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Agrobacterium tumefaciens causes crown gall disease in plants by the horizontal transfer of oncogenic DNA. The conjugation is mediated by the VirB/D4 type 4 secretion system (T4SS) that assembles an extracellular filament, the T-pilus, and is involved in mating pair formation between A. tumefaciens and the recipient plant cell. Here, we present a 3 Å cryoelectron microscopy (cryo-EM) structure of the T-pilus solved by helical reconstruction. Our structure reveals that the T-pilus is a stoichiometric assembly of the VirB2 major pilin and phosphatidylglycerol (PG) phospholipid with 5-start helical symmetry. We show that PG head groups and the positively charged Arg 91 residues of VirB2 protomers form extensive electrostatic interactions in the lumen of the T-pilus. Mutagenesis of Arg 91 abolished pilus formation. While our T-pilus structure is architecturally similar to previously published conjugative pili structures, the T-pilus lumen is narrower and positively charged, raising questions of whether the T-pilus is a conduit for ssDNA transfer., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The availability of a reliable prediction method for prediction of bacterial virulent proteins has several important applications in research efforts targeted aimed at finding novel drug targets, vaccine candidates, and understanding virulence mechanisms in pathogens. In this work, we have studied several feature extraction approaches for representing proteins and propose a novel bacterial virulent protein prediction method, based on an ensemble of classifiers where the features are extracted directly from the amino acid sequence and from the evolutionary information of a given protein. We have evaluated and compared several ensembles obtained by combining six feature extraction methods and several classification approaches based on two general purpose classifiers (i.e., Support Vector Machine and a variant of input decimated ensemble) and their random subspace version. An extensive evaluation was performed according to a blind testing protocol, where the parameters of the system are optimized using the training set and the system is validated in three different independent data sets, allowing selection of the most performing system and demonstrating the validity of the proposed method. Based on the results obtained using the blind test protocol, it is interesting to note that even if in each independent data set the most performing stand-alone method is not always the same, the fusion of different methods enhances prediction efficiency in all the tested independent data sets.

Secretins are a unique class of bacterial multimeric outer membrane proteins that probably differ considerably from other, less complex outer membrane proteins in their overall structure and organization, and in their requirements for outer membrane targeting and assembly factors. In this MicroCommentary, we discuss these differences with respect to the role of a specific class of lipoproteins, often referred to as pilotins, in secretin complex assembly. We compare them with other lipoproteins that play a role in Omp85/YaeT-mediated assembly of more classical outer membrane proteins. One of the examples we have chosen is the Myxococcus Xanthus lipoprotein Tgl. Coculture of cells with and without Tgl allows secretin assembly (and, hence, type IV pilus assembly) in the cells without Tgl, indicating that it can act by cell-to-cell contact or can transfer between cells.

Vibrio fischeri colonizes the squid Euprymna scolopes in a mutualistic symbiosis. Hatchling squid lack these bacterial symbionts, and V. fischeri strains must compete to occupy this privileged niche. We cloned a V. fischeri gene, designated pilA, that contributes to colonization competitiveness and encodes a protein similar to type IV-A pilins. Unlike its closest known relatives, Vibrio cholerae mshA and vcfA, pilA is monocistronic and not clustered with genes associated with pilin export or assembly. Using wild-type strain ES114 as the parent, we generated an in-frame pilA deletion mutant, as well as pilA mutants marked with a kanamycin resistance gene. In mixed inocula, marked mutants were repeatedly outcompeted by ES114 (P < 0.05) but not by an unmarked pilA mutant, for squid colonization. In contrast, the ratio of mutant to ES114 CFUs did not change during 70 generations of coculturing. The competitive defect of pilA mutants ranged from 1.7- to 10-fold and was more pronounced when inocula were within the range estimated for V. fischeri populations in Hawaiian seawater (200 to 2,000 cells/ml) than when higher densities were used. ES114 also outcompeted a pilA mutant by an average of twofold at lower inoculum densities, when only a fraction of the squid became infected, most by only one strain. V. fischeri strain ET101, which was isolated from Euprymna tasmanica and is outcompeted by ES114, lacks pilA; however, 11 other diverse V. fischeri isolates apparently possess pilA. The competitive defect of pilA mutants suggests that cell surface molecules may play important roles in the initiation of beneficial symbioses in which animals must acquire symbionts from a mixed community of environmental bacteria.

Polar pili biogenesis in Caulobacter involves the asymmetric localization of the CpaE and CpaC components of the pili-specific secretion apparatus to one pole of the predivisional cell followed by the biosynthesis of the pili filaments in the daughter swarmer cell. The histidine kinase signaling protein, PleC, that controls the temporal accumulation of the PilA pilin subunit is asymmetrically localized to the pole at which pili are assembled. Here we identify a protein, PodJ, that provides the positional information for the polar localization of both PleC and CpaE. The PodJ protein was found to exist in two forms, a truncated 90-kDa and a full-length 110-kDa form, each controlling a different aspect of polar development and each localizing to the cell poles at a specific time in the cell cycle. When active PleC is delocalized in a DeltapodJ mutant, the accumulation of PilA, the downstream target of PleC signaling, is impaired, providing evidence that the polar localization of this histidine kinase stimulates the response signaled by a two-component system.

The AraC homolog ToxT coordinately regulates virulence gene expression in Vibrio cholerae. ToxT is required for transcriptional activation of the genes encoding cholera toxin and the toxin coregulated pilus, among others. In this work we focused on the interaction of ToxT with the tcpA promoter and investigated the mechanism of ToxT-dependent transcriptional activation at tcpA. Deletion analysis showed that a region from -95 to +2 was sufficient for ToxT binding and activation, both of which were simultaneously lost when the deletion was extended to -63. A collection of point mutations generated by error-prone PCR revealed two small regions required for ToxT-dependent transactivation. Binding studies performed with representative mutations showed that the two regions define sites at which ToxT binds to the tcpA promoter region, most likely as a dimer. Results obtained by using a rpoA truncation mutation showed that ToxT-dependent activation at tcpA involves the C-terminal domain of the RNA polymerase alpha subunit. A model of ToxT-dependent transcriptional activation at tcpA is proposed, in which ToxT interacts with two A-rich regions of tcpA centered at -72 and -51 and requires the alpha C-terminal domain of RNA polymerase.

