Arcobacter spp. are a common contaminant of food and water, and some species, primarily A. butzleri and A. cryaerophilus, have been isolated increasingly from human diarrheal stool samples. Here, we describe the first Arcobacter multilocus sequence typing (MLST) method for A. butzleri, A. cryaerophilus, A. skirrowii, A. cibarius and A. thereius.A. butzleri, 72 A. cryaerophilus, 15 A. skirrowii and 8 A. cibarius isolates from a wide variety of geographic locations and sources, was typed in this study. Additionally, this sample set contained four strains representing a new Arcobacter species, A. thereius. The seven loci used in the four-species Arcobacter MLST method are the same as those employed previously in C. jejuni, C. coli, C. helveticus and C. fetus , glnA, gltA, glyA, pgm and tkt). A large number of alleles were identified at each locus with the majority of isolates containing a unique sequence type. All Arcobacter isolates typed in this study contain two glyA genes, one linked to lysS (glyA1) and the other linked to ada (glyA2). glyA1 was incorporated into the Arcobacter MLST method while glyA2 was not because it did not increase substantially the level of discrimination.A sample set of 374 arcobacters, including 275 Arcobacter strain discrimination and in epidemiological studies of sporadic Arcobacter-related gastroenteritis. A new Arcobacter MLST database was created http://pubmlst.org/arcobacter/; allele and ST data generated in this study were deposited in this database and are available online.No association of MLST alleles or sequence types with host or geographical source was observed with this sample set. Nevertheless, the large number of identified alleles and sequence types indicate that this MLST method will prove useful in both  Arcobacter is a member of the Gram-negative, ε-Proteobacterial subdivision. The majority of isolated arcobacters belong to one of three species: Arcobacter butzleri, A. cryaerophilus or A. skirrowii. Additional members of this taxon include: A. cibarius, isolated from broiler carcasses [A. nitrofigilis, a nitrogen-fixing organism isolated originally from estuarine plant roots [A. halophilus, isolated from a hypersaline lagoon [Candidatus Arcobacter sulfidicus, a sulfide-oxidizing marine organism [A. mytili sp. nov., isolated from mussels [A. thereius sp. nov, isolated from pigs and ducks [A. marinus sp. nov [Arcobacter butzleri, A. cryaerophilus, A. skirrowii and A. cibarius have been isolated often from both animals [The genus arcasses ; A. nitrnt roots ; A. haloe lagoon ; Candidaorganism ; A. myti mussels ; A. thernd ducks  and A. m sp. nov . Arcobac animals -10 and f animals , water a animals -16, and  animals .Arcobacter-associated human illness. Arcobacter spp., primarily A. butzleri and A. cryaerophilus, have been isolated from human diarrheal stool samples [Arcobacter-contaminated food or water and human illness has been established, although it is likely that transmission of arcobacters occur via these routes. Arcobacter spp. have been isolated also from the stools of healthy humans [A. butzleri and A. cryaerophilus strains are non-pathogenic and are human commensals. The presence of a subset of non-pathogenic strains alongside pathogenic strains within A. butzleri, A. cryaerophilus and perhaps the other food- and water-associated Arcobacter species, such as A. skirrowii and A. cibarius, would indicate a need for an accurate typing method to distinguish human-pathogenic and human-commensal arcobacters. Arcobacter typing methodology would also be useful in studies of transmission routes and source tracking during outbreak and extended epidemiological investigations. Typing of Arcobacter strains using such methods as enterobacterial repetitive intergenic consensus (ERIC)-PCR, pulsed field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP) analysis has been reported .C. jejuni [C. jejuni epidemiology , provided useful information for the development of an MLST method suitable for typing of Arcobacter species.Multilocus sequence typing (MLST), a typing method based on partial, yet defined, sequence information at seven housekeeping loci, was developed first within the ε-Proteobacteria for . jejuni . It has iewed in ). Within C. coli ,27, C. lC. fetus  and C. iaenigrae . The exiaenigrae . Develop genomes . Constru existed . Similar8 genome , as wellArcobacter species, including the three most frequently-isolated Arcobacter spp., A. butzleri, A. cryaerophilus and A. skirrowii. The Arcobacter MLST gene set is identical to the C. jejuni gene set , glnA, gltA, glyA, pgm and tkt), permitting phylogenetic comparison of data across the two genera. A sample set of 374 isolates of diverse geographic origin and source was typed in this study. Almost 300 sequence types and 1176 alleles across seven loci were identified.Here, we describe a new MLST method for multiple atpA(uncA), glnA, glyA and tkt) common to the Campylobacter MLST methods described previously [aspA, gltA and pgm) present within the C. jejuni method [Arcobacter MLST method. For optimal phylogenetic comparison, the same allelic endpoints were considered. Development of the Arcobacter MLST method was assisted by the concurrent completion of the A. butzleri strain RM4018 genome sequence [C. jejuni MLST loci were extracted, where applicable, from the existing Arcobacter and thermotolerant Campylobacter genome sequences, and aligned. Degenerate primers, situated approximately 300 bp upstream and downstream from the allelic endpoints, were designed and 94 Arcobacter strains  were amplified and sequenced. Sequence information from this sample set was aligned and used to construct the butzleri-specific primers listed in Table S1 [see additional file butzleri species, some loci did not amplify efficiently, using primers based on the Campylobacter/Arcobacter alignments. For these loci, improved primer pairs were constructed by incorporating sequences from the draft A. halophilus genome  into the Campylobacter/Arcobacter alignments. These improved primer pairs efficiently amplified the seven MLST loci  of A. cryaerophilus and A. skirrowii [see additional file In order to optimize cross-species and cross-genus comparisons, the four genes  linked to lysS and the other (glyA2) to ada. Therefore, to eliminate generation of mixed traces, amplification primers were designed within the lysS and ada genes. PCRs using the lysS and glyA reverse primers amplified specifically glyA1 and PCRs using the ada and glyA forward primers amplified specifically glyA2. All Arcobacter isolates typed in this study contained at least two glyA genes, suggesting that the presence of multiple glyA genes is an unusual feature common to the genus. The glyA locus in other Campylobacter MLST methods is also linked to lysS. For this reason, and for the fact that the glyA2 locus is less discriminatory than glyA1 (see below), the lysS-linked glyA1 locus was incorporated into the Arcobacter typing method.Initial typing of the 8 genome . In thisArcobacter MLST method to amplify successfully as many A. butzleri strains as possible, we wanted a large sample set with broad geographic origins and sources. A description of the Arcobacter isolates by geographic origin and source is listed in Tables A. butzleri isolates were typed, as well as an additional 99 isolates representing other Arcobacter species, e.g. A. cryaerophilus and A. skirrowii [see additional file A. butzleri, A. cryaerophilus, A. skirrowii and A. thereius isolates amplified and sequenced successfully with one or more of the primer pairs listed in Table S1 [see additional file Arcobacter cibarius demonstrated variable tkt amplification results, i.e. weak amplification of some loci with each primer pair and no primer pair amplifying all loci [see additional file To address the ability of the Arcobacter MLST alleles and sequence types (STs) were identified in this study (Table glnA locus to 63% (236/374) at the glyA1 locus. The high density of alleles translated also into a large number of STs  contained unique STs. A large percentage of variable sites were identified at all of the Arcobacter MLST loci  was substantially < 1 for all of the MLST loci characterized in this study , suggesting that A. butzleri STs are relatively stable, even after passage through a food animal.The presence of a large number of MLST alleles within the aspA, atpA, glnA, gltA, pgm and tkt loci, phylogenetically discrete clusters were identified that associated with species (data not shown). An example is illustrated in Figure atp locus, showing that the A. butzleri, A. skirrowii, A. thereius and A. cryaerophilus alleles form distinct groups. However, for the latter species two separate clusters, termed here 'group 1' and 'group 2' were observed. Two phylogenetically-distinct clusters of A. cryaerophilus alleles were identified also at the glnA, gltA, pgm and tkt loci [see additional file aspA locus that formed only one cluster. The existence of species-associated clustering at these six loci permits tentative identification of lateral transfer events. These events were not observed in A. butzleri because no alleles related phylogenetically to other species were identified, however, alleles related phylogenetically to those identified in A. butzleri were identified within A. cibarius and A. skirrowii . Similarly, A. skirrowii alleles were identified within A. cryaerophilus and A. thereius (e.g. aspA-125 and glnA-95), and an A. thereius allele was identified in A. cryaerophilus  is noticeably different from clustering at the other six loci  of gene conversion events between the two glyA genes , and only one putative lateral transfer event was identified at glyA. Therefore, inclusion of glyA1 in the typing scheme is merited, since the large number of glyA alleles identified in this study would substantially enhance Arcobacter strain discrimination.The presence of two highly-similar Arcobacter MLST method described here is useful for strain discrimination for the three major Arcobacter species, i.e. A. butzleri, A. cryaerophilus and A. skirrowii, as well as two additional Arcobacter species, A. thereius and A. cibarius. Additional genomic sequence data should permit revision and expansion of this typing method into additional Arcobacter species. No association, with either host or geographical source, of Arcobacter alleles or STs was observed in this study; however, the large suite of alleles and STs present within this sample set make identification of such associations difficult, since most alleles and STs were observed infrequently. Typing of additional Arcobacter isolates, thereby increasing potentially the numbers of each allele and ST, may reveal heretofore undetected association patterns within this genus. The increasing association of arcobacters with human illness, transmitted potentially by contaminated food or water, makes this method a valuable addition to Arcobacter typing. This method should prove useful in investigations of sporadic and outbreak arcobacterioses and Arcobacter epidemiology.The large number of MLST alleles and STs identified in this study indicates that the A. butzleri set typed in this study consisted of 275 isolates from 16 countries across four continents , and from a wide variety of food sources and animals  and A. skirrowii (N = 15), obtained predominantly from cattle and swine; the remainder included eight A. cibarius strains and four A. thereius strains. A large number of strains in the Arcobacter strain set were of unknown origin .The Arcobacter strains were cultured routinely under aerobic conditions at 30°C on Brain Heart Infusion agar  supplemented with 5% (v/v) laked horse blood . Arcobacter halophilus was grown on Brain Heart Infusion -blood media supplemented with 4% (w/v) NaCl. PCR enzymes and reagents were purchased from New England Biolabs  or Epicentre . All chemicals were purchased from Sigma-Aldrich Chemicals  or Fisher Scientific . DNA sequencing chemicals and capillaries were purchased from Applied Biosystems . PCR and sequencing oligonucleotides were purchased from MWG-Biotech .All 2, 250 μM (each) dNTPs, 50 pmol each primer, and 1 U Taq polymerase . PCRs for MLST were performed on a Tetrad thermocycler  with the following settings: 30 cycles of 94°C