects (9).Most prokaryote diversity measures assume log normal or log series OTU dominance density distributions (5). The kitchen winter sample, however, showed deviations from these patterns by being correlated to geometric distributions (in addition to the log series and truncated log normal distributions for the high taxonomic level). This sample also showed a much greater species richness than the other samples. A possible explanation is that the species richness of the tap water microbiota can be linked to usage and that the kitchen tap is driven toward a founder microbiota by high usage.Since our work indicates an overrepresentation of Legionella in the low-usage tap, it would be of high interest to determine whether the processes for local Legionella colonization can be related to tap usage. Understanding the ecological forces affecting Legionella and other pathogens are of great importance for human health. At the Akerhus University Hospital, this was exemplified by a Pseudomonas aeruginosa outbreak in an intensive care unit, where the outbreak could be traced back to a single tap (2).  相似文献   

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To determine whether and which spirochetes are cleared from Ixodes ricinus ticks during feeding on ruminants, ticks were removed from goats and cattle grazing on tick-infested pastures. Although about a quarter of ticks questing on the pasture were infected by spirochetes, no molted ticks that had previously engorged to repletion on ruminants harbored Lyme disease spirochetes. Borrelia miyamotoi spirochetes, however, appear not to be eliminated. Thus, the more subadult ticks are diverted from reservoir-competent hosts to zooprophylactic ruminants, the smaller the risk of infection by Lyme disease spirochetes is.Various vertebrates serve as reservoir hosts for the tick-borne agents of Lyme disease. A competent reservoir host acquires Lyme disease spirochetes when an infected tick feeds on it and maintains them to become and remain infectious for feeding ticks (10). It appears that each of the seven genospecies of Borrelia burgdorferi sensu lato prevalent in Central Europe is associated with particular reservoir hosts. Whereas rodents serve as a reservoir for B. afzelii and the recently differentiated but not yet validated “Borrelia bavariensis,” birds maintain B. garinii and B. valaisiana (3, 4). B. lusitaniae and B. spielmanii, on the other hand, seem to be limited to lizards and dormice, respectively (9, 12, 13). Ticks harboring rodent-associated spirochetes from their larval blood meal may lose the infectious burden when feeding as nymphs on a bird and vice versa (5). It appears that solely B. burgdorferi sensu stricto constitutes an intermediate position, since it may be perpetuated by birds and rodents (10, 11). As a generalist, B. burgdorferi sensu stricto appears to be less efficiently adapted to rodents than is the specialist B. afzelii. A host that is competent for one genospecies seems less competent or incompetent for another.The Central European vector tick, Ixodes ricinus, not only feeds on small animals. Wild ruminants, such as red, roe, and fallow deer, are frequently infested by all three stages of this tick (2, 6, 15). Interestingly, virtually no spirochetes were detected microscopically in ticks recovered from shot deer. On pastures, where domesticated ruminants graze at an extensive density, spirochetal infection in questing ticks is less prevalent than in nearby nonpastured sites (8). These ruminants appear to exert a zooprophylactic effect. Ruminants, although feeding numerous ticks, appear to be incompetent hosts for Lyme disease spirochetes. It is not known whether the incompetence of ruminants eliminates spirochetes in the feeding tick and whether it extends to each of the Lyme disease genospecies.To determine whether and which spirochetes are cleared from ticks feeding on ruminants, ticks were removed from goats and cattle grazing on tick-infested pastures and examined at various developmental stages for Lyme disease genospecies and B. miyamotoi. Infection rates in ruminant-derived ticks were compared to that in ticks questing on the pastures.The cattle study site was located southwest of the city of Flensburg, Germany, at the German-Danish border. The former training area of the German armed forces is used as low-intensity pasture, covering about 400 ha. Galloway cattle, in herds of mother cows, and Konik horses are allowed to graze year-round and are rotated on grazing patches. Most cattle which were examined for feeding ticks grazed in a 40-ha area which has been pastured since October 2004 and from which cattle and horses are excluded each year from April through June to permit rare plants to bloom and seed. The approximate grazing density of 0.25 livestock units (LU)/ha throughout the rest of the year fails to keep the vegetation short. The goat site was located about 50 km southeast of Stuttgart, Germany, near the village of Gruibingen in the Swabian highlands. Beech and juniper heath characterize the southern-facing mountain slopes, where goats were allowed to graze in a rotating regime during the vegetation period. The sites were in use as pastures for different lengths of time, with the oldest dating back to 2004.To obtain feeding ticks from cattle and goats, two approaches were used. For the yearly blood sampling in the spring, cattle were corralled into squeeze chutes. The head of each animal was examined for ticks. Feeding ticks were carefully removed with forceps, and replete ticks were gently rubbed off onto a sheet of fabric positioned under the cow''s head. From April through October 2006 and 2007 and in May of 2008, tame goats were examined individually for feeding ticks monthly and feeding ticks were removed with forceps. Ticks recovered from an individual animal were confined in screened vials and stored at 22°C to permit molting and/or until they were examined for spirochetes. Questing ticks were collected monthly from April through October 2008 in the cattle site and from April through October 2005 through 2007 at the goat site. They were collected by means of a flannel flag, identified to stage and species by microscopy, and preserved in 80% ethanol. To detect and identify the various spirochetes that may be present in questing or host-derived ticks, DNA from individual ticks was isolated, and a 600-nucleotide fragment of the gene carrying the 16S rRNA gene was amplified by nested PCR and sequenced as described previously (12). This method detects as little as a single spirochete even in the presence of tick and ruminant DNA. Each resulting sequence was compared with sequences of the same gene fragment representing various spirochetal genospecies. The following sequences were used for comparison: GenBank accession numbers X85196 and X85203 for B. burgdorferi sensu stricto, X85190, X85192, and X85194 for B. afzelii, X85193, X85199, and M64311 for B. garinii, X98228 and X98229 for B. lusitaniae, X98232 and X98233 for B. valaisiana, AY147008 for B. spielmanii, and AY253149 for B. miyamotoi. A complete match, permitting no more than two nucleotide changes, was required.Ticks removed while feeding on cattle or goats were examined for spirochetal DNA by nested PCR. Nineteen larvae were obtained during their feeding on goats, but none of the 17 engorged larvae and 2 resulting nymphs contained spirochetal DNA (Table (Table1).1). Of the 416 nymphal ticks that were obtained from 80 goats, only 9% developed to the adult stage, because most of the nymphs were only partially fed. None of the 37 resulting adults contained spirochetal DNA. However, three partially fed nymphal ticks were infected by Lyme disease spirochetes (0.8%), one each by B. afzelii, B. valaisiana, and B. lusitaniae. In three additional nymphs (0.8%), DNA of B. miyamotoi was detected. Of the 415 engorged nymphal ticks obtained from 42 cattle, as many as 319 (77%) molted to the adult stage, because mostly replete ticks had been collected from the cattle''s heads. None of the cattle-derived molted ticks harbored DNA of Lyme disease spirochetes. Four ticks, a nymph and three adults (one male and two females), contained DNA of B. miyamotoi. Of 306 partially engorged females removed from 68 goats, spirochetal DNA was detected in 9 females (2.9%); three harbored B. afzelii, four B. miyamotoi, and one each B. garinii and B. lusitaniae. In addition, 30 females which had fully engorged on cattle were tested for spirochetal DNA after egg-laying. None of these contained spirochetal DNA. Although DNA of Lyme disease spirochetes was detected in a rare partially fed tick, no molted tick that had previously engorged to repletion on a ruminant was infected by Lyme disease spirochetes. In contrast, B. miyamotoi appears to be present in ruminant-fed ticks regardless of their feeding state.

TABLE 1.