The study of how bacteria respond to and obtain divalent metal ions provides insight into the regulation of virulence factors in the host environment. Regulation of metal permease operons in gram-positive bacteria may involve the binding of metal-responsive repressors to palindromic domains in their control regions. The Streptococcus parasanguis fimA operon, which encodes an ATP-binding cassette (ABC) transporter system with sequence homology to the LraI family of metal transporters, possesses a palindromic regulatory region with high homology to that of the Streptococcus gordonii ScaR binding domain. Mapping of the promoter and regulatory regions of fimA and the divergently transcribed pepO gene, which encodes a zinc metalloendopeptidase, indicated that their promoter and regulatory elements overlap. fimA had one transcriptional start site, whereas pepO had three. Analysis of truncated versions of the pepO promoter suggested that all three transcriptional start sites are functional. Analysis of promoter activity under various environmental conditions indicated that the fimA operon promoter and the pepO promoter are not coordinately regulated. The fimA operon is responsive to changes in Mn(2+) concentration, but the pepO promoter is not. A S. parasanguis fimA mutant showed a growth deficiency under conditions of limiting Mn(2+). This deficiency was not alleviated by compensation with either Mg(2+) or Fe(3+). Wild-type S. parasanguis could take up Mn(2+) and Fe(3+), while the fimA mutant showed a marked reduction in this ability. These data suggested that FimA is a component of a metal transporter system capable of transporting both Mn(2+) and Fe(3+). FimA expression itself was shown to be responsive to Mn(2+) concentration, but not to availability of Fe(3+) or Mg(2+).

Each cell division in Caulobacter crescentus is asymmetric, yielding a swarmer cell with several polar pili and a non-piliated stalked cell. To identify factors contributing to the asymmetric biogenesis of polar pili, cytological studies of pilus assembly components were performed. We show here that the CpaC protein, which is thought to form the outer membrane pilus secretion channel, and its assembly factor, CpaE, are localized to the cell pole prior to the polymerization of the pilus filament. We demonstrate that the PleC histidine kinase, a two-component signal transduction protein shown previously to localize to the piliated cell pole before and during pilus assembly, controls the accumulation of the pilin subunit, PilA. Using an inactive form of PleC (PleCH610A) that lacks the catalytic histidine residue, we provide evidence that PleC activity is responsible for the asymmetric distribution of CpaE and itself to only one of the two cell poles. Thus, a polar signal transduction protein controls its own asymmetric location as well as that of a factor assembling a polar organelle.

Pathogenic bacteria are specifically adapted to bind to their customary host. Disease is then caused by subsequent colonization and/or invasion of the local environmental niche. Initial binding of Haemophilus influenzae type b to the human nasopharynx is facilitated by Hib pili, filaments expressed on the bacterial surface. With three-dimensional reconstruction of electron micrograph images, we show that Hib pili comprise a helix 70 A in diameter with threefold symmetry. The Hib pilus filament has 3.0 subunits per turn, with each set of three subunits translated 26.9 A along and rotated 53 degrees about the helical axis. Amino acid sequence analysis of pilins from Hib pili and from P-pili expressed on uropathogenic Escherichia coli were used to predict the physical location of the highly variable and immunogenic region of the HifA pilin in the Hib pilus structure. Structural differences between Hib pili and P-pili suggest a difference in the strategies by which bacteria remain bound to their host cells: P-pili were shown to be capable of unwinding to five times their original length (E. Bullitt and L. Makowski, Nature 373:164-167, 1995), while damage to Hib pili occurs by slight shearing of subunits with respect to those further along the helical axis. This capacity to resist unwinding may be important for continued adherence of H. influenzae type b to the nasopharynx, where the three-stranded Hib pilus filaments provide a robust tether to withstand coughs and sneezes.

In uropathogenic Escherichia coli, P pili (Pap) facilitate binding to host epithelial cells and subsequent colonization. Whereas P pili can be produced at 37 degrees C, the expression of these fimbriae is suppressed at 23 degrees C. Previously, insertion mutations in rimJ, a gene encoding the N-terminal acetyltransferase of ribosomal protein S5, were shown to disrupt this thermoregulatory response, allowing papBA transcription at low temperature. In this study, we created an in-frame deletion of rimJ. This deletion relieved the repressive effects not only of low temperature but also of rich (Luria-Bertani [LB]) medium and glucose on papBA transcription, indicating that RimJ modulates papBA transcription in response to multiple environmental stimuli. papI transcription was also shown to be regulated by RimJ. papBA transcription is also controlled by a phase variation mechanism. We demonstrated that the regulators necessary to establish a phase ON state--PapI, PapB, Dam, Lrp, and cyclic AMP-CAP-are still required for papBA transcription in a rimJ mutant strain. rimJ mutations increase the rate at which bacteria transition into the phase ON state, indicating that RimJ inhibits the phase OFF-->ON transition. A DeltarimJ hns651 mutant is viable on LB medium but not on minimal medium. This synthetic lethality, along with transcriptional analyses, indicates that RimJ and H-NS work through separate pathways to control papBA transcription. Mutations in rimJ do not greatly influence the transcription of the fan, daa, or fim operon, suggesting that RimJ may be a pap-specific regulator. Overexpression of rimJ under conditions repressive for papBA transcription complements the DeltarimJ mutation but has little effect on transcription under activating conditions, indicating that the ability of RimJ to regulate transcription is environmentally controlled.

Oral immunization of mice with a Salmonella vaccine expressing colonization factor antigen I (CFA/I) from enterotoxigenic Escherichia coli results in the rapid onset of interleukin-4 (IL-4) and IL-5 production, which explains the observed elevations in mucosal immunoglobulin A (IgA) and serum IgG1 antibodies. In contrast, oral immunization with the Salmonella vector does not result in the production of Th2-type cytokines. To begin to assess why such differences exist between the two strains, it should be noted that in vitro infection of RAW 264.7 macrophages resulted in the absence of nitric oxide (NO) production in cells infected with the Salmonella-CFA/I vaccine. This observation suggests differential proinflammatory cytokine production by these isogenic Salmonella strains. Upon measurement of proinflammatory cytokines, minimal to no tumor necrosis factor alpha (TNF-alpha), IL-1alpha, IL-1beta, or IL-6 was produced by Salmonella-CFA/I-infected RAW 264.7 or peritoneal macrophages, but production was greatly induced in Salmonella vector-infected macrophages. Only minute levels of IL-12 p70 were induced by Salmonella vector-infected macrophages, and none was induced by Salmonella-CFA/I-infected macrophages. The absence of IL-12 was not due to overt increases in production of either IL-12 p40 or IL-10. CFU measurements taken at 8 h postinfection showed no differences in colonization in RAW 264.7 cells infected with either Salmonella construct, but there were differences in peritoneal macrophages. However, after 24 h, the Salmonella vector strain colonized to a greater extent in RAW 264.7 cells than in peritoneal macrophages. Infection of RAW 264.7 cells or peritoneal macrophages with either Salmonella construct showed no difference in macrophage viabilities. This evidence shows that the expression of CFA/I fimbriae alters how macrophages recognize or process salmonellae and prevents the rapid onset of proinflammatory cytokines which is typical during Salmonella infections.