for 30 sec, 53°C for 30 sec, and 72°C for 2 min. Amplicons were purified on a BioRobot 8000 workstation . Cycle sequencing reactions were performed on a Tetrad thermocycler, using the ABI PRISM BigDye terminator cycle sequencing kit  and standard protocols. Cycle sequencing extension products were purified using BigDye XTerminator (Applied Biosystems). DNA sequencing was performed on an ABI PRISM 3730 DNA Analyzer (Applied Biosystems), using POP-7 polymer and ABI PRISM Genetic Analyzer Data Collection and ABI PRISM Genetic Analyzer Sequencing Analysis software.MLST primer sets are listed in Table S1 [see additional file C. fetus, C. insulaenigrae and the novel Arcobacter MLST schemes described in this study, in addition to the original MLST schemes for C. jejuni, C. coli, C. lari, C. upsaliensis and C. helveticus; 2) automatic association of allele with species, based on phylogenetic analyses of the ten MLST loci present in the different Campylobacter/Arcobacter MLST methods, permitting immediate identification of chimeras; and 3) automatic assignment of sequence type (ST), based on the profile of seven MLST alleles. Novel alleles and STs are flagged by MLSTparser3 and assigned an arbitrary number.The Perl program MLSTparser  was modiArcobacter strain typed in this study. A new Arcobacter MLST database was created http://pubmlst.org/arcobacter/; allele and ST data generated in this study were deposited in this database and are available online.MLSTparser3 was used to identify the MLST alleles and ST of each dn/ds ratios were performed using START2 http://pubmlst.org/software/analysis/. A dendrogram of unique Arcobacter STs was constructed by concatenating the allele sequences comprising each ST. Allele sequences for each strain were concatenated in the order aspA-atpA-glnA-gltA-glyA-pgm-tkt for a final composite length of 3341 bp; in addition, the MLST alleles of the A. halophilus strain LA31B were extracted from the draft genome , concatenated and incorporated into the ST analysis. Sequence alignments were performed using CLUSTALX , and dendrograms were constructed using the neighbor-joining method with the Kimura 2-parameter distance estimation method. Phylogenetic analyses were performed using MEGA version 4 [Variable sites and calculation of the ersion 4 .Arcobacter genomic DNA, and GW and EY performed the multilocus sequence typing. IVW, SLWO, KH, FM, AS and CJM isolated the Arcobacter strains and performed the initial characterization/speciation of the isolates. All authors approved and read the final manuscript.WGM designed the research project. WGM designed the MLST primer sets, analyzed the MLST data, performed the phylogenetic analyses and was the principal author of the manuscript. GW and EY isolated the Arcobacter spp. MLST genesPrimers for amplification and sequencing of the seven . Primer pairs used for amplifying the MLST loci of A. butzleri, A. cryaerophilus, A. skirrowii, A. cibarius and A. thereius are listed. For each MLST locus, the allele size is given and for each primer pair the expected amplicon size is provided.Click here for fileArcobacter allele numbers and sequence types. List of allele numbers and sequence types for the 374 arcobacters typed in this study. For each strain, the source and geographic origin is provided (if known).Click here for file


Escherichia coli. This PCR is specific, sensitive, and rapid in detecting target isolates in stool and food. Because of its simplicity, economy, and efficiency, this protocol warrants further evaluation in large, prospective studies of polymicrobial substances.We developed and tested a single multiplex polymerase chain reaction (PCR) that detects enterotoxigenic, enteropathogenic, enteroinvasive, and Shiga-toxin–producing  Escherichia coli causes disease in humans through diverse mechanisms (E. coli group include enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAggEC), and Shiga-toxin–producing E. coli (STEC), also called verocytotoxin-producing or enterohemorrhagic E. coli. ETEC produce secretory toxins (enterotoxins); EPEC adhere intimately to epithelial cells and induce host cell transmembrane signaling; EIEC invade eukaryotic cells; and STEC produce Shiga toxins.E. coli in the polymicrobial milieus of stool and food poses challenges. Occasionally, economically detectable phenotypes distinguish such organisms when they are abundant in human stools. For example, sorbitol- and lactose-nonfermenting colonies are typical of E. coli O157:H7 and EIEC . Applying such assays to enteric microbiologic diagnosis is cumbersome.Identifying diarrheagenic E. coli in stools and in food. We describe the development of a multiplex PCR that detects four categories of diarrheagenic E. coli and the application of the assay to human diarrheal stools and food in Mexico City.Nucleic acid hybridization techniques, exploited by colony hybridizations or polymerase chain reaction (PCR), apply a single detection method to a diversity of organisms. The application of nucleic acid amplifications requires selecting appropriate oligonucleotide primers and optimizing conditions to maximize sensitivity and specificity. The inclusion of reactions and conditions that apply to a variety of virulence loci so that multiple candidate pathogens can be sought in a single reaction makes this technology more efficient and economical. Such multiplex detection is an appropriate solution to the challenge of finding diarrheagenic We developed a single multiplex PCR reaction to detect ETEC, EPEC, EIEC, and STEC, using specific previously described . BecauseE. coli O86:H18 was the negative control in all assays. Each PCR tube contained 23 µL of reaction mix, comprised  of Tris-HCl , KCl (50 mM), MgCl2 (2 mM), gelatin (100 µg/mL), glycerol (5% v/v), dATP, dCTP, dGTP, and dTTP (200 µM each), AmpliTaq polymerase (GIBCO-BRL) (0.5 U/23 µL), a mixture of the 14 primers (E. coli (E. coli 3030 (O86:H18) strain was used as a negative control during the characterization. In all further experiments, the DNA mix from the four prototype E. coli served as the positive control. The multiplex PCR was further characterized by using three additional reference strains for each category , boiling them 1 min, and then freezing them until needed.  primers , and 2 µ primers  of the f were selected from standard and sorbitol MacConkey agar plates, respectively, speciated biochemically, and then subjected to multiplex PCR.Stools from 58 children <5 years of age hospitalized for diarrhea in July, August, and September, 1999, at the three main hospitals of the Instituto Mexicano del Seguro Social, Mexico City, were studied. The Institutional Review Board of the Institute approved this study, and parental informed consent was obtained for each patient. Standard diagnostic evaluations on these stools included culture for E. coli  and diarrheagenic E. coli, without enrichment. One gram of food was added to 1 mL of 0.85% sterile saline and vortexed, and serial 10-fold dilutions were prepared. To enumerate candidate E. coli, and identify diarrheagenic E. coli, 100 µL of each sample and dilutions were plated on MacConkey and sorbitol MacConkey agar plates. Five pink colonies from MacConkey and five colorless colonies from sorbitol MacConkey agar were tested for indole positivity and the lactose-fermenting phenotype (if selected from the sorbitol plate). Only indole-positive, lactose-fermenting colonies isolated from both media were then subjected to the multiplex PCR. STEC from patients and food were tested to determine if they expressed the O157 lipopolysaccharide antigen by using latex particle agglutination .Because of our concern about food safety, we purchased 52 food items (hot chili sauces and taco dressings) from street vendors in Mexico City in July, August, and September, 1999, and analyzed them for the presence of E. coli O157:H7.Multiplex PCR detected the appropriate loci in each positive control strain; extraneous bands were not produced . When DNE. coli in their stools (E. coli colonies (E. coli containing the target loci in their stools. Twenty-two (42%) of the 52 food samples contained E. coli, and 7 (13%) contained candidate diarrheagenic E. coli (eae negative.Eleven (19%) of the 58 patients had candidate diarrheagenic r stools . In 6 . Furthermore, the signals from colony hybridizations are sometimes equivocal, in contrast to the unambiguous data obtained from our assay.This multiplex PCR specifically and sensitively detected a diversity of loci in We believe that multiplex nucleic acid amplification to detect a panel of putatively pathogen traits should be considered as a replacement for tedious, less sensitive, and less specific detection technologies in clinical and food microbiologic analyses. This method should also be considered to be a more parsimonious use of PCR reagents than the individual locus PCR testing protocols described by others (Comparing our protocol’s sensitivity to that reported in other protocols is difficult because of differences in methods. Specifically, other techniques seek amplicons directly from stool cultures (E. coli isolated were the causes of the diarrhea in the children studied. However, in some samples, the PCR-positive organisms were well represented among the aerobic coliform flora selected for analysis. Such organisms were also well represented among the food isolates. Because these E. coli indicate fecal contamination, our findings present a disconcerting picture of the hygienic status of street-vended food in Mexico City. In fact, our colony selection protocol was biased towards high-frequency organisms because we sampled only five such strains. Surveys that examine several hundred colonies (E. coli remain unclear, multiple studies have demonstrated that consumption of food sold by street vendors is a risk factor for acquiring diarrhea in Mexico (Without a more extensive epidemiologic analysis, we cannot state with certainty that the positive E. coli with relative ease. Such organisms are found in food vended in Mexico City and in local children with diarrhea. This feasible technology should be evaluated in larger, controlled, prospective studies of human diarrhea and in microbiologic studies of food to establish the current epidemiology of these pathogens, including the emerging strains of STEC.We have demonstrated for the first time that multiplex PCR can detect a variety of diarrheagenic


Zaprionus genus shares evolutionary features with the melanogaster subgroup, such as space and time of origin. Although little information about the transposable element content in the Zaprionus genus had been accumulated, some of their elements appear to be more closely related with those of the melanogaster subgroup, indicating that these two groups of species were involved in horizontal transfer events during their evolution. Among these elements, the Gypsy and the Micropia retroelements were chosen for screening in seven species of the two Zaprionus subgenera, Anaprionus and Zaprionus.The Gypsy and Micropia retroelements only in species of the Zaprionus subgenus, showing that they are transcriptionally active in the sampled species. The sequences of each retroelement were closely related to those of the melanogaster species subgroup, and the most parsimonious hypothesis would be that 15 horizontal transfer events shaped their evolution. The Gypsy retroelement of the melanogaster subgroup probably invaded the Zaprionus genomes about 11 MYA. In contrast, the Micropia retroelement may have been introduced into the Zaprionus subgenus and the melanogaster subgroup from an unknown donor more recently (~3 MYA).Screening allowed the identification of diverse Gypsy and Micropia of Zaprionus and melanogaster species share similar evolutionary patterns. The sharing of evolutionary, ecological and ethological features probably allowed these species to pass through a permissive period of transposable element invasion, explaining the proposed waves of horizontal transfers. Zaprionus genus  is composed by two subgenera (Anaprionus s.s. and Zaprionus s.s.) which seem to be originated in Asia about 14 MYA dCTP labelled. The nylon membranes were hybridized at 65°C and washed twice with 2× SSC and 0.1% SDS, and once with 0.2× SSC and 0.1% SDS for 20 min at the