Borrelia genospecies detected in I. ricinus ticks that had engorged as larvae, nymphs, or adults on goats or cattle
HostNo. of host animalsTicks
% of infected ticks harboring Borrelia sp.b
StageStateNo. examined% infected% infected by LDa spirocheteafzgarvallusmiy
Goats10LarvaEngorged170.00.0
2NymphMolted20.00.0
80NymphEngorged3791.60.816.70.016.716.750.0
22AdultMolted370.00.0
68AdultEngorged3062.91.633.311.10.011.144.4
Cattle36NymphEngorged961.00.00.00.00.00.0100
42AdultMolted3190.90.00.00.00.00.0100
10AdultEngorged300.00.0
Total144c1,1861.40.723.55.95.911.852.9
Open in a separate windowaLD, Lyme disease.bafz, B. afzelii; gar, B. garinii; val, B. valaisiana; lus, B. lusitaniae; miy, B. miyamotoi.cValue is not the sum of the above numbers because individual hosts were infested by various tick stages.The prevalence of spirochetal infection was determined in questing ticks collected on the pastures on which the cattle or the goats had roamed. A third of the nymphs and nearly a fifth of the adult ticks that quested on the cattle pasture in northern Germany contained spirochetal DNA (Table (Table2).2). The majority of these nymphs and half the infected adults were infected by B. afzelii. About a fifth of the nymphs and a quarter of the adult ticks questing on the goat pastures in southern Germany were infected by spirochetes. B. afzelii and B. lusitaniae infected most of these ticks. Thus, the cattle and goats in the study sites must have been exposed to numerous vector ticks infected by spirochetes.

TABLE 2.

Relative prevalences of Borrelia genospecies in questing nymphal and adult I. ricinus ticks sampled on goat or cattle pastures in Germany
Grazing animals on pastureTicks
% of infected ticks harboring Borrelia sp.a
StageNo. examined% infectedafzgarvalburlusspibismiy>1 genospecies
GoatsNymph55717.241.76.35.24.236.50.00.08.32.1
Adult51125.013.37.06.33.961.70.80.010.23.1
CattleNymph41332.490.30.70.70.00.00.04.56.02.2
Adult6717.950.016.78.30.00.00.016.78.30.0
Open in a separate windowaafz, B. afzelii; gar, B. garinii; val, B. valaisiana; bur, B. burgdorferi; lus, B. lusitaniae; spi, B. spielmanii; bis, B. bissettii-like (1, 7); miy, B. miyamotoi.Ticks infected by Lyme disease spirochetes appear to lose their infection when feeding on goats or cattle. If the blood meal on ruminants had no effect on the spirochetal burden, about 130 and 70 of the analyzed nymphs derived from cattle and goats, respectively, should have contained spirochetal DNA. The two infected cattle-derived ticks harbored solely spirochetes not related to those causing Lyme disease. Most of the ticks removed from goats were partially fed and appeared to be somewhat more likely to contain spirochetal DNA. Whether the detected DNA indicates viable spirochetes is not known. Either feeding on goats fails to eliminate spirochetes as effectively as does a blood meal on cattle or, more plausibly, engorgement to repletion is required for a complete elimination of DNA from Lyme disease spirochetes. If no spirochetal DNA is detected, the tick cannot contain viable spirochetes and thus is not infectious in its host-seeking stage.Wild and domestic ruminants appear to be reservoir incompetent for Lyme disease spirochetes. They do not constitute reservoirs for this pathogen, because no larval tick feeding on them acquires Lyme disease spirochetes. Of 176 engorged I. ricinus larvae or resulting nymphs that had been collected from roe, fallow, red deer, and wild sheep in a Central European site in an earlier study, spirochetes were detected by dark-field microscopy in only two ticks (6). Similarly, only 2 of nearly 200 Ixodes dammini nymphs resulting from larvae that had engorged on white-tailed deer in northeast America contained spirochetes detectable by direct immunofluorescence (15). No spirochetes were detected by phase-contrast microscopy in more than 200 Swedish nymphs that derived from roe-deer-fed larvae (2). Considering that B. miyamotoi morphologically resembles Lyme disease spirochetes, it is likely that all of the spirochetes detected microscopically in ruminant-derived ticks during these earlier studies were not related to B. burgdorferi sensu lato. Not only do larvae fail to acquire Lyme disease spirochetes from ruminants, but infected nymphs also appear to lose their spirochetal load when feeding on these animals, as the present study demonstrates. In ticks that had fully engorged on cattle, the only spirochetal DNA that was detected was that of B. miyamotoi. Also, the American strain of B. miyamotoi was discovered in larvae resulting from field-collected adult females that had routinely been fed on sheep (14). The previous observation that the prevalence of B. miyamotoi in a cattle pasture was not significantly reduced compared to that in the nonpastured site nearby further exemplifies the differential effect of ruminants on these two kinds of spirochetes (8). Whereas Lyme disease spirochetes are eliminated when their tick vector feeds on a ruminant, B. miyamotoi appears not to be affected by such a blood meal.Ruminants reduce the prevalence of infected ticks on a pasture. For the present study, sites were chosen that had only recently come into use as pastures and where cattle were excluded during the peak season of tick activity. The spirochetal prevalence was thus similar to that in the surrounding areas where no domestic ruminants roamed (data not shown) and permitted us to compare infection rates before and after the blood meal on ruminants. The effect of the grazing schedule and of the grazing duration that is required to result in reduced prevalence still needs to be determined. Domestic ruminants employed in landscape management appear to exert their zooprophylactic effect in multiple ways, by eliminating spirochetes from vector ticks feeding on them and by reducing the ecotonal vegetation, thereby limiting coverage and food sources of reservoir hosts while simultaneously rendering the microclimate less suitable for vector ticks. This study''s observations indicate that Lyme disease spirochetes are eliminated from the tick during its blood meal on a ruminant. The mechanism by which Lyme disease spirochetes are cleared during the tick''s blood meal is under investigation. Evidently, Lyme disease spirochetes are destroyed in a way that renders their DNA no longer detectable by means of nested PCR. A simulation model indicates that the availability of incompetent hosts for subadult tick stages would reduce the prevalence of infection (16). Therefore, the more subadult ticks are diverted from reservoir competent birds or mice to incompetent ruminants, the smaller the risk of infection with the agent of Lyme disease is.  相似文献   