The pilin glycoprotein (PilE) is the main building block of the pilus of Neisseria gonorrhoeae (gonococcus [GC]). GC pilin is known to carry a disaccharide O-glycan, which has an alphaGal attached to the O-linked GlcNAc by a 1-3 glycosidic bond. In this report, we describe the cloning and characterization of the GC gene, pilus glycosyl transferase A (pgtA), which encodes the galactosyl transferase that catalyzes the synthesis of this Gal-GlcNAc bond of pilin glycan. A homopolymeric tract of Gs (poly-G) is present in the pgtA gene of many GC strains, and this pgtA with poly-G can undergo phase variation (Pv). However, in many other GC, pgtA lacks the poly-G and is expressed constitutively without Pv. Furthermore, by screening a large number of clinical isolates, a significant correlation was observed between the presence of poly-G in pgtA and the dissemination of GC infection. Poly-G was found in pgtA in all (24 out of 24) of the isolates from patients with disseminated gonococcal infection (DGI). In contrast, for the vast majority (20 out of 28) of GC isolated from uncomplicated gonorrhea (UG) patients, pgtA lacked the poly-G. These results indicate that Pv of pgtA is likely to be involved in the conversion of UG to DGI.

Synthesis of the plasminogen activator streptokinase (SK) by group A streptococci (GAS) has recently been shown to be subject to control by two two-component regulators, covRS (or csrRS) and fasBCA. In independent studies, response regulator CovR proved to act as the repressor, whereas FasA was found to act indirectly as the activator by controlling the expression of a stimulatory RNA, fasX. In an attempt at understanding the regulation of SK production in the human group C streptococcal (GCS) strain H46A, the strongest SK producer known yet, we provide here physical and functional evidence for the presence of the cov and fas systems in GCS as well and, using a mutational approach, compare the balance between their opposing actions in H46A and GAS strain NZ131. Sequence analysis combined with Southern hybridization revealed that the covRS and fasCAX operons are preserved at high levels of primary structure identity between the corresponding GAS and GCS genes, with the exception of fasB, encoding a second sensor kinase that is not a member of the GCS fas operon. This analysis also showed that wild-type H46A is actually a derepressed mutant for SK and streptolysin S (SLS) synthesis, carrying a K102 amber mutation in covR. Using cov and fas mutations in various combinations together with strain constructs allowing complementation in trans, we found that, in H46A, cov and fas contribute to approximately equal negative and positive extents, respectively, to constitutive SK and SLS activity. The amounts of SK paralleled the level of skc(H46A) transcription. The most profound difference between H46A and NZ131 regarding the relative activities of the cov and fas systems consisted in significantly higher activity of a functional CovR repressor in NZ131 than in H46A. In NZ131, CovR decreased SK activity in a Fas(+) background about sevenfold, compared to a 1.9-fold reduction of SK activity in H46A. Combined with the very short-lived nature of covR mRNA (decay rate, 1.39/min), such differences may contribute to strain-specific peculiarities of the expression of two prominent streptococcal virulence factors in response to environmental changes.

Isolates of Bordetella bronchiseptica associated with different animal hosts were analysed by fim3 and flaA genotyping by temporal temperature gradient gel electrophoresis (TTGE). All the isolates from cats (n = 138), dogs (n = 42) and pigs (n = 13) could be assigned to one of two fim3 and one of three flaA TTGE sequence types, respectively. Two avian isolates and a marmoset isolate exhibited novel fim3 sequence types. Dominant but different TTGE sequence types were apparent in isolates from dogs and pigs for both fim3 (87.5% and 100%, respectively, of isolates were the dominant type) and flaA (95% and 92%, respectively, of isolates were the dominant type). There was a wider distribution of TTGE sequence types amongst cat isolates. As both fimbriae and flagella have been implicated in adherence of bordetellae to host cells, sequence variations in fimbrial proteins and FlaA may have a role to play in host preferences.

We characterized 100 Escherichia coli urosepsis isolates from adult patients according to host compromise status by means of ribotyping, PCR phylogenetic grouping, and PCR detection of papG alleles and the virulence-related genes sfa/foc, fyuA, irp-2, aer, hly, cnf-1 and hra. We also tested these strains for copies of pap and hly and their direct physical linkage with other virulence genes in an attempt to look for pathogenicity islands (PAIs) described for the archetypal uropathogenic strains J96, CFT073, and 536. Most of the isolates belonged to E. coli phylogenetic groups B2 and D and bore papG allele II, aer, and fyuA/irp-2. papG allele II-bearing strains were more common in noncompromised patients, while papG allele-negative strains were significantly more frequent in compromised patients. Fifteen ribotypes were identified. The three archetypal strains harbored different ribotypes, and only one-third of our urosepsis strains were genetically related to one of the archetypal strains. Three and 18 strains harbored three and two copies of pap, respectively, and 5 strains harbored two copies of hly. papGIII was physically linked to hly, cnf-1, and hra (reported to be PAI II(J96)-like genetic elements) in 14% of the strains. The PAI II(J96)-like domain was inserted within pheR tRNA in 11 strains and near leuX tRNA in 3 strains. Moreover, the colocalized genes cnf-1, hra, and hly were physically linked to papGII in four strains and to no pap gene in three strains. papGII and hly (reported to be PAI I(CFT073)-like genetic elements) were physically linked in 16 strains, pointing to a PAI I(CFT073)-like domain. Three strains contained both a PAI II(J96)-like domain and a PAI I(CFTO73)-like domain. Forty-two strains harbored papGII but not hly, in keeping with the presence of a PAI II(CFT073)-like domain. Only one strain harbored a PAI I(536)-like domain (hly only), and none harbored a PAI I(J96)-like domain (papGI plus hly) or a PAI II(536)-like domain (papGIII plus hly). This study provides new data on the prevalence and variability of physical genetic linkage between pap and certain virulence-associated genes that are consistent with their colocalization on archetypal PAIs.