hybridization temperature. mel: D. melanogaster, mul: Z. multistriatus, tub: Z. tuberculatus, cam: Z. camerounensis, dav: Z. davidi, gab: Z. gabonicus, afr: Z. africanus, ind: Z. indianus.Click here for fileDrosophila genome searchesDescription of the genomic sequences from the 12 . Chromosome and nucleotide location of the Gypsy and Micropia insertions from the Drosophila genomes.Click here for fileGypsy retroelement in the melanogaster subgroup genomesStructure of the . Gypsy insertions in D. melanogaster (mel), D. simulans (sim), D. sechellia (sec), D. yakuba (yak) and D. erecta (ere). The sequences represented have at least 80% identity with the canonical element of D. melanogaster (AF033821), which is the first schematic representation. Black rectangles - long terminal repeats. Gray rectangles - coding regions. Asterisk - genomic sequences interrupted by the scaffold ends or Ns. Striped rectangles - region used in the phylogenetic analyses.Click here for fileGypsy sequences of Zaprionus, melanogaster, D. willistoni and S. latisfasciaeformisPairwise genetic distance among . Distances calculated by the MCL method as implemented by MEGA 4.1. The sequences were clustered according the phylogenetic clades.Click here for fileZaprionus and melanogaster Gypsy sequencesdN (below) and dS (above) values of pairwise comparisons among . Distances calculated by Nei-Gojobori method (Jukes-Cantor's correction), as implemented by MEGA 4.1.Click here for fileGypsy sequences of Zaprionus and melanogaster speciesZ values (above) and significance P-values (below) for the Codon-Based Z-test of neutrality between . Test performed using alternative hypothesis of non-neutrality (dN ≠ dS) and Nei-Gojobori distance (Jukes-Cantor's correction). Gray cells correspond to significant (p < 0.05) pairwise comparisons.Click here for fileMicropia retroelement in the melanogaster subgroup genomesStructure of the . Micropia insertions in D. melanogaster (mel), D. simulans (sim), D. sechellia (sec) and D. yakuba (yak). The sequences represented have at least 80% identity with the canonical element of D. melanogaster (X14037), which is the first schematic representation. Black rectangles - long terminal repeats. Gray rectangles - coding regions. Asterisk - genomic sequences interrupted by the scaffold ends or Ns. Striped rectangles - region used in the phylogenetic analyses.Click here for fileMicropia sequences of Zaprionus, melanogaster and repleta speciesPairwise genetic distance among . Distances calculated by the MCL method as implemented by MEGA 4.1.Click here for fileMicropia sequences of Zaprionus and melanogaster speciesdN (below) and dS (above) values of pairwise comparisons between . Distances calculated by the Nei-Gojobori method with Jukes-Cantor's correction as implemented by MEGA 4.1.Click here for fileMicropia sequences of Zaprionus and melanogaster speciesZ values (above) and significance P-values (below) for the Codon-Based Z-test of selection between . Test performed using alternative hypothesis of non-neutrality (dN ≠ dS) and Nei-Gojobori distance (Jukes-Cantor's correction). Gray cells correspond to significant (p < 0.05) pairwise comparisons.Click here for file


Leptospira spirochetes, which affects not only humans but also animals. It has long been expected to find an effective vaccine to prevent leptospirosis through immunization of high risk humans or animals. Although some leptospirosis vaccines have been obtained, the vaccination is relatively unsuccessful in clinical application despite decades of research and millions of dollars spent. In this review, the recent advancements of recombinant outer membrane protein (OMP) vaccines, lipopolysaccharide (LPS) vaccines, inactivated vaccines, attenuated vaccines and DNA vaccines against leptospirosis are reviewed. A comparison of these vaccines may lead to development of new potential methods to combat leptospirosis and facilitate the leptospirosis vaccine research. Moreover, a vaccine ontology database was built for the scientists working on the leptospirosis vaccines as a starting tool.Leptospirosis is a serious infection disease caused by pathogenic strains of the  Leptospira, which affects almost all mammals [Leptospira has over 200 pathogenic serovars, and divides into 25 serogroups, and many different strains with small antigenic differences can be found in some serovars [Leptospirosis is a widespread disease , caused  mammals -13. Lept mammals ,14. It i mammals -18  vaccines, inactivated and attenuated vaccines, and DNA vaccines for reviewing the current advancements in leptospirosis vaccine research.Recombinant protein vaccines have a great potential against leptospirosis. Several leptospiral recombinant protein vaccines have been constructed with modern biotechnological methods ,61,70-75Leptospira, was reported [Leptospira infection, contrary to adenovirus-mediated Hap1, which induced a significant protection [L. interrogans was described [lig gene was shown to be useful in the detection of pathogenic Leptospira [The protective characteristics of several recombinant OMP vaccines have been tested, including leptospiral outer membrane protein OmpL1, lipoprotein LipL41 , hemolysreported . 6 yearsreported . Howeverotection . In 2002escribed . Then, tescribed ,73,74. Tescribed -81. Moreptospira .Leptospira as a factor involved in virulence [L. interrogans [Several leptospiral outer membrane proteins, e.g. LAg42, Loa22, Lk73.5, have been recognized as leptospirosis vaccine candidates, but they were not tested in animal models for vaccine development. LAg42 is a 42 kDa inner-membrane protein. It was identified in pathogenic irulence . Loa22 wirulence . Lk73.5 errogans . AlthougLeptospira species. LipL45 is produced as a 45-kDa lipoprotein and it is processed to a 31-kDa C-terminal form, P31LipL45 [Lipoproteins are important proteins in leptospires. These proteins are abundant in the outer membrane, to which they are attached through fatty acids. Because of difficulty in production of lipoproteins in heterologous expression systems, only LipL41 was reported as a potential vaccine. However, many lipoproteins  could be suitable as vaccine candidates. LipL32  and LipL31LipL45 . LipL45 31LipL45 .L. interrogans or nonpathogenic L. biflexa was shown to induce production of the tumor necrosis factor, interleukin-10 and CD69 [Moreover, the lipoprotein-like complex glycolipoproteins (GLPs) were suggested as vaccine candidates. A glycolipoprotein (GLP) extracted from either pathogenic and CD69 . Obviousin vivo [On the other hand, lipoprotein LipL36 was found not suitable as leptospirosis vaccine. LipL36 is a 36 kDa leptospiral outer membrane lipoprotein , which iin vivo ,86. Prodin vivo .Leptospira [Only a few papers were reported to identify leptospiral virulence factors, including FlaA, FlaB , Hsp58 , SphH 8,90 and Cptospira . In mostptospira .flaA::cat, flaA::kan, flaB1::kan, flaB2::cat and flaB3::cat mutants, it was shown that these strains were less motile than the wild-type strain [Leptospira. The gene encoding the FlaB virulence factor, flaB, can be amplified from the genomic DNA of several pathogenic serovars. Cloning and sequence analysis indicated that flaB is suitable in the detection of infection by pathogenic leptospires [Using e strain . These rtospires .L. interrogans chp locus, which consists of two genes: chpK and chpI. Expression of chpK in Escherichia coli results in inhibition of bacterial growth. Coexpression of chpI neutralizes ChpK toxicity. The chp locus was found in all representative pathogenic strains of L. interrogans [The virulence factor Hsp58 is a major target for the vaccine design . The virerrogans . All virLeptospira in animals. Many recently reported outer membrane proteins , lipoproteins  and newly discovered virulence factors  can help us to find more suitable vaccine candidates [L. interrogans serovar Icterohaemorrhagiae Lai [L. interrogans serovar Copenhageni [Only Lig, LipL41 and Hap1 proteins were approved as vaccines against ndidates ,11-13,94giae Lai  and L. ienhageni  were repenhageni . The basLeptospira is similar to that in other Gram-negative bacteria [Analysis of LPSs should open new avenues for vaccine developments ,66. The bacteria ,96-98. Lbacteria ,99,100. bacteria ,101-103.L. biflexa serovar Patoc can effectively protect hamsters against L. interrogans serovar Manilae. In this case, the protective effect strongly depended on the dose and administration times of LPS vaccine prepared from L. biflexa serovar Patoc [It has been found that a LPS vaccine may be serovar-independent. For example, LPS vaccine prepared from ar Patoc . Howeverar Patoc . ObviousL. interrogans serovar Copenhageni, maximum titers were observed approximately 6 weeks after immunization. The protection was achieved by immunization with as little as 2.5 μg of LPS or PS [In 1989, a pioneer research on leptospiral LPS vaccines was reported . ImmunizPS or PS . The immPS or PS . The proPS or PS . Total ePS or PS .L. interrogans serovar Pomona was reported [Moreover, an immunogenicity of oligosaccharide fraction from the LPS of reported . The olireported .Leptospira antigens [L. interrogans with a chloroform-methanol-water solution and partially purified by silica gel column chromatography. This antigen exhibited a protective activity in hamsters infected with lethal doses of L. interrogans [L. interrogans by the hot phenol-water method could enhance the immunological response in vivo [Several lipopolysaccharide-like substances (LSSs) were reported as antigens ,106,107. in vivo .L. borgpetersenii subtype Hardjobovis and L. interrogans subtype Hardjoprajitno belong to different species, they are serologically indistinguishable, and thus classified as serovar Hardjo. This is because the LPSs of these subtypes are identical [Since LPSs are major antigens involved in serological response , these mdentical . Hence, Although LPSs have been tested as leptospirosis vaccines for almost 20 years, and promising results have been obtained, there are still have many problems to be solved. Between others, details of compositions and structures of leptospiral LPSs have to be determined.L. interrogans serovar Hardjo or serovar Pomona contained IgM specific to both serovars [L. interrogans followed the second vaccination, a high rate of protection against L. interrogans was observed and duration of immunity was at least 1-year [The inactivated and attenuated vaccines have been reported for more than 50 years. Some inactivated or attenuated leptospirosis vaccines were successfully tested in cattle -116 and serovars . Althougt 1-year . The efft 1-year . Hydrostt 1-year . The inat 1-year .The results discussed above show that inactivated or attenuated vaccines are suitable as the leptospirosis vaccines ,8-11,13.Leptospira was achieved [flaB2 in experiments with guinea pigs [DNA vaccines have been used against different diseases . These vachieved . Anothernea pigs . ObviousWith the development of leptospirosis vaccines, it is now possible to construct a database for leptospirosis vaccines. We have constructed a leptospirosis vaccine ontology database, which is available at the website . In thisVaccines are administered to a large number of healthy humans and animals to make them resistant to diseases, therefore, vaccines must be of high safety ,145. Therfb) might result in attenuation a leptospiral strain. Careful analysis of genes coding for the virulence factors of Leptospira should allow us to construct attenuated strains for leptospirosis vaccines.The attenuated vaccines were achieved by propagation of the microbe under conditions different from those in the infected host and, hopefully, unfavorable to its growth in the host. Obviously, this method could not guarantee enough safety. It requires many tests to ensure the safety, but the safety still remains an issue. A more rational approach to attenuate the microbe would be to inactivate leptospiral components or genes known to code virulence factors. For example, an attenuated leptospiral vaccine could be developed by elimination of the leptospiral O antigen. Analogously, inactivation of the LPS biosynthetic loci  declare that they have no competing interests.All authors have contributed to the content of the article.