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10.
GTP cyclohydrolase I (GCYH-I) is an essential Zn2+-dependent enzyme that catalyzes the first step of the de novo folate biosynthetic pathway in bacteria and plants, the 7-deazapurine biosynthetic pathway in Bacteria and Archaea, and the biopterin pathway in mammals. We recently reported the discovery of a new prokaryotic-specific GCYH-I (GCYH-IB) that displays no sequence identity to the canonical enzyme and is present in ∼25% of bacteria, the majority of which lack the canonical GCYH-I (renamed GCYH-IA). Genomic and genetic analyses indicate that in those organisms possessing both enzymes, e.g., Bacillus subtilis, GCYH-IA and -IB are functionally redundant, but differentially expressed. Whereas GCYH-IA is constitutively expressed, GCYH-IB is expressed only under Zn2+-limiting conditions. These observations are consistent with the hypothesis that GCYH-IB functions to allow folate biosynthesis during Zn2+ starvation. Here, we present biochemical and structural data showing that bacterial GCYH-IB, like GCYH-IA, belongs to the tunneling-fold (T-fold) superfamily. However, the GCYH-IA and -IB enzymes exhibit significant differences in global structure and active-site architecture. While GCYH-IA is a unimodular, homodecameric, Zn2+-dependent enzyme, GCYH-IB is a bimodular, homotetrameric enzyme activated by a variety of divalent cations. The structure of GCYH-IB and the broad metal dependence exhibited by this enzyme further underscore the mechanistic plasticity that is emerging for the T-fold superfamily. Notably, while humans possess the canonical GCYH-IA enzyme, many clinically important human pathogens possess only the GCYH-IB enzyme, suggesting that this enzyme is a potential new molecular target for antibacterial development.The Zn2+-dependent enzyme GTP cyclohydrolase I (GCYH-I; EC 3.5.4.16) is the first enzyme of the de novo tetrahydrofolate (THF) biosynthesis pathway (Fig. (Fig.1)1) (38). THF is an essential cofactor in one-carbon transfer reactions in the synthesis of purines, thymidylate, pantothenate, glycine, serine, and methionine in all kingdoms of life (38), and formylmethionyl-tRNA in bacteria (7). Recently, it has also been shown that GCYH-I is required for the biosynthesis of the 7-deazaguanosine-modified tRNA nucleosides queuosine and archaeosine produced in Bacteria and Archaea (44), respectively, as well as the 7-deazaadenosine metabolites produced in some Streptomyces species (33). GCYH-I is encoded in Escherichia coli by the folE gene (28) and catalyzes the conversion of GTP to 7,8-dihydroneopterin triphosphate (55), a complex reaction that begins with hydrolytic opening of the purine ring at C-8 of GTP to generate an N-formyl intermediate, followed by deformylation and subsequent rearrangement and cyclization of the ribosyl moiety to generate the pterin ring in THF (Fig. (Fig.1).1). Notably, the enzyme is dependent on an essential active-site Zn2+ that serves to activate a water molecule for nucleophilic attack at C-8 in the first step of the reaction (2).Open in a separate windowFIG. 1.Reaction catalyzed by GCYH-I, and metabolic fate of 7,8-dihydroneopterin triphosphate.A homologous GCYH-I is found in mammals and other higher eukaryotes, where it catalyzes the first step of the biopterin (BH4) pathway (Fig. (Fig.1),1), an essential cofactor in the biosynthesis of tyrosine and neurotransmitters, such as serotonin and l-3,4-dihydroxyphenylalanine (3, 52). Recently, a distinct class of GCYH-I enzymes, GCYH-IB (encoded by the folE2 gene), was discovered in microbes (26% of sequenced Bacteria and most Archaea) (12), including several clinically important human pathogens, e.g., Neisseria and Staphylococcus species. Notably, GCYH-IB is absent in eukaryotes.The distribution of folE (gene product renamed GCYH-IA) and folE2 (GCYH-IB) in bacteria is diverse (12). The majority of organisms possess either a folE (65%; e.g., Escherichia coli) or a folE2 (14%; e.g., Neisseria gonorrhoeae) gene. A significant number (12%; e.g., B. subtilis) possess both genes (a subset of 50 bacterial species is shown in Table Table1),1), and 9% lack both genes, although members of the latter group are mainly intracellular or symbiotic bacteria that rely on external sources of folate. The majority of Archaea possess only a folE2 gene, and the encoded GCYH-IB appears to be necessary only for the biosynthesis of the modified tRNA nucleoside archaeosine (44) except in the few halophilic Archaea that are known to synthesize folates, such as Haloferax volcanii, where GCYH-IB is involved in both archaeosine and folate formation (13, 44).

TABLE 1.

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

11.
12.
Melioidosis has been considered an emerging disease in Brazil since the first cases were reported to occur in the northeast region. This study investigated two municipalities in Ceará state where melioidosis cases have been confirmed to occur. Burkholderia pseudomallei was isolated in 26 (4.3%) of 600 samples in the dry and rainy seasons.Melioidosis is an endemic disease in Southeast Asia and northern Australia (2, 4) and also occurs sporadically in other parts of the world (3, 7). Human melioidosis was reported to occur in Brazil only in 2003, when a family outbreak afflicted four sisters in the rural part of the municipality of Tejuçuoca, Ceará state (14). After this episode, there was one reported case of melioidosis in 2004 in the rural area of Banabuiú, Ceará (14). And in 2005, a case of melioidosis associated with near drowning after a car accident was confirmed to occur in Aracoiaba, Ceará (11).The goal of this study was to investigate the Tejuçuoca and Banabuiú municipalities, where human cases of melioidosis have been confirmed to occur, and to gain a better understanding of the ecology of Burkholderia pseudomallei in this region.We chose as central points of the study the residences and surrounding areas of the melioidosis patients in the rural areas of Banabuiú (5°18′35″S, 38°55′14″W) and Tejuçuoca (03°59′20″S, 39°34′50′W) (Fig. (Fig.1).1). There are two well-defined seasons in each of these locations: one rainy (running from January to May) and one dry (from June to December). A total of 600 samples were collected at five sites in Tejuçuoca (T1, T2, T3, T4, and T5) and five in Banabuiú (B1, B2, B3, B4, and B5), distributed as follows (Fig. (Fig.2):2): backyards (B1 and T1), places shaded by trees (B2 and T2), water courses (B3 and T3), wet places (B4 and T4), and stock breeding areas (B5 and T5).Open in a separate windowFIG. 1.Municipalities of Banabuiú (5°18′35″S, 38°55′14″W) and Tejuçuoca (03°59′20″S, 39°34′50″W).Open in a separate windowFIG. 2.Soil sampling sites in Banabuiú and Tejuçuoca.Once a month for 12 months (a complete dry/rainy cycle), five samples were gathered at five different depths: at the surface and at 10, 20, 30 and 40 cm (Table (Table1).1). The samples were gathered according to the method used by Inglis et al. (9). Additionally, the sample processing and B. pseudomallei identification were carried out as previously reported (1, 8, 9).

TABLE 1.

Distribution of samples with isolates by site and soil depth
Sitesa and depth (cm)No. of B. pseudomallei isolates in samples from:
Banabuiú (n = 300)Tejuçuoca (n = 300)Total (n = 600)
B1/T13
    Surface2
    10
    201
    30
    40
B2/T21
    Surface1
    10
    20
    30
    40
B3/T315
    Surface2
    102
    204
    303
    404
B4/T45
    Surface
    101
    201
    3011
    401
B5/T52
    Surface
    10
    20
    302
    40
Total62026
Open in a separate windowaSites designated with B are in Banabuiú, and sites designated with T are in Tejuçuoca. See the text for details.The data on weather and soil composition were obtained from specialized government institutions, such as FUNCEME, IPECE, and EMBRAPA. The average annual temperature in both municipalities is between 26 and 28°C. In 2007, the annual rainfall in Tejuçuoca was 496.8 mm, and that in Banabuiú was 766.8 mm. There are a range of soil types in both Tejuçuoca and Banabuiú: noncalcic brown, sodic planossolic, red-yellow podzolic, and litholic. In Banabuiú, there are also alluvial and cambisol soils. The characteristic vegetation in both municipalities is caatinga (scrublands).There were isolates of B. pseudomallei in 26 (4.3%) of the 600 samples collected. The bacterium was isolated at a rate (3%) similar to that previously reported (9). The bacterium isolation occurred in both the dry (53.8%) and the rainy (46.2%) seasons. Tejuçuoca represented 76.9% (20/26) of the strains isolated. Four sites in Tejuçuoca (T1, T3, T4, and T5) and three in Banabuiú (B1, B2, and B4) presented isolates of the bacterium (Table (Table1).1). The isolation of the B. pseudomallei strains varied from the surface down to 40 cm. However, 17 of the 26 positive samples (65.3%) were found at depths between 20 and 40 cm (Table (Table1).1). Only two isolates were found at the surface during the dry season.A study in Vietnam (13) and one in Australia (9) reported the presence of B. pseudomallei near the houses of melioidosis patients. In our study, the same thing happened. Site T3 (15/26; 57.6%) was located 290 m from the patient''s house, as reported by the Rolim group (14).B. pseudomallei was isolated from a sheep paddock in Australia, where animals sought shelter below mango and fig trees (17). In our study, the bacterium was isolated at site T5, a goat corral alongside the house where the outbreak occurred in Tejuçuoca. Four sites in places shaded by trees yielded positive samples (30.7%) in both Tejuçuoca (palm trees) and Banabuiú (mango trees). Additionally, B. pseudomallei was isolated on three occasions from a cornfield (site 4B) located alongside the house of the melioidosis patient in Banabuiú.In the main areas of endemicity, the disease is more prevalent in the rainy season (4, 5, 16). The outbreak in Tejuçuoca was related to rainfall (14). Besides the association of cases of the disease with rainfall itself, the isolation of B. pseudomallei in soil and water was also demonstrated during the dry season (12, 15). An Australian study isolated strains from soil and water during the dry and rainy seasons (17). A Thai study also reported B. pseudomallei in the dry season (18). In our study, the isolation of B. pseudomallei took place either at the end of the wet season or in the dry months. Fourteen of the positive samples (53.8%) were collected during the dry season, albeit near a river or reservoir (sites T3 and B4).Physical, biological, and chemical soil features appear to influence the survival of B. pseudomallei (6, 10). In the present study, the soil was classified as litholic with sandy or clayey textures. It is susceptible to erosion, and when there is a lack of water, it is subject to salinization. During the dry season, the clay layer becomes dried, cracked, and very hard. During the rainy season, it becomes soggy and sticky. The isolation of B. pseudomallei in the dry season is possibly related to the capacity for adaptation of this soil, since the extreme conditions of lithosols do not prevent the bacterial growth and survival.It has been shown that B. pseudomallei is more often isolated at depths between 25 and 45 cm (17). In our study, 65.3% of the positive samples were taken at depths between 20 and 40 cm. Moreover, of these 17 samples, 10 (58.8%) were collected during the dry months. Also, unlike in other regions, two positive samples were taken from the surface in the period without rainfall.The rainfall in Tejuçuoca and Banabuiú is generally low, and temperatures do not vary significantly during the year. Therefore, the isolation of B. pseudomallei in these places occurs outside the rainfall, temperature, and moisture conditions observed in other regions of endemicity. Our data thus suggest that peculiar environmental features, such as soil composition, might favor the multiplication of B. pseudomallei in northeast Brazil.  相似文献   