Xylitol is transported by Streptococcus mutans via a constitutive phosphoenolpyruvate:fructose phosphotransferase system (PTS) composed of a IIABC protein. Spontaneous xylitol-resistant strains are depleted in constitutive fructose-PTS activity, exhibit additional phenotypes, and are associated with the caries-preventive properties of xylitol. Polymerase chain-reactions and chromosome walking were used to clone the fxp operon that codes for the constitutive fructose/xylitol-PTS. The operon contained three open reading frames: fxpA, which coded for a putative regulatory protein of the deoxyribose repressor (DeoR) family, fxpB, which coded for a 1-phosphofructokinase, and fxpC, which coded for a IIABC protein of the fructose-PTS family. Northern blot analysis revealed that these genes were co-transcribed into a 4.4-kb mRNA even in the absence of fructose. Inactivation of the fxpC gene conferred resistance to xylitol, confirming its function. The fxp operon is also present in the genomes of other xylitol-sensitive streptococci, which could explain their sensitivity to xylitol.

The first step in the colonization of the human urinary tract by pathogenic Escherichia coli is the mannose-sensitive binding of FimH, the adhesin present at the tip of type 1 pili, to the bladder epithelium. We elucidated crystallographically the interactions of FimH with D-mannose. The unique site binding pocket occupied by D-mannose was probed using site-directed mutagenesis. All but one of the mutants examined had greatly diminished mannose-binding activity and had also lost the ability to bind human bladder cells. The binding activity of the mono-saccharide D-mannose was delineated from this of mannotriose (Man(alpha 1-3)[Man(alpha 1-6)]Man) by generating mutants that abolished D-mannose binding but retained mannotriose binding activity. Our structure/function analysis demonstrated that the binding of the monosaccharide alpha-D-mannose is the primary bladder cell receptor for uropathogenic E. coli and that this event requires a highly conserved FimH binding pocket. The residues in the FimH mannose-binding pocket were sequenced and found to be invariant in over 200 uropathogenic strains of E. coli. Only enterohaemorrhagic E. coli (EHEC) possess a sequence variation within the mannose-binding pocket of FimH, suggesting a naturally occurring mechanism of attenuation in EHEC bacteria that would prevent them from being targeted to the urinary tract.

The unicellular motile cyanobacterium Synechocystis sp. PCC 6803 exhibits phototactic motility that depends on the type IV-like thick pilus structure. By gene disruption analysis, we showed that a gene cluster of slr1041, slr1042, slr1043 and slr1044, whose predicted products are homologous to PatA, CheY, CheW and MCP, respectively, was more or less required for pilus assembly, motility and natural transformation competency with extraneous DNA. By sequence homology, the missing cheA-like gene in this cluster was identified as novel split genes, slr0073 and slr0322, at separate loci on the genome. This was confirmed by non-motile phenotype of their disruptants. Unique hyperpiliation was observed in the slr1042 and slr0073 disruptants, suggestive of their specific interaction with pilT1. The genes, thus identified as pil genes in this study, were designated pilG (slr1041), pilH (slr1042), pilI (slr1043), pilJ (slr1044), pilL-N (slr0073) and pilL-C (slr0322).

Agrobacterium tumefaciens T pili are long semi-rigid, flexuous filaments of 10 nm diameter that are primarily composed of T pilin cyclized protein subunits. The cyclic character of T pilin apparently confers a high level of structural stability on the T pilus. Purified T pili subjected to extreme environmental conditions such as acid and alkali, including glycerol remained relatively unaffected morphologically. T pili lost their semi-rigidity when subjected to high temperatures and high pH, and dissociated into donut shaped subunits when exposed to Triton X-100. Sodium dodecyl sulfate increased the uptake of uranyl acetate exposing a 2 nm wide lumen running the length of the T pilus filament.

The differential expression of virulence genes be-tween the two disease-causing biotypes of Vibrio cholerae, classical and El Tor, is primarily due to a single basepair change in the tcpPH promoter, which strongly influences the ability of the LysR regulator AphB to activate transcription in response to environmental conditions. We show here that this single basepair change influences virulence gene expression by dramatically altering the affinity of AphB for its recognition site in the tcpPH promoter. AphB binds greater than 10-fold more efficiently to a wild-type classical tcpPH promoter fragment with an A at -65 relative to a wild-type El Tor fragment that has a G at this position. As this single basepair change is located within the left arm of the LysR recognition motif (5'-TGCAA-N7-TTGCA), which extends from -69 to -53, a systematic mutagenesis of the other positions within this site was carried out to assess their influence on AphB binding in vitro and transcriptional activation in vivo. This analysis revealed that the left and right arms of the interrupted dyad display a high degree of symmetry with respect to their role in AphB binding. The right promoter proximal arm also plays a role in transcriptional activation that is distinct from its role in AphB binding. A second AphB binding site (5'-TGCAA-N7-TGTCA) was identified upstream of the aphB gene itself, which extends from +17 to +33 relative to the start of transcription and functions in autorepression. Although the sequences of the AphB binding sites at the tcpPH and aphB promoters are highly conserved, important differences exist in the way that AphB functions at each of these sites.

LT and ST are the main enterotoxins of enterotoxigenic Escherichia coli (ETEC) found in clinical isolates, and CS3 (the common antigen in the CFA/II family of fimbrial antigens) is one of the most prevalent antigens of colonization factors. The genetic determinants encoding CS3 and LT-B/ST fusion toxin were manipulated so that these important antigens are expressed simultaneously in attenuated Salmonella typhimurium oral vaccine strain X4072. These antigens produced by X4072 (pXZL88) could be recognized with monospecific CS3, LT or ST antibodies respectively. The specific antibodies against CS3, LT and ST could be detected. In the sera of immunized mice via oral route with the live bacteria. Significantly, the antibody to ST was able to neutralize the biological activity of native ST. This prototype construct may be proved to be useful in investigating the live vector approach to immunoprophylaxis of ETEC diarrhea disease.