Brugia malayi DNA in vector (Aedes aegypti- Liverpool) and non-vector (Culex pipiens) mosquitoes at different times after ingestion of infected blood.The purpose of this study was to extend prior studies of molecular detection of Ae. aegypti- Liverpool. Infectious L3s were seen in the head, thorax and abdomen of vector mosquitoes 14 days after Mf ingestion. In contrast, parasites were only detected by histology shortly after the blood meal in Cx. pipiens, and these were not labeled by the antibody.Parasite DNA was detected over a two week time course in 96% of pooled thoraces of vector mosquitoes. In contrast, parasite DNA was detected in only 24% of thorax pools from non-vectors; parasite DNA was detected in 56% of midgut pools and 47% of abdomen pools from non-vectors. Parasite DNA was detected in vectors in the head immediately after the blood meal and after 14 days. Parasite DNA was also detected in feces and excreta of the vector and non-vector mosquitoes which could potentially confound results obtained with field samples. However, co-housing experiments failed to demonstrate transfer of parasite DNA from infected to non-infected mosquitoes. Parasites were also visualized in mosquito tissues by immunohistololgy using an antibody to the recombinant filarial antigen Bm14. Parasite larvae were detected consistently after mf ingestion in This study provides new information on the distribution of filarial parasites and parasite DNA in vector and non-vector mosquitoes. This information should be useful for those involved in designing and interpreting molecular xenomonitoring studies. Wuchereria bancrofti, Brugia malayi, and B. timori. These parasites are currently targeted for elimination by the Global Program for the Elimination of Lymphatic Filariasis (GPELF), and workers in this program have reported both achievements and future challenges to eliminating parasite transmission in endemic areas [Human lymphatic filiarasis (LF) is caused by the mosquito-borne filarial nematodes ic areas -3. One iic areas . Moleculic areas . Moleculic areas -9.B. malayi and W. bancrofti in mosquitoes [B. malayi infected (a constitutive parasite transcript) and infective mosquitoes (a L3-specific transcript) [Anopheles spp. have been divided into two body regions (head/thorax and abdomen) to provide better estimates of mosquitoes infected with Plasmodium sporozoites and/or pre-sporozoite stages [Onchocerca DNA, which is more likely to provide a better estimate of infective-stage parasites because other developmental stages generally reside outside of the head [The detection of parasite DNA in mosquito samples is a valuable tool for molecular xenomonitoring (MX), but this does not differentiate parasite developmental stages or distinguish whether the DNA is from living or dead parasites -12. Recesquitoes ,14, incle stages -17 and tthe head ,19.The studies conducted herein follow our previous work, which demonstrated that DNA-based diagnostics are unable to distinguish the developmental stage of LF parasites or whether parasites are living or dead in the mosquito . DespiteAe. aegypti-LVP, 73.6% of the recovered B. malayi mf at 2 h post ingestion (PI) had successfully penetrated the midgut, with 49.4% located in the thorax. At 14 days post ingestion of microfilaremic blood (DPI), 80% of Ae. aegypti harbored L3s. In contrast, from Cx. pipiens only mf were recovered and 93% of them were found in the midgut lumen.Table Ae. aegypti-LVP pooled body region samples (n = 300) and 36% of Cx. pipiens (n = 300) pooled body regions tested. These differences were highly significant (P < 0.0001). In Ae. aegypti-LVP, parasite DNA was detected in all four body regions with 43, 71, 88, and 96% of heads, midguts, abdomens, and thoraces (n = 75 for each) positive by qPCR, respectively. Parasite DNA also was detected in all Cx. pipiens body regions, with 17, 24, 47, and 56% of heads, thoraces, abdomens, and midguts (n = 75 for each) positive, respectively. The differences in the percentage of B. malayi DNA positive samples were significant between mosquito species in all body regions  except the midguts (P = 0.09). The detection of parasite DNA within certain mosquito body regions was positively or negatively correlated with time. Specifically, the detection of B. malayi DNA was negatively correlated with time in 'whole body' Cx. pipiens  and Cx. pipiens midguts , and positively correlated with time in Ae. aegypti-LVP heads .PCR results are summarized in Figure Ae. aegypti-LVP body regions  or between pooled and individual Cx. pipiens body regions .Individual mosquitoes that were separated into body regions for DNA detection assays were compared to results of the pooled mosquito body regions Fig. . FollowiB. malayi DNA in individual housed mosquitoes and their voided excreta and feces. All mosquitoes were positive for parasite DNA immediately (2 hr) after ingesting microfilaremic blood. From 1-4 DPI all Ae. aegypti-LVP tested positive for parasite DNA, and three of these (15%) mosquitoes had detectible B. malayi DNA in their feces. In contrast, 60% of Cx. pipiens were DNA negative at 4 DPI, but B. malayi DNA was detected in 100% of Cx. pipiens feces tested at 3-4 DPI. Of the twenty samples of feces collected over the entire observation period of 1-4 DPI from each species, B. malayi DNA was detected in 15 and 65% of Ae. aegypti-LVP and Cx. pipiens fecal samples, respectively (P = 0.003).Figure Ae. aegypti-LVP together with Cx. pipiens that had fed on a microfilaremic gerbil. After 7 days, mosquitoes were collected and pooled by species. None of the 17 Ae. aegypti-LVP pools (with 10 mosquitoes per pool) were positive by real-time PCR. In contrast, 17 of the 21 Cx. pipiens pools (with 5 mosquitoes each) were positive. Most of these samples had relatively high Ct values indicating small amounts of B. malayi DNA, but 5 pools had higher Ct values ranging between 29 and 35. Although B. malayi DNA can be detected in feces of infected mosquitoes, feces did not cause false positive DNA signals from uninfected mosquitoes after co-housing.In order to test the possibility that infected mosquitoes contaminate uninfected mosquitoes while they are together in the same trap or sampling tube, we housed uninfected B. malayi in vector and non-vector mosquitoes. In Ae. aegypti-LVP, unlabeled mf were detected within the midgut directly after the bloodmeal , and (2) the detection of parasite proteins as larvae develop in susceptible and refractory mosquitoes.The application of molecular assays to detect parasites within vectors is influenced by the vector-parasite interaction. In these studies, we analyzed the detection of filarial worms in susceptible and refractory mosquitoes by targeting parasite DNA or protein in molecular assays. The three mosquito strains examined have very different interactions with squitoes . The folAe. aegypti-LVP and non-vector Cx. pipiens, the head region had the lowest parasite DNA detection rates (43 and 17% respectively) when all time points are considered. However, there was a striking difference at 14 days when nearly 100% of pools of Ae. aegypti-LVP heads and very few pools of Cx. pipiens heads contained parasite DNA. This finding correlates well with infectivity rates at that time point. Although DNA detection in mosquito heads might provide a better estimate of infectivity rates than DNA detection in whole mosquitoes  prior to PCR assays provided further details on the location of detectible parasite DNA within mosquitoes. In both vector oes Fig. , we do nB. malayi DNA also was not restricted to the midgut in Cx. pipiens. Although most mf do not leave the Cx. pipiens midgut lumen, mf are sometimes detected outside of the midgut by dissection  after ingestion [Brugia--Cx. pipiens and certain Wuchereria--Culex spp. interactions) [Ae. aegypti and Cx. pipiens were examined for parasite DNA. B. malayi DNA was detected in voided materials of both species, but significantly more Cx. pipiens had detectible parasite DNA in their excreta and feces following a microfilaremic blood meal  -25, parameal Fig . AlthougHowever, to prevent cross-contamination, the following steps are recommended for processing wild-caught mosquitoes: (1) Immediately kill mosquitoes to limit mosquito-mosquito contact, especially if blood engorged mosquitoes are present; (2) When killing mosquitoes, avoid methods of slow death in which the mosquito host is killed and parasites are not, because it is possible for L3s to escape the body of living, dead, and dying mosquitoes, especially if mosquitoes come in contact with aqueous fluids -28; (3) Plasmodium circumsporozoite protein (CSP) [B. malayi L3s (NEB-D1E5) is specific to a B. malayi L3 surface antigen and distinguishes B. malayi L3s from infective-stage larvae of other filarial worms apart from B. timori [B. malayi antigen Bm14 [Ae. aegypti-LVP. In contrast, the protein was not detected in parasites present in non-vector Cx. pipiens (harboring mf in the midgut), or Ae. aegypti-RKF . These results suggest that Bm14 may be a specific biomarker for viable filarial parasites in mosquitoes.Immunohistochemistry provides an alternative approach to detecting and studying parasite migration and development in mosquitoes; several studies have used this approach with antibodies to in (CSP) ,29-31. Min (CSP) ,33, and in (CSP) ,34,35. I. timori ,37. In tgen Bm14 ,39 were B. malayi, the findings may be of interest to scientists and programs that use molecular techniques to detect other pathogens  in vectors.Improved methods are needed for assessing changes in mosquito infection and infectivity rates in the context of LF control/elimination programs. As infection rates in humans and vectors decrease following MDA, increased numbers of mosquitoes must be tested to accurately estimate parasite prevalence . This maAedes aegypti , Ae. aegypti  and Culex pipiens pipiens (Iowa strain) maintained at the University of Wisconsin-Madison, as previously described [B. malayi. Ae. aegypti-LVP support the development of B. malayi from mf to L3s, but parasites do not develop in Cx. pipiens, because mf do not penetrate the midgut epithelium. In Ae. aegypti-RKF, mf penetrate the mosquito midgut and migrate into thoracic muscles where they fail to develop to L2s. This mosquito strain was only used for comparison in the immunohistology experiments. Four- to seven-day-old mosquitoes were sucrose starved ~14 h prior to blood feeding. Mosquitoes were exposed to B. malayi by blood feeding on microfilaremic cat blood in a water-jacketed membrane feeder fitted with a parafilm membrane [Meriones unguiculatus) to serve as parasite-negative, blood-fed controls. Engorged mosquitoes were sorted and maintained in the laboratory.Mosquitoes used for these studies were obtained from colonies of escribed ,41. Thesmembrane . MosquitB. malayi-infected cat blood obtained from the NIAID Filariasis Research Reagent Repository Center http://www.filariasiscenter.org used in these studies ranged from 24-191 mf/20 μl blood.Laboratory animals were handled according to guidelines approved by the Animal Care Committee at the University of Wisconsin-Madison. The mf densities of B. malayi development. Individual mosquitoes were separated into head, thorax, midgut, and abdomen, and each body region was teased apart and individually examined for parasites by microscopy as previously described [Five mosquitoes were dissected at 2 hr, 7 d, and 14 d post ingestion of microfilaremic blood (PI) to estimate the mean intensity of infection, and to record the stage of escribed .Ae. aegypti-LVP and Cx. pipiens were separated into body regions  and placed, separately or in pools of four, into 2.0 ml microcentrifuge tubes for parasite DNA detection. To create a pooled sample, four mosquitoes were separated into body regions, and the body regions were combined by type into tubes. For example, four mosquitoes were used to produce one pool of four heads, one pool of four thoraces, one pool of four abdomens, and one pool of four midguts. Five pooled samples were prepared at 2 h, 1, 3, 7, and 14 d PI; thus, a total of 20 mosquitoes were collected at each time point for pooled samples. In addition to creating pooled samples, individual mosquitoes were dissected into body regions as described above and then placed individually into tubes. Five individuals were dissected at each time point to create twenty samples: five tubes contained individual heads, five contained a single thorax, five contained an abdomen, and five contained a midgut. These samples were screened for B. malayi DNA to compare detection results between individuals and pooled samples. All samples were cataloged to track a given body region back to the particular mosquito or pool of mosquitoes, allowing the assay results from each body region to be combined into results for the 'whole mosquito.' For example, if at least one of the body region samples was positive; then the 'whole mosquito' was considered positive and only if all body regions tested negative; then the 'whole mosquito' was considered negative.To examine possible sources of parasite DNA contamination, mosquitoes were individually housed to collect material voided by excretion  and defecation  . ImmediaAe aegypti were housed together with B. malayi-infected Cx. pipiens that had fed on two B. malayi-infected gerbils with microfilaremias of 68 and 146 mf/20 μl blood. Engorged Cx. pipiens were sorted from non-blood fed mosquitoes, and the engorged mosquitoes were mixed with non-blood fed Ae. aegypti. Four cartons were studied with each containing ~60 uninfected Ae. aegypti and ~40 B. malayi-infected Cx. pipiens. At 7 DPI, mosquitoes were collected for qPCR detection of parasite DNA. Cold anesthetized mosquitoes were sorted by species and placed in pools of five individuals for Cx. pipiens and pools of ten individuals for Ae. aegypti. Mosquitoes were flash frozen on dry ice and stored at -80°C.Uninfected Brugia HhaI repeat [B. malayi was used as positive control.Genomic DNA from pooled mosquitoes or body parts was extracted using a commercial column method as described previously . QuantitI repeat . In all B. malayi larvae were stained using the alkaline phosphatase anti-alkaline phosphatase method as described previously [Five mosquitoes of each species were collected at 2 h, 1, 3, 7, and 14 d PI  and stored in 80% ethanol at room temperature until embedding. Mosquitoes were embedded in paraffin, and eviously . A polyceviously .http://www.graphpad.com. Fisher's exact tests, with two-tailed P-values, were used to compare parasite development and DNA detection between Ae. aegypti-LVP and Cx. pipiens. Pearson correlation tests were used to test for trends in parasite DNA detection over time. Statistical results were considered significant at P ≤ 0.05.Data were graphed and analyzed with GraphPad Prism 5.0 MF: microfilariae; L1, L2, L3: first- to third-stage larva; PI: post ingestion of microfilaremic blood; DPI: days post ingestion of microfilaremic blood; MX: molecular xenomonitoring.The authors declare that they have no competing interests.SE participated in the conception of the study including its design and organization, performed mosquito exposures and sample collection, data interpretation, statistical analysis, and drafted the manuscript. KF carried out the DNA detection and immunohistology assays, and data interpretation. GW participated in data interpretation and critical manuscript revisions. BC participated in study design, data interpretation, and critical manuscript revisions. PF participated in the conception of the study including its design and organization, data interpretation, and helped draft the manuscript. All authors read and approved the final manuscript.


Helicobacter pylori contain Lewis antigens, mimicking glycan structures produced by human cells. The interaction of Lewis antigens with human dendritic cells induces a modulation of the immune response, contributing to the H. pylori virulence. The amount and position of Lewis antigens in the LPS varies among H. pylori isolates, indicating an adaptation to the host. In contrast to most bacteria, the genes for H. pylori O antigen biosynthesis are spread throughout the chromosome, which likely contributed to the fact that the LPS assembly pathway remained uncharacterized. In this study, two enzymes typically involved in LPS biosynthesis were found encoded in the H. pylori genome; the initiating glycosyltransferase WecA, and the O antigen ligase WaaL. Fluorescence microscopy and analysis of LPS from H. pylori mutants revealed that WecA and WaaL are involved in LPS production. Activity of WecA was additionally demonstrated with complementation experiments in Escherichia coli. WaaL ligase activity was shown in vitro. Analysis of the H. pylori genome failed to detect a flippase typically involved in O antigen synthesis. Instead, we identified a homolog of a flippase involved in protein N-glycosylation in other bacteria, although this pathway is not present in H. pylori. This flippase named Wzk was essential for O antigen display in H. pylori and was able to transport various glycans in E. coli. Whereas the O antigen mutants showed normal swimming motility and injection of the toxin CagA into host cells, the uptake of DNA seemed to be affected. We conclude that H. pylori uses a novel LPS biosynthetic pathway, evolutionarily connected to bacterial protein N-glycosylation.Lipopolysaccharide (LPS) is a major component on the surface of Gram negative bacteria and is composed of lipid A-core and the O antigen polysaccharide. O polysaccharides of the gastric pathogen  Helicobacter pylori exposes lipopolysaccharide (LPS) containing Lewis antigens that mimic human glycan structures. H. pylori alters its Lewis antigen display in adaptation to the individual host. Lewis antigens can interact with human dendritic cells, thereby inducing a suppression of the immune response and facilitating a chronic H. pylori infection. Whereas three general LPS biosynthesis pathways are known, the route of LPS assembly in H. pylori remained to be elucidated. We identified and characterized two components of the H. pylori LPS pathway, WecA and WaaL, which demonstrated that, as in other bacteria, the glycan is initially assembled onto a polyprenoid lipid carrier. This intermediate then has to cross a membrane barrier, requiring specialized translocases. H. pylori does not employ a translocase from common LPS pathways. We show that instead H. pylori uses a translocase named Wzk, which is involved in protein N-glycosylation in other bacteria. Wzk was able to translocate various glycan structures. The identification of Wzk as the H. pylori translocase involved in LPS biosynthesis indicates an evolutionary connection between LPS and glycoprotein biosynthesis pathways.Bacterial surfaces are decorated with glycans. The human stomach pathogen  Lipopolysaccharide (LPS) is a prevalent macromolecule in the outer membrane of Gram negative bacteria and represents an important virulence factor. LPS is composed of three parts: lipid A which is embedded in the outer membrane, the core oligosaccharide, and the O antigen Helicobacter pylori contributes in several respects to the virulence of this human gastric pathogen, which is recognized by the World Health Organization as a Type 1 carcinogen H. pylori mimics carbohydrate structures present on human epithelial cells, blood cells, and in secretions, by incorporating Lewis antigens on its O chains x) and Lewis y (Ley), can be found in certain regions of the O antigen. Some strains also display Lewis a (Lea) and b (Leb) antigens or can have alternative O antigen structures H. pylori profits from this molecular mimicry, as Lex and Ley interact with the C-type lectin DC-SIGN on dendritic cells, which signals the immune system to down-regulate an inflammatory response H. pylori O polysaccharide are variable, differing between strains and also between cells from the same isolate H. pylori fucosyltransferases, enzymes required for the synthesis of Lewis antigens H. pylori strains are adapted to the individual human host, enabling the establishment of a chronic infection The O antigen of H. pylori are not arranged in a single cluster, but rather found in various locations distributed throughout the chromosome. Nevertheless, many enzymes required for H. pylori LPS biosynthesis have been identified and characterized. These include glycosyltransferases responsible for the addition of the monosaccharide building blocks in the assembly of the O polysaccharide H. pylori remained uncharacterized.Unlike most bacteria, the genes involved in LPS biosynthesis in In all characterized LPS biosynthetic pathways, the O polysaccharide is assembled onto the undecaprenyl phosphate (UndP) lipid carrier by specific glycosyltransferases