13.
Twelve cluster groups of Escherichia coli O26 isolates found in three cattle farms were monitored in space and time. Cluster analysis suggests that only some O26:H11 strains had the potential for long-term persistence in hosts and farms. As judged by their virulence markers, bovine enterohemorrhagic O26:H11 isolates may represent a considerable risk for human infection.Shiga toxin (Stx)-producing Escherichia coli (STEC) strains comprise a group of zoonotic enteric pathogens (42). In humans, infections with some STEC serotypes result in hemorrhagic or nonhemorrhagic diarrhea, which can be complicated by hemolytic-uremic syndrome (HUS) (49). These STEC strains are also designated “enterohemorrhagic E. coli” (EHEC). Consequently, EHEC strains represent a subgroup of STEC with a high pathogenic potential for humans. Strains of the E. coli serogroup O26 were originally classified as enteropathogenic E. coli due to their association with outbreaks of infantile diarrhea in the 1940s. In 1977, Konowalchuk et al. (37) recognized that these bacteria produced Stx, and 10 years later, the Stx-producing E. coli O26:H11/H− strains were classified as EHEC. EHEC O26 strains constitute the most common non-O157 EHEC group associated with diarrhea and HUS in Europe (12, 21, 23, 24, 26, 27, 55, 60). Reports on an association between EHEC O26 and HUS or diarrhea from North America including the United States (15, 30, 33), South America (51, 57), Australia (22), and Asia (31, 32) provide further evidence for the worldwide spread of these organisms. Studies in Germany and Austria (26, 27) on sporadic HUS cases between 1996 and 2003 found that EHEC O26 accounted for 14% of all EHEC strains and for ∼40% of non-O157 EHEC strains obtained from these patients. A proportion of 11% EHEC O26 strains was detected in a case-control study in Germany (59) between 2001 and 2003. In the age group <3 years, the number of EHEC O26 cases was nearly equal to that of EHEC O157 cases, although the incidence of EHEC O26-associated disease is probably underestimated because of diagnostic limitations in comparison to the diagnosis of O157:H7/H− (18, 34). Moreover, EHEC O26 has spread globally (35). Beutin (6) described EHEC O26:H11/H−, among O103:H2, O111:H, O145:H28/H−, and O157:H7/H−, as the well-known pathogenic “gang of five,” and Bettelheim (5) warned that we ignore the non-O157 STEC strains at our peril.EHEC O26 strains produce Stx1, Stx2, or both (15, 63). Moreover, these strains contain the intimin-encoding eae gene (11, 63), a characteristic feature of EHEC (44). In addition, EHEC strains possess other markers associated with virulence, such as a large plasmid that carries further potential virulence genes, e.g., genes coding for EHEC hemolysin (EHEC-hlyA), a catalase-peroxidase (katP), and an extracellular serine protease (espP) (17, 52). The efa1 (E. coli factor for adherence 1) gene was identified as an intestinal colonization factor in EHEC (43). EHEC O26 represents a highly dynamic group of organisms that rapidly generate new pathogenic clones (7, 8, 63).Ruminants, especially cattle, are considered the primary reservoir for human infections with EHEC. Therefore, the aim of this study was the molecular characterization of bovine E. coli field isolates of serogroup O26 using a panel of typical virulence markers. The epidemiological situation in the beef herds from which the isolates were obtained and the spatial and temporal behavior of the clonal distribution of E. coli serogroup O26 were analyzed during the observation period. The potential risk of the isolates inducing disease in humans was assessed.In our study, 56 bovine E. coli O26:H11 isolates and one bovine O26:H32 isolate were analyzed for EHEC virulence-associated factors. The isolates had been obtained from three different beef farms during a long-term study. They were detected in eight different cattle in farm A over a period of 15 months (detected on 10 sampling days), in 3 different animals in farm C over a period of 8 months (detected on 3 sampling days), and in one cow on one sampling day in farm D (Table (Table1)1) (28).

TABLE 1.

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

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Specific therapy is not available for hantavirus cardiopulmonary syndrome caused by Andes virus (ANDV). Peptides capable of blocking ANDV infection in vitro were identified using antibodies against ANDV surface glycoproteins Gn and Gc to competitively elute a cyclic nonapeptide-bearing phage display library from purified ANDV particles. Phage was examined for ANDV infection inhibition in vitro, and nonapeptides were synthesized based on the most-potent phage sequences. Three peptides showed levels of viral inhibition which were significantly increased by combination treatment with anti-Gn- and anti-Gc-targeting peptides. These peptides will be valuable tools for further development of both peptide and nonpeptide therapeutic agents.Andes virus (ANDV), an NIAID category A agent linked to hantavirus cardiopulmonary syndrome (HCPS), belongs to the family Bunyaviridae and the genus Hantavirus and is carried by Oligoryzomys longicaudatus rodents (11). HCPS is characterized by pulmonary edema caused by capillary leak, with death often resulting from cardiogenic shock (9, 16). ANDV HCPS has a case fatality rate approaching 40%, and ANDV is the only hantavirus demonstrated to be capable of direct person-to-person transmission (15, 21). There is currently no specific therapy available for treatment of ANDV infection and HCPS.Peptide ligands that target a specific protein surface can have broad applications as therapeutics by blocking specific protein-protein interactions, such as preventing viral engagement of host cell receptors and thus preventing infection. Phage display libraries provide a powerful and inexpensive tool to identify such peptides. Here, we used selection of a cyclic nonapeptide-bearing phage library to identify peptides capable of binding the transmembrane surface glycoproteins of ANDV, Gn and Gc, and blocking infection in vitro.To identify peptide sequences capable of recognizing ANDV, we panned a cysteine-constrained cyclic nonapeptide-bearing phage display library (New England Biolabs) against density gradient-purified, UV-treated ANDV strain CHI-7913 (a gift from Hector Galeno, Santiago, Chile) (17, 18). To increase the specificity of the peptides identified, we eluted phage by using monoclonal antibodies (Austral Biologicals) prepared against recombinant fragments of ANDV Gn (residues 1 to 353) or Gc (residues 182 to 491) glycoproteins (antibodies 6B9/F5 and 6C5/D12, respectively). Peptide sequences were determined for phage from iterative rounds of panning, and the ability of phage to inhibit ANDV infection of Vero E6 cells was determined by immunofluorescent assay (IFA) (7). Primary IFA detection antibodies were rabbit polyclonal anti-Sin Nombre hantavirus (SNV) nucleoprotein (N) antibodies which exhibit potent cross-reactivity against other hantavirus N antigens (3). ReoPro, a commercially available Fab fragment which partially blocks infection of hantaviruses in vitro by binding the entry receptor integrin β3 (5), was used as a positive control (80 μg/ml) along with the original antibody used for phage elution (5 μg/ml). As the maximum effectiveness of ReoPro in inhibiting hantavirus entry approaches 80%, we set this as a threshold for maximal expected efficacy for normalization. The most-potent phage identified by elution with the anti-Gn antibody 6B9/F5 bore the peptide CPSNVNNIC and inhibited hantavirus entry by greater than 60% (61%) (Table (Table1).1). From phage eluted with the anti-Gc antibody 6C5/D12, those bearing peptides CPMSQNPTC and CPKLHPGGC also inhibited entry by greater than 60% (66% and 72%, respectively).