Fimbrial adhesins mediate the attachment of pathogenic Escherichia coli to various host tissues leading to the development of disease. The Dr hemagglutinin and F1845 fimbriae belong to the Dr family of adhesins, which is associated with urinary tract infections and diarrheal disease. These adhesins bind to the Dr(a) blood-group antigen present on decay-accelerating factor (DAF). The Dr hemagglutinin is unique in this family since it also binds to type IV collagen and its binding is inhibited by the presence of chloramphenicol. We have purified the major structural subunits of Dr and F1845 fimbriae, DraE and DaaE, as fusions to maltose-binding protein and to oligohistidine tags and examined their binding to erythrocytes, Chinese hamster ovary cell transfectants expressing DAF, and a DAF fusion protein. The DraE and DaaE fusion proteins bind to the DAF receptor in a specific manner resembling the distinct phenotypes of the corresponding Dr and F1845 fimbriae. In contrast to binding studies with the DAF receptor, the DraE fusion proteins did not bind to type IV collagen.

The nucleotide sequences of five open reading frames within the Vibrio cholerae NAGV14 type IV pilus gene cluster were determined. The genes showed high homology to the mannose-sensitive hemagglutinin (MSHA) pilus genes mshB, mshC, mshD, mshO and mshP. PCR analysis showed that a MSHA-like gene cluster is highly conserved among different V. cholerae strains, with the exception of the previously reported major pilin subunit. Recombinant MshB and MshO proteins were purified and specific antiserum was raised to each of them. Western blotting analyses showed that these antisera reacted with purified NAGV14 and MSHA pili. The results suggested that MshB and MshO are minor components of the pilus fiber. Although there was no cross-reaction between the major pilin subunits of NAGV14 and MSHA pili, minor components seemed to be highly homologous and immunologically cross-reactive.

Virulence of enterotoxicogenic Escherichia coli is mediated by rodlike, rigid, highly hydrophobic proteins designated fimbriae or colonization factors (CFs). More than 20 different colonization factors have been described so far using predominantly immunological and genetic methods. To characterize these hydrophobic proteins by liquid chromatography-mass spectrometry (LC-MS), different methodologies were explored. A novel LC-MS method was developed using hexafluoroisopropanol to maintain the hydrophobic proteins in solution. In addition, these proteins were digested with cyanogen bromide and peptide mapping by LC-MS was established. This technique was particularly useful in identification of closely related CFs. Both LC-MS and peptide mapping methodologies were found to be useful in characterizing highly hydrophobic CFs of E. coli. To search for molecular weights of mature proteins in the National Center for Biotechnology Information (NCBI) database, a new feature was developed and its applicability tested. The identification of a class of pathogenic virulence proteins, either intact or digested, is possible with molecular weight database searching.

The initial attachment of Neisseria meningitidis to the target cell surface appears to be largely pilus depend-ent in capsulated bacteria. Intimate adhesion subsequently occurs to permit colonization. We recently reported that insertional inactivation of the crgA gene, which encodes a transcriptional regulator belonging to the LysR family, decreased meningococcal adhesion to epithelial cells and abolished intimate adhesion. In this report, we analyse expression of the pilE and sia genes, which are involved in the biosynthesis of pili and capsule respectively, during bacteria-host cell interactions. Western blotting, transcriptional fusion and reverse transcriptase polymerase chain reaction (RT-PCR) analysis showed that the expression of these genes was downregulated during intimate adhesion. DNA-binding assays, footprinting and RT-PCR analysis indicated that this downregulation was directly mediated by the CrgA protein. The pilE and sia promoters were found to have a CrgA binding motif in common. These results strongly suggest that N. meningitidis displays an adaptive response upon cell contact. CrgA may play a central regulatory role in meningococcal adhesion, particularly in switching from initial to intimate adhesion by downregulating the bacterial surface structures that hinder this adhesion.

A comparative study was performed to determine the effects of pH, osmolarity, and human urine on the transcription of several fim genes, as well as the overall expression of type 1 pili. Several fim-lacZYA fusions were constructed on single-copy plasmids to test a range of pHs and a range of osmolarities. Growth in acidic medium slightly reduced expression from all of the fim promoters (fimA, fimB, and fimE). Increased osmolarity in neutral-pH medium repressed fimA and fimB transcription by approximately 50% when 400 mM NaCl was used and nearly threefold when 800 mM NaCl was used, whereas fimE transcription rose slightly as the osmolarity increased. This effect was more pronounced in high-osmolarity acidic media; fimB and fimA expression decreased fivefold in growth media containing 800 mM NaCl compared to expression in growth media without added NaCl. Moreover, fimE expression doubled under the same high-osmolarity conditions compared to expression in a low-osmolarity acidic environment. When a fimB-lacZ or fimE-lacZ fusion was inserted into the chromosome of strain AAEC189, fimE expression changed slightly as the osmolarity increased, but fimB expression decreased by 50% in a low-pH high-osmolarity environment. When strain AAEC189 with either a plasmid-borne fimB-lacZ fusion or a plasmid-borne fimE-lacZ fusion was grown in human urine, similar changes in the levels of fimB and fimE expression were observed. Limiting-dilution reverse transcription-PCR confirmed that these changes in fim expression occurred in clinical isolates of uropathogenic Escherichia coli grown in media with different pHs and different osmolarities. Furthermore, the invertible switch region in uropathogenic strain NU149 shifted from favoring the phase-on position in a neutral-pH low-osmolarity environment to favoring the phase-off position in a low-pH high-osmolarity environment. Results obtained with an ompR mutant strain demonstrated that fimB expression was derepressed and that OmpR may neutralize repression by an acid response regulator of fimE expression in a low-pH environment. In addition, H-NS was verified to be important in regulation of fimB, but it had only a slight effect on fimE under the specific pH and osmotic growth conditions tested. Enzyme immunoassays with anti-type 1 pilus antibody and hemagglutination assays showed that fewer type 1 pili were detected with cells in a low-pH high-osmolarity environment. Together, these observations demonstrate that a combination of low pH and high osmolarity regulates the transcription of fim genes, which favors a shift in the invertible element to the phase-off orientation and a loss of type 1 pilus expression. Taken together, our data suggest that the environmental cues that we tested may regulate expression of type 1 pili in specific in vivo niches, such as murine kidneys and possibly human kidneys.