located in the cytoplasmic compartment of the bacteria Three LPS biosynthesis pathways are known to date Campylobacter jejuni which is closely related to H. pylori, the cell surface is covered with lipooligosaccharides (LOS) instead of LPS In some Gram negative bacteria, including H. pylori for the assembly of the Lewis antigens onto the lipid A-core. We found that these polysaccharides are assembled as typical O antigens onto the UndP carrier. Surprisingly, for the membrane translocation of the lipid-linked glycan, H. pylori employs an enzyme which has not been previously found to be involved in LPS synthesis, but instead is used by other bacteria in the biosynthesis of N-glycoproteins. This translocase, named Wzk, has no strict structural requirement for its substrates, a characteristic that enables H. pylori to produce O antigens of various structures and lengths according to the phenotype of the infected host.The goal of this investigation was to determine the pathway used by H. pylori LPS biosynthesis, a genome search was performed using the sequences of enzymes known to participate in LPS biosynthetic pathways. The search resulted in the identification of homologs of the E. coli wecA and waaL genes. The proteins encoded in the H. pylori genes JHP1488 in strain J99 and HPG27_1518 in strain G27 are 22% identical to E. coli WecA and 94% identical to each other. The genes JHP0385 and HPG27_389 encode polypeptides which are 19% identical to the O antigen ligase WaaL and 95% identical to each other. The overall homology between WecA and WaaL sequences from different organisms is low. Nevertheless these proteins, including the H. pylori homologs identified, share similar membrane topologies and a few conserved key residues. Alignments of the protein sequences are shown in H. pylori genome seemed to lack homologs of wzx, wzt, wzm or wbbF, which encode the flippase proteins involved in O antigen translocation in the known pathways. Furthermore, sequences encoding an O antigen polymerase Wzy, or a chain length regulator Wzz could not be found. Thus, the canonical LPS pathways are incomplete in H. pylori.In order to identify genes possibly involved in H. pylori LPS biosynthesis, mutants in both H. pylori strains, J99 and G27, were constructed by inserting a chloramphenicol-resistance cassette into the putative wecA and waaL open reading frames. For the generation of complemented strains, each gene was re-introduced into the recA gene on the chromosome of the corresponding mutant strain. This location in the genome was selected to prevent further recombinations, a procedure expected to stabilize the mutations. We took advantage of the H. pylori natural competence for the construction of the mutant strains. Interestingly, this procedure was not successful for the generation of complemented strains, as no colonies were recovered on the selective plates after transformation. However, complemented cells were efficiently obtained by electroporation.To address whether the identified genes were indeed involved in et al. H. pylori LPS biosynthesis.Monoclonal antibodies reacting with Lewis antigens were used to visualize the presence or absence of O antigens on the cell surfaces by fluorescence microscopy . Not allH. pylori LPS biosynthesis, the LPS of all strains was purified, separated by SDS-PAGE and visualized by silver staining . As shown in HPwecA complemented O antigen synthesis in the E. coli wecA mutant, which confirms its role as a UDP-GlcNAc: undecaprenyl-phosphate GlcNAc-1-phosphate transferase.The availability of HP could be expressed in E. coli, it was unable to restore smooth LPS production in an E. coli waaL mutant (data not shown). The transfer of O antigens onto the lipid A-core generally requires a strain specific core structure, and heterologous expression of O antigen ligases is therefore often not functional E. coli and H. pylori core regions are structurally different HP in E. coli.On the contrary, although WaaLHPH. pylori waaL mutant strain could not be achieved and the enzyme was not functional with the E. coli lipid A-core as an acceptor, we tested O antigen ligation activity by performing an in vitro assay. WaaLHP containing a C-terminal deca-His tag (encoded in pIH52) was expressed in the E. coli waaL mutant strain CLM24 E. coli O antigen ligase. Membranes containing the enzyme were solubilized with detergents and WaaLHP was purified by nickel affinity chromatography as described in E. coli co-purified with the H. pylori ligase. Lipid A-core from the HPH. pylori waaL mutant strain was purified and used as an acceptor structure. O antigen ligases typically show relaxed specificity towards the structure of the O polysaccharide in vitro assay. Due to the presence of multiple bands, LPS containing a polymerized O antigen is often indistinguishable from the corresponding UndPP-bound O antigen in SDS-PAGE and Western blot analysis. We reasoned that the use of a short oligosaccharide of defined length would facilitate the interpretation of results. Therefore, the UndPP-linked heptasaccharide derived from the C. jejuni N-linked protein glycosylation pathway was selected as substrate. This lipid-linked glycan can be synthesized in E. coli cells carrying the plasmid pACYCpglBmutin vitro glycosylation As full complementation of the C. jejuni heptasaccharide  which encodes a flippase involved in the translocation of the UndPP-linked heptasaccharide during protein N-glycosylation in C. jejuniH. pylori genome. The genes annotated as JHP1129 in strain J99 and HPG27_1153 in strain G27 encode polypeptide sequences which are 97% identical to each other and share 37% identity with C. jejuni PglK . It was concluded that the absence of O antigens in the H. pylori LPS has no adverse effect on flagellar function in vitro.Our fluorescence microscopy experiments allowed us to confirm the presence of LPS on the membranous sheaths covering H. pylori O antigen mutants. The natural competence of H. pylori depends on the ComB type IV secretion system H. pylori possesses additional type IV secretion systems, we examined if the presence of O antigens on the bacterial surface might be required for the function of these machineries. One additional H. pylori type IV secretion system is encoded in the cag pathogenicity island H. pylori correlates with enhanced virulence, as the Cag type IV secretion system builds a needle-like device for the injection of a single known effector protein, CagA, directly into the host cells. Within the host, CagA is tyrosine phosphorylated and interferes with cell signaling pathways As mentioned above, the protocol for natural DNA uptake was not successful in the construction of the complemented strains. One possible reason is a reduced natural competence of the H. pylori strains. AGS gastric epithelial cells were infected with H. pylori cells from an overnight grown liquid culture. Cells were harvested four hours after infection, when many epithelial cells displayed an elongated morphology, a typical effect following CagA translocation H. pylori strains injected similar amounts of CagA. We concluded that mutations interfering with the synthesis of smooth LPS in H. pylori may reduce natural competence, but do not affect type IV secretion systems in general.We compared the efficiency of CagA injection into human gastric epithelial cells between wild type and O antigen mutant H. pylori colonization. The fact that the genes involved in LPS biosynthesis are not grouped in a single locus in the H. pylori chromosome is probably one of the reasons why the pathway for the biosynthesis of this key macromolecule remained to be determined. The objective of this investigation was to elucidate the LPS biosynthetic pathway in H. pylori. Genomic analysis revealed that none of the previously characterized pathways for LPS biosynthesis is complete in H. pylori, suggesting that this bacterium uses an alternative strategy. One possibility was that the synthesis of LPS took place directly on the lipid A-core as it occurs with the LOS biosynthesis in C. jejuni and Neisseria spp. H. pylori LPS biosynthesis. WecAHP initiates this pathway by transferring a GlcNAc-phosphate from UDP-GlcNAc to UndP. The resulting molecule, UndPP-GlcNAc, serves as an acceptor for the assembly of the O chain backbone, composed of alternating GlcNAc and Gal residues. Some of these linear polysaccharides are decorated at selected locations through the activity of various fucosyltransferases, producing the Lewis antigens HP.With the display of Lewis antigens on the O chains, the LPS plays a unique role in H. pylori LPS biosynthesis follows a novel pathway, differing from all the established LPS pathways in the translocation of the O chain. We found that this step is accomplished by Wzk, which is not related to any described translocase involved in O antigen synthesis. Instead, Wzk is homolog to C. jejuni PglK, responsible for UndPP-heptasaccharide flipping during protein N-glycosylation. Wzk and PglK are related to the lipid A-core flippase MsbA, with the closest sequence similarity among known ATP transporters. ATPase activity of PglK has been reported by Alaimo et al.H. pylori, the flippase Wzk is the only polypeptide required and sufficient for translocation of UndPP-linked glycans. The ability of Wzk to translocate Lewis antigens, the C. jejuni heptasaccharide, as well as the E. coli O16 antigen, demonstrates that Wzk activity is independent of the length or the composition of the translocated UndPP-linked sugars.H. pylori, are located in separate loci dispersed along the chromosome. H. pylori exhibits a high rate of DNA uptake and genetic variability H. pylori by allowing more diversity and flexibility in the LPS structure. It is particularly intriguing that the position of the H. pylori wzk gene is located in close proximity to tRNA genes, which are known to be hot-spots for the insertion of mobile genetic elements C. jejuni, PglK is encoded as part of the pgl-cluster, responsible for N-linked protein glycosylation pgl-locus, has homologs in eukaryotes and archaea but not in H. pylori, which does not possess this general glycosylation machinery H. pylori may have discarded its pgl-cluster, yet retained wzk to act on the synthesis of LPS. Alternatively, the wzk gene could have been adopted by other epsilon- or delta proteobacteria to produce N-glycoproteins. Subsequently, some of these organisms, like