TABLE 1.

Peptide-bearing phage eluted from ANDV
Phage% Inhibition (SD)aP valueb
Phage bearing the following peptides eluted with anti-Gn antibody 6B9/F5
    Group 1 (<30% inhibition)
        CDQRTTRLC8.45 (15.34)0.0002
        CPHDPNHPC9.94 (7.72)0.333
        CQSQTRNHC11.76 (13.25)0.0001
        CLQDMRQFC13.26 (9.92)0.0014
        CLPTDPIQC15.70 (14.05)0.0005
        CPDHPFLRC16.65 (15.22)0.8523
        CSTRAENQC17.56 (16.50)0.0004
        CPSHLDAFC18.98 (20.06)0.0017
        CKTGHMRIC20.84 (7.47)0.0563
        CVRTPTHHC20.89 (27.07)0.1483
        CSGVINTTC21.57 (19.61)0.0643
        CPLASTRTC21.65 (5.98)0.004
        CSQFPPRLC22.19 (8.26)0.0004
        CLLNKQNAC22.34 (7.78)0.001
        CKFPLNAAC22.89 (6.15)0.0001
        CSLTPHRSC23.63 (16.74)0.0563
        CKPWPMYSC23.71 (6.68)0.0643
        CLQHDALNC24.01 (7.60)1
        CNANKPKMC24.67 (11.67)0.0004
        CPKHVLKVC25.30 (28.36)0.0003
        CTPDKKSFC26.91 (11.15)0.399
        CHGKAALAC27.22 (32.53)0.005
        CNLMGNPHC28.08 (21.35)0.0011
        CLKNWFQPC28.64 (18.49)0.0016
        CKEYGRQMC28.76 (29.33)0.0362
        CQPSDPHLC29.44 (31.22)0.0183
        CSHLPPNRC29.70 (17.37)0.0061
    Group 2 (30-59% inhibition)
        CSPLLRTVC33.05 (20.26)0.0023
        CHKGHTWNC34.17 (12.50)0.0795
        CINASHAHC35.62 (13.03)0.3193
        CWPPSSRTC36.75 (26.95)0.0006
        CPSSPFNHC37.78 (7.11)0.0001
        CEHLSHAAC38.47 (7.60)0.0115
        CQDRKTSQC38.74 (9.12)0.1802
        CTDVYRPTC38.90 (25.03)0.006
        CGEKSAQLC39.11 (27.52)0.0013
        CSAAERLNC40.13 (6.33)0.0033
        CFRTLEHLC42.07 (5.01)0.0608
        CEKLHTASC43.60 (27.92)0.1684
        CSLHSHKGC45.11 (49.81)0.0864
        CNSHSPVHC45.40 (28.80)0.0115
        CMQSAAAHC48.88 (44.40)0.5794
        CPAASHPRC51.84 (17.09)0.1935
        CKSLGSSQC53.90 (13.34)0.0145
    Group 3 (60-79% inhibition)
        CPSNVNNIC61.11 (25.41)0.1245
Negative control0 (6.15)
6B9/F5 (5 μg/ml)26.77 (5.33)
ReoPro (80 μg/ml)79.86 (4.88)
Phage bearing the following peptides eluted with anti-Gc antibody 6C5/D12
    Group 1 (<30% inhibition)
        CHPGSSSRC1.01 (7.03)0.0557
        CSLSPLGRC10.56 (13.62)0.7895
        CTARYTQHC12.86 (3.83)0.3193
        CHGVYALHC12.91 (7.32)0.0003
        CLQHNEREC16.79 (13.72)0.0958
        CHPSTHRYC17.23 (14.53)0.0011
        CPGNWWSTC19.34(9.91)0.1483
        CGMLNWNRC19.48 (19.42)0.0777
        CPHTQFWQC20.44 (13.65)0.0008
        CTPTMHNHC20.92 (11.68)0.0001
        CDQVAGYSC21.79 (23.60)0.0063
        CIPMMTEFC24.33 (9.28)0.2999
        CERPYSRLC24.38 (9.09)0.0041
        CPSLHTREC25.06 (22.78)0.1202
        CSPLQIPYC26.30 (34.29)0.4673
        CTTMTRMTC (×2)29.27 (8.65)0.0001
    Group 2 (30-59% inhibition)
        CNKPFSLPC30.09 (5.59)0.4384
        CHNLESGTC31.63 (26.67)0.751
        CNSVPPYQC31.96 (6.51)0.0903
        CSDSWLPRC32.95 (28.54)0.259
        CSAPFTKSC33.40 (10.64)0.0052
        CEGLPNIDC35.63 (19.90)0.0853
        CTSTHTKTC36.28 (13.42)0.132
        CLSIHSSVC36.40 (16.44)0.8981
        CPWSTQYAC36.81 (32.81)0.5725
        CTGSNLPIC36.83 (31.64)0.0307
        CSLAPANTC39.73 (4.03)0.1664
        CGLKTNPAC39.75 (16.98)0.2084
        CRDTTPWWC40.08 (18.52)0.0004
        CHTNASPHC40.26 (4.77)0.5904
        CTSMAYHHC41.89 (8.61)0.259
        CSLSSPRIC42.13 (29.75)0.2463
        CVSLEHQNC45.54 (6.55)0.5065
        CRVTQTHTC46.55 (8.45)0.3676
        CPTTKSNVC49.28 (14.00)0.3898
        CSPGPHRVC49.50 (42.60)0.0115
        CKSTSNVYC51.20 (4.60)0.0611
        CTVGPTRSC57.30 (11.31)0.0176
    Group 3 (60-79% inhibition)
        CPMSQNPTC65.60 (13.49)0.014
        CPKLHPGGC71.88 (27.11)0.0059
Negative control0.26 (4.53)
6C5/D12 (5 μg/ml)22.62 (8.40)
ReoPro (80 μg/ml)80.02 (76.64)
Open in a separate windowaStandard deviations of four experiments are shown in parentheses. Peptide-bearing phage were added at 109 phage/μl.bP values for the pairwise amino acid alignment score of each peptide versus that of integrin β3 were determined using an unpaired Student''s t test. P values considered statistically significant are shown in bold.To determine whether the peptide sequences of any of the identified inhibitory phage showed homology to integrin β3, a known entry receptor for pathogenic hantaviruses (6, 7), we used the Gap program to perform a pairwise amino acid alignment of each peptide versus the extracellular portion of integrin β3 and determined P values for the alignments. Of 45 phage eluted with the anti-Gn antibody, 6B9/F5, 27 of the peptide sequences showed homology to integrin β3 (P < 0.05), and 9 were highly significant (P ≤ 0.0005) (Fig. (Fig.1A).1A). Of the latter, CKFPLNAAC and CSQFPPRLC map to the hybrid domain (Fig. (Fig.1B),1B), which is proximal to the plexin-semaphorin-integrin domain (PSI) containing residue D39, shown to be critical for viral entry in vitro (19). Five sequences (CPSSPFNH, CPKHVLKVC, CNANKPKMC, CQSQTRNHC, and CDQRTTRLC) map to the I-like (or βA) domain near the binding site of ReoPro (2). Finally, CLPTDPIQC maps to the epidermal growth factor 4 (EGF-4) domain, and CSTRAENQC aligns to a portion of β3 untraceable in the crystal structure, specifically the linker region between the hybrid domain and EGF-1. Although this represents a disordered portion of the protein (22), the location of this loop proximal to the PSI domain is worth noting, due to the role of the PSI domain in facilitating viral entry (19). Therefore, 60% of phage eluted with the anti-Gn antibody showed some homology to integrin β3, and those with highly significant P values predominantly mapped to or proximal to regions of known interest in viral entry.Open in a separate windowFIG. 1.Inhibitory peptides identified through phage panning against ANDV show homology to integrin β3. (A) Alignment of phage peptide sequences with P values for integrin β3 pairwise alignment of less than 0.05. Residues comprising the signal peptide, transmembrane, and cytoplasmic domains, which were not included during pairwise alignment, are underlined. Residues 461 to 548, which are missing in the crystal structure, are italicized. Residues involved in the ReoPro binding site are highlighted in green (2). Residue D39 of the PSI domain is highlighted in yellow (19). Peptides are shown above the sequence of integrin β3, with antibody 6C5/D12-eluted sequences shown in blue text and sequences eluted with antibody 6B9/F5 shown in red. Peptide sequences with alignment P values of ≤0.0005 are highlighted in yellow. Percent inhibition of the peptide-bearing phage is shown in parentheses. (B) View of integrin αvβ3 (PDB ID 1U8C [23]). αv is shown in blue ribbon diagram, and β3 is shown in salmon-colored surface representation, with specific domains circled. Residues corresponding to the ReoPro binding site are shown in green, as in panel A, and D39 is shown in yellow. Regions corresponding to 6C5/D12-eluted peptides with P values of ≤0.0005 for alignment with integrin β3 (highlighted in panel A) are shown in blue, and those corresponding to 6B9/F5-eluted peptides with P values of ≤0.0005 for alignment with integrin β3 are shown in red. Alignment of peptide PLASTRT (P value of 0.0040) adjacent to D39 of the PSI domain is shown in magenta. Graphics were prepared using Pymol (DeLano Scientific LLC, San Carlos, CA).Of the 41 peptide-bearing phage eluted with the anti-Gc antibody 6C5/D12, 14 showed sequence homology to integrin β3 (P < 0.05), 4 of which had P values of ≤0.0005 (Fig. (Fig.1A).1A). Of the latter, sequence CTTMTRMTC mapped to the base of the I-like domain (Fig. (Fig.1B),1B), while CHGVYALHC and CRDTTPWWC mapped to the EGF-3 domain. Finally, sequence CTPTMHNHC mapped to the linker region untraceable in the crystal structure. Therefore, in contrast to peptide sequences identified by competition with the anti-Gn antibody, sequences identified by competition with the anti-Gc antibody 6C5/D12 appear to be mostly unrelated to integrin β3.As a low level of pathogenic hantavirus infection can be seen in cells lacking integrin β3, such as CHO cells (19), we asked if any of the identified peptide sequences could represent a previously unidentified receptor. We used the Basic Local Alignment Search Tool to search a current database of human protein sequences for potential alternate receptors represented by these peptides. However, none of the alignments identified proteins that are expressed at the cell surface, eliminating them as potential candidates for alternate viral entry receptors. This suggests that the majority of the peptides identified here likely represent novel sequences for binding ANDV surface glycoproteins.To determine whether synthetic peptides would also block infection, we synthesized cyclic peptides based on the 10 most-potent peptide-bearing phage. These peptides, in the context of phage presentation, showed levels of inhibition ranging from 44 to 72% (Table (Table2).2). When tested by IFA at 1 mM, four of the synthetic peptides showed inhibition levels significantly lower than those of the same peptide presented in the context of phage. This is not surprising, as steric factors due to the size of the phage and the multivalent presentation of peptide in the context of phage may both contribute to infection inhibition (8). However, there was no significant difference in inhibition by synthetic peptide versus peptide-bearing phage for six of the sequences, implying that inhibition in the context of phage was due solely to the nature of the peptide itself and not to steric factors or valency considerations contributed by the phage, which contrasts with our previous results, determined by using phage directed against αvβ3 integrin (10).