The paradigm of homologous recombination comes from Escherichia coli, where the genes involved have been segregated into pathways. In the human pathogen Neisseria gonorrhoeae (the gonococcus), the pathways of homologous recombination are being delineated. To investigate the roles of the gonococcal recN and recJ genes in the recombination-based processes of the gonococcus, these genes were inactivated in the N. gonorrhoeae strain FA1090. We report that both recN and recJ loss-of-function mutants show decreased DNA repair ability. In addition, the recJ mutant was decreased in pilus-dependent colony morphology variation frequency but not DNA transformation efficiency, while the recN mutant was decreased in DNA transformation efficiency but not pilus-dependent variation frequency. We were able to complement all of these deficiencies by supplying an ectopic functional copy of either recJ or recN at an irrelevant locus. These results describe the role of recJ and recN in the recombination-dependent processes of the gonococcus and further define the pathways of homologous recombination in this organism.

Pseudomonas aeruginosa is an opportunistic pathogen that may cause severe infections in humans and other vertebrates. In addition, a human clinical isolate of P. aeruginosa, strain PA14, also causes disease in a variety of nonvertebrate hosts, including plants, Caenorhabditis elegans, and the greater wax moth, Galleria mellonella. This has led to the development of a multihost pathogenesis system in which plants, nematodes, and insects have been used as adjuncts to animal models for the identification of P. aeruginosa virulence factors. Another approach to identifying virulence genes in bacteria is to take advantage of the natural differences in pathogenicity between isolates of the same species and to use a subtractive hybridization technique to recover relevant genomic differences. The sequenced strain of P. aeruginosa, strain PAO1, has substantial differences in virulence from strain PA14 in several of the multihost models of pathogenicity, and we have utilized the technique of representational difference analysis (RDA) to directly identify genomic differences between P. aeruginosa strains PA14 and PAO1. We have found that the pilC, pilA, and uvrD genes in strain PA14 differ substantially from their counterparts in strain PAO1. In addition, we have recovered a gene homologous to the ybtQ gene from Yersinia, which is specifically present in strain PA14 but absent in strain PAO1. Mutation of the ybtQ homolog in P. aeruginosa strain PA14 significantly attenuates the virulence of this strain in both G. mellonella and a burned mouse model of sepsis to levels comparable to those seen with PAO1. This suggests that the increased virulence of P. aeruginosa strain PA14 compared to PAO1 may relate to specific genomic differences identifiable by RDA.

The longus type IV pilus structural gene (lngA) was sought among 217 clinical enterotoxigenic Escherichia coli (ETEC) strains isolated in Argentina. lngA was present in 20.7% of the isolates and was highly associated with ETEC producing heat-stable toxin and the most common colonization factors. The prevalence of longus among ETEC strains in Argentina was comparable to that of colonization factor antigen I (CFA/I), CFA/II, and CFA/IV in other regions of the world.

We have previously shown that the pilL, pilN, pilQ, pilS, pilU, and pilV genes of plasmid R64 encode outer membrane lipoprotein, secretin, cytoplasmic ATPase, major pilin, prepilin peptidase, and minor pilin, respectively, which are required for thin-pilus formation. In this work, we characterized the products of the remaining essential genes, pilK, pilM, pilO, pilP, pilR, and pilT, with regard to their localization and processing. Overexpression systems containing pilM, pilO, and pilP genes fused with N-terminal glutathione S-transferase (GST) or a His tag were constructed. Overproduced proteins were purified and used to raise specific antibodies. Localization of PilM, PilO, and PilP proteins was performed by Western blot analysis with anti-GST-PilM, anti-PilO, and anti-PilP antibodies, respectively. The pilK, pilR, and pilT products were produced with a C-terminal His tag and then detected by anti-His tag antibody. Subcellular fractionation experiments with Escherichia coli cells producing R64 thin pili revealed that PilK, PilM, and PilR are inner membrane proteins, and PilP and PilT are periplasmic proteins. PilO protein was localized to the outer membrane in the presence of other Pil proteins, whereas it was localized to the cytoplasm in the absence of these proteins. Furthermore, the cleavage site of PilP protein was determined by N-terminal amino acid sequencing of purified mature PilP protein. We predict that PilK, PilM, PilO, PilP, and PilT proteins function as the components of the pilin transport apparatus and thin-pilus basal body.

The FimA protein of Streptococcus parasanguis is a virulence factor in the rat model of endocarditis, and immunization with FimA protects rats against homologous bacterial challenge. Because FimA-like proteins are widespread among the oral streptococci, the leading cause of native valve endocarditis, we evaluated the ability of this vaccinogen to protect rats when challenged by other streptococcal species. Here we report that FimA vaccination produced antibodies that cross-reacted with and protected against challenge by the oral streptococci S. mitis, S. mutans, and S. salivarius. FimA thus has promise as a vaccinogen to control infective endocarditis caused by oral streptococci.

Streptococcus parasanguis is a primary colonizer of the tooth surface and plays a pivotal role in the formation of dental plaque. The fimbriae of S. parasanguis are important in mediating adhesion to saliva-coated hydroxylapatite (SHA), an in vitro tooth adhesion model. The Fap1 adhesin has been identified as the major fimbrial subunit, and recent studies suggest that Fap1 is a glycoprotein. Monosaccharide analysis of Fap1 purified from the culture supernatant of S. parasanguis indicated the presence of rhamnose, glucose, galactose, N-acetylglucosamine and N-acetylgalactosamine. A glycopeptide moiety was isolated from a pronase digest of Fap1 and purified by immunoaffinity chromatography. The monosaccharide composition of the purified glycopeptide was similar to that of the intact molecule. The functionality of the glycan moiety was determined using monoclonal antibodies (MAbs) specific for the intact Fap1 glycoprotein. These antibodies were grouped into two categories based on their ability to block adhesion of S. parasanguis to SHA and their corresponding specificity for either protein or glycan epitopes of the Fap1 protein. 'Non-blocking' MAb epitopes were mapped to unique protein sequences in the N-terminus of the Fap1 protein using non-glycosylated recombinant Fap1 proteins (rFap1 and drFap1) expressed in Escherichia coli. In contrast, the 'blocking' antibodies did not bind to the recombinant Fap1 proteins, and were effectively competed by the binding to the purified glycopeptide. These data suggest that the 'blocking' antibodies are specific for the glycan moiety and that the adhesion of S. parasanguis is mediated by sugar residues associated with Fap1.