C. jejuni, may have lost their LPS cluster, producing LOS instead. In either case, bacteria appear to favor the dedication of their lipid-linked glycans exclusively to one biosynthetic pathway, either protein glycosylation or LPS biosynthesis.In most bacteria, the genes involved in O antigen biosynthesis are clustered in a single locus, which facilitates horizontal gene exchange and regulation of O antigen synthesis H. pylori cells and electroporation was required for the construction of strains for complementation experiments. One possible explanation is a loss of natural competence for DNA uptake. A similar observation has been reported for a C. jejuni LOS mutant strain C. jejuni LOS and capsule mutants displayed increased DNA uptake ability H. pylori natural competence remains unclear, we demonstrated that the presence of O antigens does not have a general inhibitory effect on H. pylori type IV secretion systems, because the type IV secretion apparatus encoded in the cag pathogenicity island was functional in O chain deficient H. pylori mutants. Alternatively, the lack of O antigens may induce a stress response, resulting in the induction of DNA restriction enzymes, which could digest the foreign DNA before or after uptake into the cells.We encountered difficulties with natural transformation of O antigen mutant H. pylori flagella, we investigated the possible role of O antigens in motility. H. pylori mutant strains were not defective in in vitro swimming motility compared to wild type strains. However, the presence of LPS on the flagellar surface may still play a role in vivo. The O antigens may have a protective function by shielding the flagella against components of the host immune defense, and by actively down-regulating flagellin-specific activation of the innate immune system via the interaction between Lewis antigens and DC-SIGN.Due to the presence of LPS on the H. pylori pathogenicity is their interaction with DC-SIGN, which results in modulation of the host immune defense H. pylori maintains a balance between activation and repression of the host immune system H. pylori and possibly C. jejuni infections.A central role of the Lewis antigens in N-glycosylation, supporting an evolutionary connection between these pathways.Interestingly, Wzk is the first protein common to both, LPS biosynthesis and protein H. pylori genome. Global sequence identities were calculated with LALIGN  (http://www.ch.embnet.org/software/LALIGN_form.html).NCBI BLASTP (default settings) was used for the search of putative WecA, WaaL and lipid-linked glycan translocase polypeptide sequences encoded in the Oligonucleotides used for DNA amplification are listed in HPwecA was amplified by PCR, using the primers WecAHPEcoRIfw and WecAHPH6XbaIrv and genomic DNA from H. pylori J99 as template. The HPwecA PCR product was cloned into pEXT20 HP in E. coli.For the construction of pIH22, HPwecA from H. pylori J99, which was cloned into pGEM-T (Promega), generating pGEM-HPwecA. The obtained plasmid was digested with SgfI (Promega), which is cutting the open reading frame of HPwecA. Blunt ends were generated with a Klenow fragment (New England Biolabs). Insertion of a chloramphenicol-resistance cassette (CAT), derived from plasmid DT3072 H. pylori wecA::cat.The primers WecA_forward and WecA_reverse were used to amplify HPwecA gene was amplified with the primers NdeIHPJ99wecAfw and HPJ99wecAH6BamHIrev. The PCR product was ligated into vector pGEM-T easy (Promega), resulting in plasmid pIH27, which was subsequently digested with NdeI and BamHI and cloned into vector pSK+recxorf8-flag H. pylori wecA::cat recA::wecA.The J99 H. pylori J99 was used as template for the PCR amplification of HPwaaL using Ligase_forward and Ligase_reverse primers. The amplified DNA sequence was cloned into pGEM-T, generating pGEM-HPwaaL. The HPwaaL gene was subsequently cut with NheI (Invitrogen) and blunt ends were generated using a polymerase Klenow fragment. The CAT cassette was excised from DT3072 using HincII restriction enzyme and inserted into the HPwaaL gene of pGEM-HPwaaL, generating pGEM-HPwaaL-CAT, which was used for the construction of H. pylori waaL::cat.Genomic DNA from HPwaaL from H. pylori G27 was amplified with primers NdeIG27waaLfw and G27waaLH10BamHIrv. The PCR product and pSK+recxorf8-flag were digested with NdeI and BamHI and ligated. The resulting plasmid pIH53 was used for the construction of H. pylori waaL::cat recA::waaL.For the construction of pIH53, HP in E. coli.Plasmid pIH53 was digested with NdeI, whereas pEXT20 was digested with EcoRI. Blunt ends were generated with a Klenow fragment and the resulting DNA fragments were further digested with BamHI. Subsequent ligation of the DNA fragments resulted in pIH52, which was used for the expression of WaaLH. pylori J99 DNA using the primers KpnIHPpglKfw and HPpglKH6XbaIrev. The PCR product and pEXT20 were digested with KpnI and XbaI. Ligation of the DNA fragments resulted in pIH23, which was used for the expression of Wzk in E. coli.The gene encoding the flippase Wzk was amplified from genomic wzk in pIH23 was cut with PsiI, and the CAT cassette from HincII-digested DT3072 was inserted by ligation. The resulting plasmid pIH40 was used for the construction of H. pylori wzk::cat.The gene wzk gene was excised from pIH23 through digestion with KpnI and PstI restriction enzymes. Plasmid pSK+recxorf8-flag was digested with BamHI and NdeI. After treatment with a Klenow fragment the restriction products were ligated to generate pIH43, which was used for the construction of H. pylori wzk::cat recA::wzk.The C. jejuni pglK, with the plasmid pACYCpglH. pylori wzk::cat recA::pglK.Oligonucleotides NdeICj81116pglKfw and Cj81116pglKXbaIrv were used for the PCR amplification of acrA encoding the glycosylation acceptor protein AcrA from C. jejuni was excised from plasmid pWA2 E. coli.The gene H. pylori strains J99 H. pylori mutant strains were generated through natural transformation with the plasmids pGEM-HPwecA-CAT for wecA mutants, pGEM-HPwaaL-CAT for waaL mutants and pIH40 for wzk mutants, respectively, resulting in the disruption of the targeted genes through insertion of a chloramphenicol resistance cassette by homologous recombination. Mutant strains were recovered as single colonies after growth on selective plates containing chloramphenicol. Complementation was achieved through electroporation of the mutant strains for the uptake of the plasmids pIH42 for wecA complementation, pIH53 for waaL complementation and pIH43 for wzk complementation, respectively. Following homologous recombination, the complemented genes were inserted into the chromosome of the H. pylori mutant strains, disrupting the recA gene. Complemented colonies were selected on plates containing chloramphenicol and kanamycin. All strains were verified with PCR analysis.H. pylori strains were grown on brucella broth agar plates, supplemented with 10% heat inactivated fetal bovine serum, or on brain heart infusion agar with 10% horse serum. The antibiotics vancomycin (5 µg/ml), cycloheximide (100 µg/ml), trimethoprim (10 µg/ml) and amphotericin B (8 µg/ml) were added and cells were incubated at 37°C under micro-aerobic conditions, obtained by adding a CampyGen gas pack (Oxoid) to an anaerobic jar. O antigen mutant strains were selected with chloramphenicol (25 µg/ml). Kanamycin (20 µg/ml) was added for the selection of complemented strains. Liquid cultures were grown overnight in brucella broth supplemented with 10% heat inactivated fetal bovine serum and the appropriate antibiotics at 37°C with 160 rpm rotation. E. coli strains were grown overnight in LB broth at 37°C with rotation at 200 rpm.H. pylori cultures were adjusted to an optical density at 600 nm wave length (OD600) of 1.0 per ml. Cells were washed with PBS and an equivalent of 0.4 OD600 was applied to each cover glass. After 30 min of incubation on ice, the cell suspension was removed and cells were fixed with 4% paraformaldehyde for 10 min at room temperature. Staining and microscopy procedures were conducted as described by Couturier and Stein y antibody (1/200)  and a secondary Alexa Fluor 488 goat anti-mouse antibody (1/500) (Molecular Probes) were used for staining.Microscope cover glasses were prepared for the attachment of cells using (3-aminopropyl)triethoxysilane (Sigma) according to Strähle and coworkers et al.x and anti-Ley antibodies , or rabbit anti-O16 antigen antiserum . After incubation with a secondary goat anti-mouse IgM IRDye-800 antibody or a goat anti-rabbit IRDye-800 antibody, respectively (LI-COR Biosciences), the blots were scanned with an Odyssey infrared imaging system (LI-COR Biosciences).Small scale LPS extraction using hot phenol was performed following the procedure described by Marolda E. coli serogroup O16 laboratory strains produce rough LPS without O antigen due to a mutation inactivating the rhamnosyltransferase responsible for the addition of the second sugar residue in the O chain assembly. Smooth LPS production can be restored with the addition of a plasmid pMF19 containing the rhamnosyltransferase gene E. coli W3110 transformed with pMF19 served as a positive control, producing long chain O16 LPS. E. coli strain CLM37 has a mutation in ECwecA, and therefore is unable to assemble O chains HP activity. WecAHP activity was examined in CLM37 transformed with pMF19 and pIH22.E. coli O antigen ligase mutant strain CLM24 was transformed with pMF19 and either pIH52 or pEXT20 for the analysis of WaaLHP activity or for the corresponding negative control, respectively.The H. pylori Wzk to flip UndPP-linked glycans in an N-glycosylation pathway, E. coli strain SCM7 wzk), pIH18 (encoding the acceptor protein AcrA) and pACYCpglKmut (encoding the C. jejuni glycosylation machinery with a mutation in the translocase gene pglKpgl (containing the intact C. jejuni glycosylation machinery) instead of pACYCpglKmut. To further examine if Wzk has O antigen translocase activity, the E. coli flippase mutant strain CLM17 To test the ability of C. jejuni glycan, were applied. After incubation with a secondary goat anti-rabbit IRDye680 antibody (LI-COR Biosciences), bands were visualized with an Odyssey imaging system (LI-COR Biosciences).LPS profiles were analyzed by silver staining as described above. Western blotting was used to determine the glycosylation status in the Wzk activity tests. As primary antibodies, either