TABLE 2.

Synthetic cyclic peptides inhibit ANDV infection
TargetSample% Inhibition bya:
Peptide-bearing phageSynthetic peptide
GnCMQSAAAHC48.88 (44.40)59.66 (11.17)
GcCTVGPTRSC57.30 (11.31)46.47 (7.61)
GnCPSNVNNIC61.11 (25.41)44.14 (10.74)
GnCEKLHTASC43.60 (27.92)34.87 (9.26)
GcCPKLHPGGC71.88 (27.11)30.95 (7.73)b
GnCSLHSHKGC45.11 (49.81)29.79 (9.34)
GcCPMSQNPTC65.60 (13.49)18.19 (8.55)b
GnCKSLGSSQC53.90 (13.34)18.10 (7.55)b
GnCNSHSPVHC45.40 (28.80)15.52 (10.48)
GnCPAASHPRC51.84 (17.09)0 (10.72)b
Integrin β3ReoPro80.10 (7.72)
Gn6B9/F5 antibody42.72 (6.75)
Gc6C5/D12 antibody31.04 (7.81)
Open in a separate windowaStandard deviations of the results of at least four experiments are shown in parentheses.bMean percent inhibition between phage and synthetic peptide differs significantly (P < 0.05).The three most-potent synthetic peptides were examined for their ability to inhibit ANDV entry in a dose-dependent manner. The concentration of each peptide that produces 50% of its maximum potential inhibitory effect was determined. As shown in Fig. Fig.2A,2A, the 50% inhibitory concentration for each of the peptides was in the range of 10 μM, which from our experience is a reasonable potency for a lead compound to take forward for optimization.Open in a separate windowFIG. 2.Activities of synthetic peptides in inhibition of ANDV infection in vitro. (A) Peptides were examined for their ability to block ANDV infection of Vero E6 cells in a dose-dependent manner by IFA. (B) Peptides were tested in parallel for the ability to block infection of Vero E6 cells by ANDV, SNV, HTNV, and PHV. (C) Peptides were tested, singly or in combination, for the ability to block ANDV infection of Vero E6 cells. For all experiments, controls included media, ReoPro at 80 μg/ml, and monoclonal antibodies 6C5/D12 and 6B9/F5 at 5 μg/ml. All peptides were used at 1 mM. Data points represent n = 2 to 6, with error bars showing the standard errors of the means. Statistical analyses were performed on replicate samples using an unpaired Student''s t test.In order to determine the specificity of the three most-potent synthetic cyclic peptides in blocking ANDV, we examined them for inhibition of ANDV infection versus two other pathogenic hantaviruses, SNV and Hantaan virus (HTNV), or the nonpathogenic hantavirus Prospect Hill virus (PHV). As shown in Fig. Fig.2B,2B, ReoPro, which binds integrin β3, showed inhibition of infection by each of the pathogenic hantavirus strains, known to enter cells via β3, but not the nonpathogenic PHV, which enters via integrin β1 (6, 7). In contrast, peptides selected for the ability to bind ANDV were highly specific inhibitors of ANDV versus SNV, HTNV, or PHV. The specificities of peptides eluted by the anti-Gn monoclonal antibody are not surprising, as they are likely due to global differences in the Gn amino acid sequence. Specifically, sequence homologies between ANDV and SNV, HTNV, and PHV are 61%, 36%, and 51%, respectively, for the region corresponding to the immunogen for antibody 6B9/F5. Although homology between the immunogen for antibody 6C5/D12 and the corresponding Gc region of these viruses is somewhat higher (82% with SNV, 63% with HTNV, and 71% with PHV), the possibility that the monoclonal antibody used here recognizes a three-dimensional epitope lends itself to the high specificity of the peptides.The current model for cellular infection by hantaviruses (14) is as follows. Viral binding of the host cell surface target integrin is followed by receptor-mediated endocytosis and endosome acidification. Lowered pH induces conformational changes in Gn and/or Gc, which facilitate membrane fusion and viral release into the cytosol. As there is currently little information available about whether one glycoprotein is dominant in mediating infection, and as neutralizing epitopes have been found on both Gn and Gc glycoproteins (1, 4, 12, 13, 20), we examined whether combining anti-Gn- and anti-Gc-targeted synthetic peptides would lead to an increased infection blockade compared to those for single treatments. As shown in Fig. Fig.2C,2C, the combination of anti-Gn and anti-Gc peptides CMQSAAAHC and CTVGPTRSC resulted in a significant increase in infection inhibition (P = 0.0207 for CMQSAAAHC, and P = 0.0308 for CTVGPTRSC) compared to that resulting from single treatments. Although the high specificity of the peptides for ANDV makes it unlikely that this combination treatment will lead to more cross-reactivity with other pathogenic hantaviruses, this can be determined only by additional testing. Regardless, these data suggest a unique role for each of these viral proteins in the infection process as well as the benefits of targeting multiple viral epitopes for preventing infection.To our knowledge, the peptides reported here are the first identified that directly target ANDV, and this work further illustrates the power of coupling phage display and selective elution techniques in the identification of novel peptide sequences capable of specific protein-protein interactions from a large, random pool of peptide sequences. These novel peptide inhibitors (R. S. Larson, P. R. Hall, H. Njus, and B. Hjelle, U.S. patent application 61/205,211) provide leads for the development of more-potent peptide or nonpeptide organics for therapeutic use against HCPS.  相似文献   