Fimbriae of Porphyromonas gingivalis, a periodontopathogen, play an important role in its adhesion to and invasion of host cells. The fimA genes encoding fimbrillin (FimA), a subunit protein of fimbriae, have been classified into five types, types I to V, based on nucleotide sequences. We previously reported that P. gingivalis with type II fimA was strongly associated with adult periodontitis. In the present study, we compared the abilities of recombinant FimA (rFimA) types I to V to adhere to and invade human gingival fibroblasts (HGF) and a human epithelial cell line (HEp-2 cells) by using rFimA-conjugated microspheres (rFimA-MS). There were no significant differences in the abilities of the rFimA-MS to adhere to HGF; however, the adhesion of type II rFimA-MS to HEp-2 cells was significantly greater than those of other types of rFimA-MS. We also observed that type II rFimA-MS invaded epithelial cells and accumulated around the nuclei. These adhesion and invasion characteristics were eliminated by the addition of antibodies to type II rFimA and alpha5beta1-integrin. In contrast, Arg-Gly-Asp-Ser peptide and a synthetic peptide of proline-rich protein C had negligible inhibitory effects. Furthermore, P. gingivalis strain HW24D1 with type II fimA adhered to cells and invaded them more than strains with other fimA genotypes. These results suggest that type II FimA can bind to epithelial cells most efficiently through specific host receptors.

Co-ordinate expression of many virulence genes in the human pathogen Vibrio cholerae is under the direct control of the ToxT protein, including genes whose products are required for the biogenesis of the toxin-co-regulated pilus (TCP) and cholera toxin (CTX). This work examined interactions between ToxT and the promoters of ctx and tcpA genes. We found that a minimum of three direct repeats of the sequence TTTTGAT is required for ToxT-dependent activation of the ctx promoter, and that the region from -85 to -41 of the tcpA promoter contains elements that are responsive to ToxT-dependent activation. The role of H-NS in transcription of ctx and tcpA was also analysed. The level of activation of ctx-lacZ in an E. coli hns- strain was greatly increased even in the absence of ToxT, and was further enhanced in the presence of ToxT. In contrast, H-NS plays a lesser role in the regulation of the tcpA promoter. Electrophoretic mobility shift assays demonstrated that 6x His-tagged ToxT directly, and specifically, interacts with both the ctx and tcpA promoters. DNase I footprinting analysis suggests that there may be two ToxT binding sites with different affinity in the ctx promoter and that ToxT binds to -84 to -41 of the tcpA promoter. In vitro transcription experiments demonstrated that ToxT alone is able to activate transcription from both promoters. We hypothesize that under conditions appropriate for ToxT-dependent gene expression, ToxT binds to AT-rich promoters that may have a specific secondary conformation, displaces H-NS and stimulates RNA polymerase resulting in transcription activation.

Porphyromonas gingivalis is an oral pathogen that has recently been associated with chronic inflammatory diseases such as atherosclerosis. The strength of the epidemiological associations of P. gingivalis with atherosclerosis can be increased by the demonstration that P. gingivalis can initiate and sustain growth in human vascular cells. We previously established that P. gingivalis can invade aortic, heart, and human umbilical vein endothelial cells (HUVEC), that fimbriae are required for invasion of endothelial cells, and that fimbrillin peptides can induce the expression of the chemokines interleukin 8 and monocyte chemotactic protein. In this study, we examined the expression of surface-associated cell adhesion molecules on endothelial cells in response to P. gingivalis infection by fluorescence-activated cell sorting FACS analysis and confocal microscopy. Coculture of HUVEC with P. gingivalis strain 381 or A7436 resulted in the induction in the expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and P- and E-selectins, which was maximal at 48 h postinfection. In contrast, we did not observe induction of ICAM-1, VCAM-1, or P- or E-selectin expression in HUVEC cultured with the noninvasive P. gingivalis fimA mutant DPG3 or when P. gingivalis was incubated with fimbrillin peptide-specific anti-sera prior to the addition to HUVEC. Furthermore, the addition of a peptide corresponding to the N-terminal domain of fimbrillin to HUVEC resulted in an increase in ICAM-1, VCAM-1, and P- and E-selectins, which was maximal at 48 h and similar to that observed for live P. gingivalis. Treatment of P. gingivalis-infected HUVEC with cytochalsin D, which prevented P. gingivalis invasion, also resulted in the inhibition of ICAM-1, VCAM-1, or P- and E-selectin expression. Taken together, these results indicate that active P. gingivalis invasion of HUVEC mediated via the major fimbriae stimulates surface-associated cell adhesion molecule expression. Stimulation of adhesion molecules involved in the recruitment of leukocytes to sites of inflammation by P. gingivalis may play a role in the pathogenesis of systemic inflammatory diseases associated with this microorganism, including atherosclerosis.

Recent cross-sectional and prospective epidemiological studies have demonstrated an association between periodontal disease and atherosclerosis and human coronary heart disease. Previously, we have established that the periodontal pathogen Porphyromonas gingivalis is capable of invading aortic, heart, and human umbilical vein endothelial cells (HUVEC). Since atherosclerosis is a chronic inflammatory response initiated at the vascular wall, interactions of P. gingivalis with endothelial cells and the subsequent host cell response to infection may be important in the pathogenesis of atherosclerosis. In this study we examined the consequences of P. gingivalis infection of HUVEC on the expression of the chemokines interleukin-8 (IL-8) and monocyte chemotactic protein 1 (MCP-1). HUVEC were found to constitutively produce low levels of IL-8 and MCP-1. The addition of P. gingivalis fimbrillin-specific peptides, lipopolysaccharides (LPS), or heat-killed whole cell preparations to HUVEC stimulated modest IL-8 and MCP-1 responses. In contrast, coculture of HUVEC with live P. gingivalis strain A7436, 33277, or 381 abolished the IL-8 and MCP-1 responses. Inhibition of IL-8 and MCP-1 production was not dependent on bacterial adherence since similar results were obtained with the nonadherent P. gingivalis fimA mutant DPG3 or when P. gingivalis was preincubated with fimbrillin peptide antisera prior to the addition to HUVEC. Furthermore, treatment of P. gingivalis-infected HUVEC with cytochalsin D, which prevented P. gingivalis invasion, also abolished the constitutive IL-8 and MCP-1 responses. Treatment of HUVEC with E. coli LPS stimulated robust IL-8 and MCP-1 responses that were abolished when stimulated cells were cocultured with live P. gingivalis. Analysis of P. gingivalis-infected HUVEC cultures by an RNase protection assay revealed an increase in the IL-8 transcript relative to uninfected HUVEC. Pretreatment of P. gingivalis with protease inhibitors prior to the addition to HUVEC prevented the inhibition of IL-8 and MCP-1 production in P. gingivalis-infected HUVEC, indicating that the inhibition was proteolytically mediated. Coculture of HUVEC with a P. gingivalis mutant deficient in lysine-specific cysteine proteinase (gingipain K [Kgp]) resulted in an increase in both IL-8 transcription and protein expression relative to that observed in HUVEC cocultured with the P. gingivalis wild-type strain. These results indicate that P. gingivalis can temporally modulate the chemokine response in endothelial cells through both fimbriae and gingipain-mediated mechanisms.