an anti-AcrA antibody HP was excised from a coomassie stained gel  according to the supplier protocol and dissolved in 0.1% formic acid. Peptides were separated with a LC/MSD Trap XCT (Agilent Technologies) and the resulting mass spectrum was used for the identification of the protein by the Mascot search engine (www.matrixscience.com) using the NCBInr database.The protein band corresponding to the putative WaaLined gel . The proE. coli O antigen ligase mutant CLM24 was transformed with pIH52 which encodes HPwaaL with a C-terminal deca-histidine tag. Cells were grown overnight at 37°C with 0.2 mM IPTG for the production of the O antigen ligase. The protocol described by Faridmoayer et al.HP. Briefly, membrane fractions were solubilized with 2% elugent  in phosphate buffer, pH 7.2. Elugent concentration was diluted to 1% and the membrane fraction loaded unto a nickel agarose column (Qiagen) with 20 mM imidazole. The washing solution contained 50 mM imidazole and 0.5% DDM (Anatrace). Ligase was eluted with 250 mM imidazole in the presence of 0.5% DDM. The lipid A-core from HPwaaL mutant cells was obtained as described above and utilized as acceptor in the in vitro assay. UndPP-linked glycans serving as substrates were produced by E. coli CLM24 cells transformed with pACYCpglBmutet al. HP (4–5 µg), purified LPS , with or without ATP (2 mM). Reaction products were visualized by silver staining or Western blotting using the glycan specific antibody HR6. In addition, mild acid hydrolysis was performed similar to the method described by Ielpi et al.The H. pylori wild type and O antigen mutant cells from overnight cultures, grown either on plates or in liquid media, were spotted on soft brucella broth agar plates (0.3% agar) and incubated at 37°C under micro-aerobic conditions. Swimming motility was analyzed after 3–6 days by comparison of the colony growth diameters.et al. H. pylori cells with a multiplicity of infection of 100∶1 for four hours. After incubation, AGS cells were washed, harvested and fractionated. Membrane fractions were analyzed by Western blotting, using anti-CagA , and anti-phosphotyrosine  antibodies. Analysis with an anti-h-Met antibody  showing the general host membrane protein h-Met served as loading control.The procedure for the CagA translocation assay was previously described by Cendron Figure S1http://bioinfo.genotoul.fr/multalin/multalin.html). (A) Alignments of WecA sequences from sequenced H. pylori strains G27, 26695, J99, HPAG1 and P12. (B) Alignment of WecA sequences from H. pylori G27 and J99 with WecA sequences from Burkholderia cenocepacia and E. coli.WecA alignments. Alignments of WecA polypeptide sequences were done using MultiAlin ((0.51 MB GIF)Click here for additional data file.Figure S2http://bioinfo.genotoul.fr/multalin/multalin.html). (A) Alignments of WaaL sequences from sequenced H. pylori strains G27, 26695, HPAG1, P12 and J99. (B) Alignment of WaaL sequences from H. pylori G27 and J99 with WaaL sequences from E. coli, Pseudomonas aeruginosa and Salmonella typhimurium.WaaL alignments. Alignments of WaaL polypeptide sequences were done using MultiAlin ((0.51 MB GIF)Click here for additional data file.Figure S3H. pylori flagella. Fluorescence microscopy of H. pylori J99 using an anti-Lex antibody confirmed the presence of Lewis antigens on the membranous sheath covering the H. pylori flagella, indicated by an arrow.Lewis antigens are present on the (0.28 MB GIF)Click here for additional data file.Figure S4HPwaaL complementation. Purified LPS from 1: wild type H. pylori G27, 2: waaL mutant, 3: waaL complemented was analyzed by Western blot using an anti-Ley antibody. The partial production of smooth LPS in the complemented strain is indicated with an arrow. Protein marker standards were included for reference.Partial (0.34 MB GIF)Click here for additional data file.Figure S5HP-His10. WaaLHP-His10 purification fractions using nickel affinity chromatography were run on a 10% SDS-PAGE and proteins were stained with coomassie. 1: loading sample; 2: flow through; 3: washing fraction; 4–9: elution fractions 1–6 (1 ml was collected per elution fraction). The band containing WaaLHP-His10 is indicated with an arrow. Its identity was confirmed by mass spectrometry. Protein marker standards were included for reference.Purification of WaaL(0.50 MB GIF)Click here for additional data file.Figure S6E. coli LPS, (2) H. pylori G27 wild type LPS and (3) C. jejuni LLO in the same conditions as applied for the in vitro ligation assay are shown in a Western blot using anti-E. coli O16 antigen, anti-Ley and HR6 anti-C. jejuni glycan antibodies, (A) not hydrolyzed and (B) after mild acid hydrolysis. Mild acid hydrolysis affects UndPP-linked oligosaccharides (lane 3) but does not hydrolyze LPS . Protein marker standards were included for reference.Validation of the mild acid hydrolysis protocol. (1) (0.51 MB GIF)Click here for additional data file.Figure S7H. pylori WaaL in vitro activity. Ligation in vitro was performed (1) in the absence and (2) in the presence of ATP (2 mM). Reaction samples were separated with SDS-PAGE (15%) and were analyzed with (A) silver staining and  Western blotting using the HR6 anti-C. jejuni glycan antibody, whereby reaction samples were treated with mild acid in (C), hydrolyzing the UndPP-linked glycan (substrate). Protein marker standards were included for reference.ATP is not required for (0.51 MB GIF)Click here for additional data file.Figure S8http://bioinfo.genotoul.fr/multalin/multalin.html). (A) Alignments of Wzk sequences from sequenced H. pylori strains G27, 26695, J99, HPAG1 and P12. (B) Alignment of Wzk sequences from H. pylori G27 and J99 with homologous sequences from Wolinella succinogenes and Arcobacter butzleri and PglK from C. jejuni. (C) Alignment of Wzk sequences from H. pylori G27 and J99 with homologous sequences from Wolinella succinogenes and Arcobacter butzleri, PglK from C. jejuni and MsbA sequences from H. pylori J99 and E. coli.Wzk alignments. Alignments of translocase polypeptide sequences were done using MultiAlin ((0.49 MB GIF)Click here for additional data file.Figure S9H. pylori O chains containing Lewis antigens. The positions of the fucose residues can change . Shown are terminal Ley, terminal Lex and internal Lex with terminal Ley. (B) C. jejuni heptasaccharide . (C) E. coli O16 antigen .Glycan structures translocated by Wzk. Shown are the glycan structures translocated by Wzk in this study. (A) (0.17 MB GIF)Click here for additional data file.Table S1Oligonucleotides.(0.04 MB DOC)Click here for additional data file.


To the Editor: Increased risk for reactivation of tuberculosis (TB) after treatment with tumor necrosis factor (TNF) antagonists, particularly infliximab, is well documented  but was negative for acid-fast bacilli on direct examination and PCR testing for Mycobacterium tuberculosis complex. Laparoscopy on May 2, 2008, showed extensive peritoneal inflammation. Peritoneal biopsy samples contained acid-fast bacilli and caseating granulomas; PCR for M. tuberculosis complex was positive. At this time, results of a repeat QuantiFERON-TB in Tube test and a tuberculin skin test (TST) were negative, but a T-SPOT.TB  test was positive . M. bovis ssp. bovis was grown in cultures of peritoneal biopsy samples. For culture, the MGIT 960 automated culture system  was used. The isolate was identified by use of a multiplex PCR-based, solid-phase, reverse-hybridization assay , excluding M. bovis BCG  result  and an unremarkable chest radiograph. On November 30, 2007, she was given prednisone  and prescribed 3 doses of infliximab because of severe inflammation seen during colonoscopy . She was hospitalized on April 25, 2008, at which time she had ascites; the fluid contained 1.4 × 10M. bovis in a patient treated with infliximab highlights the need to be aware of local epidemiology with regard to transmissible infectious diseases. It particularly emphasizes the value of careful history taking regarding consumption of unpasteurized dairy products in assessing risk for latent TB infection (LTBI) before starting anti-TNF treatment. Some infectious diseases experts   consumption of unpasteurized dairy products may prompt preventive chemotherapy before the start of anti-TNF treatment. Thus, a potentially increasing number of human TB cases due to M. bovis may be prevented.Our patient probably acquired


Bacteroides fragilis ATCC 51477-B1, was sequenced and consisted of 44,929 bases with a G+C content of 38.7%. Forty-six putative open reading frames were identified and genes were organized into functional clusters for host specificity, lysis, replication and regulation, and packaging and structural proteins.The genome of a fecal pollution indicator phage,  Escherichia coli and Bacteroides fragilis serve as fecal pollution indicators (1). Bacteroides phages are more attractive fecal indicators than E. coli because Bacteroides are more abundant in fecal matter, provide higher host specificity, and are anaerobic and thus less likely to reproduce in aquatic environments than E. coli . Th. Th19]. B. fragilis [Four potential promoters were identified on the positive strand of the genome based on similarity to promoters found in fragilis . Two werfragilis  revealedB. fragilis surface components [Eight polymorphic regions occurring within ORFs and displaying sequence variability from 4% to 13% were identified with the extensive pyrosequencing data. These regions ranged in size from 200 bp to greater than 1,000 bp. ORF7, the putative tail fiber protein, displayed the most polymorphisms, including a pyrosequencing contig with a deletion relative to the final assembly. Tail fiber variability modifies the phage host range  and may,mponents ,24.B. fragilis phage ATCC 51477-B1 was deposited in GenBank with accession number FJ008913.The sequence for The authors declare that they have no competing interests.SAH cloned and sequenced the genome by primer walking, assisted in the pyrosequence data analysis and annotation, and drafted the manuscript. ACL analyzed pyrosequencing data, annotated the genome, and assisted in drafting the manuscript. SR organized the study and provided final editing for the manuscript. DW provided genome cloning. GSS participated in the study design and coordination. All authors read and approved the final manuscript.Phage ATCC 51477-B1 genome organization.Click here for file