19.
20.
The Bifidobacterium spp. present in 10 infant fecal samples (4 from infants with eczema and 6 from healthy infants) were quantified with both hierarchical oligonucleotide primer extension (HOPE) and fluorescence in situ hybridization-flow cytometry. The relative abundances of Bifidobacterium longum and B. catenulatum with respect to the total bifidobacteria had a poor correlation (ρ, <0.600; P value, >0.208), presumably due to differences in primer specificity and the level of hybridization stringency of both methods. In contrast, the relative abundances of organisms of the genus Bifidobacterium against the total amplified 16S rRNA genes and those of B. adolescentis, B. bifidum, and B. breve against the genus Bifidobacterium exhibited a good statistical correlation (ρ, >0.783; P value, <0.066). This good comparability supports HOPE as a method to achieve high-throughput quantitative determination of bacterial targets in a time- and cost-effective manner.The “microflora hygiene” hypothesis states that a lack of exposure to pathogens or certain commensal bacteria in early life may predispose some individuals to allergic disorders (14). However, inconsistent findings on the abundance of health-associated microbes have prevented precise conclusions as to their role in modulating host health. For example, by performing fluorescence in situ hybridization (FISH) on infant feces, Bifidobacterium spp. were found in high abundance in healthy infants (11). In contrast, certain species, like Bifidobacterium pseudocatenulatum, may be more commonly detected in infants with eczema (3). Therefore, to facilitate our understanding of microbial composition and its correlation to human health, it is essential to use a rapid and high-throughput molecular method to determine the abundances of bacterial targets in a large sample size (16). Although FISH-flow cytometry (FISH-FC) is routinely used to quantify the abundances of bacterial targets in feces (9, 11, 17), it does not suffice as a high-throughput method due to the limited range of spectrally distinct fluorophores that are available in the UV spectrum (10, 13). There is a need to develop a high-throughput technique which can complement the existing molecular methods to rapidly evaluate the relative abundance of bacterial targets.A molecular method termed hierarchical oligonucleotide primer extension (HOPE) was developed to rapidly determine the relative abundances of bacterial 16S rRNA genes among total PCR-amplified 16S rRNA genes (19). HOPE uses primers of different lengths that were designed to target bacteria at different phylogenetic levels. The primers anneal to complementary regions of the targeted bacteria and extend with a fluorophore-labeled nucleotide when the bacterial target is present. The extended primers can be differentiated on a genetic analyzer based on primer length and fluorophore color. The relative abundance of the bacterial target against a higher-level primer can then be quantified by calculating the ratio of the peak area of the extended primer with that of a higher-level primer. A subsequent study demonstrated that HOPE can be used for rapid and specific determination of Bacteroides spp. present in feces and wastewaters at different taxonomical levels (5). It also has the versatility to be expanded to include other bacterial groups. This can potentially facilitate the identification and quantification of bacterial populations that modulate the health of an individual at different temporal intervals.This study aimed to demonstrate HOPE as a time- and cost-effective method to quantify the abundances of Bifidobacterium spp. in 10 infant fecal samples (4 from infants with eczema and 6 from healthy infants) that were collected at 1, 3, and 12 months of age. The abundances of the Bifidobacterium spp. as determined, respectively, by HOPE and FISH-FC were also compared to validate the use of HOPE as a quantitative method.To obtain the total PCR-amplified 16S rRNA genes, genomic DNA of the fecal microbiota was extracted based on a previously described protocol (12) prior to 20 cycles of PCR amplification (modified 11F forward primer 5′-GTT YGA TYC TGG CTC AG-3′ and 1492R reverse primer 5′-GGY TAC CTT GTT ACG ACT T-3′) (6, 7). The amplicons were purified, and the concentrations were diluted to 10 ng/μl. For HOPE, a total of 12 primers specifically targeting six Bifidobacterium spp. at different taxonomic levels were designed based on a previously described protocol (5). The specificity of the designed primers was verified in silico against entries in RDP II (2), and the sensitivity of the primers was determined as described previously (19). FISH-FC was performed on the same set of fecal samples based on the protocol described by Lay et al. (9). Table Table11 lists the HOPE primers and FISH probes used in this study. A nonparametric Spearson ranked correlation analysis (Minitab) was performed on the abundances of Bifidobacterium spp. as quantified by FISH-FC and HOPE, respectively.

TABLE 1.

HOPE primers and FISH probes included in this study
Primer or probeTarget(s) (no. of RDP II hits)aSequence (5′-3′)Poly(A) tail length (nt)Type of ddNTPb addedHOPE reaction tube no.Reference
HOPE primers
    Bia183_speB. adolescentis (62)AAG GAC ATG CAT CCA ACT0G2This study
    Biang183_speB. angulatum (3)TTC CCA GAC CAC CAT GCG ATG GAC T0G2This study
    Bibif183_speB. bifidum (11)GAA TCT TTC CCA CAA TCA CAT GCG AT6C2This study
    Bbreve1264_speB. breve (11)CAG GGA TCC GCT CCA GCT CGC A4C2This study
    Bicat181_speB. catenulatum, B. pseudocatenulatum (41)CCA TGC GAG GAG TCG GAG CA0T2This study
    Bil181_speB. longum (12)CAT GCG ATC AAC TGG AA5C1This study
    Bifgp1120_cluB. breve, some B. longum isolates (19)ACA ATC CGC TGG CAA CAC G15G1, 2This study
    Bifgp1250_cluB. dentium, B. bifidum (14)GTC GCC ATG TCG CAT CCC GC10T1, 2This study
    Bifgp442_cluB. adolescentis, B. ruminatium (75)CCG AAG GGC TTG CTC CCA G15T1, 2This study
    Bifgp272_cluB. angulatum, B. catenulatum, B. pseudocatenulatum (49)GCC GGC TAC CCG TCG TAG GCT C8G1, 2This study
    Bif660_genMost Bifidobacterium spp. (308)CCA CCG TTA CAC CGG GAA TTC CAG0T18c
    Eub338Ia_domMost Bacteria spp. (221136)GCT GCC TCC CGT AGG AG23T119
FISH probes
    Bado434B. adolescentis (79)GCT CCC AGT CAA AAG CG17
    Bang198B. angulatum (3)AAT CTT TCC CAG ACC ACC17
    Bbif186B. bifidum (13)CCA CAA TCA CAT GCG ATC ATG17
    Bbre198B. breve (28)AAA GGC TTT CCC AAC ACA CC17
    Bcat187B. catenulatum, B. pseudocatenulatum (40)ACA CCC CAT GCG AGG AGT17
    Blon1004B. longum (48), some Spirochaeta spp. (16)AGC CGT ATC TCT ACG ACC GT17
    Bif164Most Bifidobacterium spp. (293)CAT CCG GCA TTA CCA CCC8
    Eub338Most Bacteria spp. (220802)GCT GCC TCC CGT AGG AGT1
    Non338Non-Bacteria spp. (0)ACT CCT ACG GGA GGC AGC18
Open in a separate windowaDenotes that only good-quality 16S rRNA sequences of >1,200 bp were subjected to BLAST analysis to retrieve the number of perfectly matched hits in RDP II.bddNTP, dideoxynucleoside triphosphate.cModified from reference 8.The extended HOPE primers were detected in the genetic analyzer when target DNA templates made up more than 0.10% of the total genomic DNA. The lower detection sensitivity of the Bifidobacterium-targeting primers than those obtained in previous studies (5, 19) may be due to the high GC content of DNA templates. As Bifidobacterium spp. are predominant in infant feces (4, 16), the effect of the low primer sensitivities on subsequent findings could be negligible.Our findings agree with previous studies and suggested that the genus Bifidobacterium was predominant in the infants'' fecal microbiota (Fig. (Fig.1).1). Furthermore, it was observed that the abundances varied across individuals and with time (Fig. (Fig.1).1). In most individuals, the relative abundances of the genus Bifidobacterium against the total amplified 16S rRNA genes were also highest in the fecal samples that were collected at 1 and 3 months after birth (Fig. (Fig.1).1). On average, B. adolescentis was only detected by both HOPE and FISH-FC in the fecal microbiota that was collected 12 months after birth (Fig. (Fig.2).2). In contrast, the B. catenulatum group and B. bifidum were consistently detected at all sampling times and at relatively high abundances of up to 28.5% and 16.6% of the total bifidobacteria, respectively (Fig. (Fig.2).2). Furthermore, B. breve was detected in the fecal microbiota of infants with eczema at 1 and 3 months after birth and at relative abundances that ranged from 2.8 to 7.9% of the total bifidobacteria (Fig. (Fig.2).2). In contrast, a reverse trend was observed throughout the period in healthy infants (Fig. (Fig.2).2). However, the role of B. breve in atopic eczema cannot be conclusively determined from this study as other variables such as the mode of delivery and the dietary regimen were not investigated (15).Open in a separate windowFIG. 1.Relative abundances of bifidobacteria in fecal samples obtained from 10 infants at 1, 3, and 12 months after birth. Abundances were quantified by HOPE (A) and FISH-FC (B).Open in a separate windowFIG. 2.Relative abundances of Bifidobacterium spp. and total bifidobacteria. Abundances in samples from the infants in the respective health groups and time points were averaged and quantified by HOPE (○) and FISH-FC (▵).To determine the comparability of HOPE and FISH-FC, the relative abundances of Bifidobacterium spp. quantified by both methods were statistically analyzed by nonparametric Spearson correlation analysis. The relative abundances of B. longum and the B. catenulatum group against the genus Bifidobacterium did not show a significant correlation (P values = 0.208 and 0.623, respectively) (Table (Table2),2), and the discrepancy may be due to the different specificities of the HOPE primers and FISH-FC probes. Table Table11 showed that FISH-FC probe Blon1004 was designed to target B. longum at a fourfold higher coverage than the HOPE primer (Bil181) and would understandably result in a significant difference between the abundances detected. Although the HOPE primer and the FISH-FC probe that target the B. catenulatum group have similar specificities, the discrepancy in the abundances detected may be due to the difference between the hybridization stringencies of the two methods. In this study, fluorescently labeled probes for FISH-FC were hybridized to their complementary 16S rRNA genes at 35°C (9). The low hybridization temperature may have resulted in cross-hybridization with nontargets and therefore comparably higher abundances of the B. catenulatum group than those quantified by HOPE (Fig. (Fig.11).

TABLE 2.

Nonparametric correlation analysis of the relative abundances of Bifidobacterium spp. determined by HOPE and FISH-FC
BacteriaSpearson correlation (ρ)P valueCorrelation of abundances quantified by HOPE and FISH-FC
All Bifidobacterium spp.a0.8290.042Good at 90% confidence level
B. adolescentisb0.9200.009
B. breveb0.8570.029
B. bifidumb0.7830.066Fairly good at 85% confidence level
B. catenulatum groupb0.6000.208Not significant
B. longumb0.2570.623
Open in a separate windowaThe relative abundances of Bifidobacterium spp. against the total Bacteria spp. amplified were compared in this correlation analysis.bThe relative abundances of the individual Bifidobacterium sp. shown against the total amplified Bifidobacterium spp. were compared in this correlation analysis.Despite the poor correlation of the relative abundances of B. longum and the B. catenulatum group, a fairly good correlation, ranging from 0.783 to 0.920, was obtained for the relative abundances of the bifidobacteria with respect to the total bacteria and also for the relative abundances of individual species like B. adolescentis, B. bifidum, and B. breve against the genus Bifidobacterium (average P value = 0.04) (Table (Table22).FISH-FC and quantitative PCR are two molecular methods used to examine the Bifidobacterium spp. present in the fecal microbiota. Compared to FISH-FC, the entire HOPE procedure for the identification and quantification of Bifidobacterium spp. after primary DNA extraction and PCR amplification took less than 120 min (5, 19), which was significantly shorter than that required for FISH-FC. Furthermore, we demonstrate the use of inexpensive unlabeled oligonucleotide primers to achieve up to nine-plexing per reaction. Compared to quantitative PCR, which uses fluorescently labeled PCR assays like the TaqMan, HOPE would allow a relatively more cost-effective examination of up to 864 targets in a 96-well plate format.Furthermore, HOPE is highly adaptable and allows the total number of detectable bacterial targets to be easily increased simply by adding HOPE reactions or by adding a primer(s) to individual reactions. For example, although the HOPE primer targeting B. longum is highly specific, it did not achieve satisfactory coverage of the entire B. longum group. Primers that target B. dentium and B. infantis were also not included in this study. These species may constitute the large unidentified fraction of Bifidobacterium spp. that was not accounted for. Besides profiling for these known Bifidobacterium spp., the yet-to-be-cultured Bifidobacterium spp. can also be identified by the construction of 16S rRNA gene libraries and designed with new HOPE primers that target the unrepresented Bifidobacterium spp. The current list of primer assays can be easily expanded to include these new primers so as to provide more comprehensive coverage of the bifidobacterial population that is present in infant feces.In summary, this study has demonstrated the potential of HOPE as a time- and cost-effective detection method that can examine the relative abundances of bacterial targets at various taxonomic levels. It can be used to capture possible changes in the abundances of Bifidobacterium spp. and/or other bacterial targets present in infant feces. The abundances can then be correlated with clinical disorders such as allergic diseases, and the findings will eventually assist in the elucidation of the roles played by microorganisms in the mediation of immune responses.  相似文献   

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