Twenty-five Vibrio strains belonging to nine different species, isolated in common mussels, were examined for the presence of different virulence genes: ctxA, tcpA, toxR, toxS, ace, zot and vpi previously found in pathogenic Vibrio cholerae strains. Our results suggest that there is a wide dissemination of Vibrio cholerae virulence genes among the various Vibrio species tested. This finding raises the question of whether a different approach should be taken to study "environmental" Vibrio strains.

VirB2 propilin is processed by the removal of a 47-amino-acid signal peptide to generate a 74-amino-acid peptide product in both Escherichia coli and Agrobacterium tumefaciens. The cleaved VirB2 protein is further cyclized to form the T pilin in A. tumefaciens but not in E. coli. Mutations in the signal peptidase cleavage sequence of VirB2 propilin cause the formation of aberrant T pilin and also severely attenuate virulence. No T pilus was observed in these mutants. The potential role of the exact VirB2 propilin cleavage and cyclization in T pilus biogenesis and virulence is discussed.

Type IV pili (Tfp) of Neisseria gonorrhoeae, the Gram-negative etiologic agent of gonorrhea, facilitate colonization of the human host. Tfp are assumed to play a key role in the initial adherence to human epithelial cells by virtue of the associated adhesin protein PilC. To examine the structural and functional basis for adherence in more detail, we identified potential genes encoding polypeptides sharing structural similarities to PilE (the Tfp subunit) within the N. gonorrhoeae genome sequence database. We show here that a fiber subunit-like protein, termed PilV, is essential to organelle-associated adherence but dispensable for Tfp biogenesis and other pilus-related phenotypes, including autoagglutination, competence for natural transformation, and twitching motility. The adherence defect in pilV mutants cannot be attributed to reduced levels of piliation, defects in fiber anchoring to the bacterial cell surface, or to unstable pilus expression related to organelle retraction. PilV is expressed at low levels relative to PilE and copurifies with Tfp fibers in a PilC-dependent fashion. Purified Tfp from pilV mutants contain PilC adhesin at reduced levels. Taken together, these data support a model in which PilV functions in adherence by promoting the functional display of PilC in the context of the pilus fiber.

Rats were dosed for 6 days with purified SEF 21 fimbriae of Salmonella enterica serovar Enteritidis 10360. The levels of fimbriae in gut contents associated with tissues and in the faeces were quantified by direct non-competitive ELISA. SEF 21 was distributed throughout the gut. The majority was found in the large intestine where it was primarily in the luminal contents. In contrast, a high proportion of SEF 21 detected in the ileum, the main site of salmonella colonisation and invasion, was tissue-bound. Thus, purified SEF 21 survived intestinal passage and associated with the stomach and gastrointestinal tract in a pattern similar to that found with whole Salmonella cells.

Gram-negative bacteria have surface appendages that assemble via different secretion machineries. Recently, new experimental approaches have contributed to a better understanding of the molecular mechanisms of flagellar and pilus assembly, and protein secretion. These findings can be applied to plant pathogenic bacteria, which probably transfer effector proteins directly into their eukaryotic host cells. Here, it is suggested that assembly of Hrp pili occurs in the periplasm and that unfolded effector proteins attach to pilins within the pili, thus effecting protein translocation. A two-domain structure for the HrpA pilin from Pseudomonas syringae is also predicted.

The epidemic potential of 113 V. cholerae eltor strains of different origin was determined with new diagnostic cholera bacteriophages eltor ctx+ and ctx-, as well as the test for hemolytic activity. Of these strains 50 were epidemically safe and 51 were epidemically dangerous, while the epidemic potential of 12 other strains could not be detected. Determination of genes ctxA, tcpA and toxR in the strains under study by means of the polymerase chain reaction (PCR) revealed that epidemically dangerous strains carried the whole set of the above genes in 92.2% of cases. 98.0% of epidemically safe cultures were lacking either gene ctxA, or genes ctxA and tcpA, or genes ctxA, tcpA and toxR, which confirmed their incapacity to cause cholera. The results of the differentiation of the cultures with new diagnostic cholera phages coincided with the results of PCR in 90% of cases. The most complete and reliable evaluation of the epidemic potential of individual vibrio isolates may be obtained using the two compared methods. The amplification test system gives more information when isolates with unclear epidemic potential are analyzed.

A subset of nontypeable Haemophilus influenzae (NTHI) biotype IV isolates from the human genital tract or from infected newborn infants forms a cryptic genospecies characterized by, among other features, the presence of peritrichous pili. The objective of this study was to determine the similarity of these pili to hemagglutinating, HifA- and HifE-containing pili expressed by respiratory H. influenzae isolates. For this analysis, the presence of hifA and hifE and their gene products in NTHI biotype IV strains was assessed, the binding of H. influenzae biotype IV strains to human epithelial cells was characterized, possible genital tissue tropism of these isolates was explored, and the role of HifA- and HifE-possessing pili in the adhesion of NTHI biotype IV strains to human epithelial cells was determined. None of the six biotype IV NTHI isolates tested agglutinated human red blood cells, nor could they be enriched for hemagglutinating variants. Although hifA, which encodes the major structural subunit of hemagglutinating pili, and hifE, which encodes the tip adhesin of hemagglutinating pili, were detected by PCR from six and five, respectively, of the six biotype IV strains tested, neither HifA nor HifE (the gene products of hifA and hifE) were detected in any of these strains by Western blot analysis using antisera that recognize HifA and HifE of respiratory strains. Transmission electron microscopy showed no surface pili on the two biotype IV H. influenzae isolates examined; strain 4162 containing an insertional mutation in hifA also showed no surface pili, whereas strain 1595 containing an insertional mutation in hifB showed pilus-like structures that were shorter and thicker than hemagglutinating pili of the respiratory strains AAr176 and M43. In enzyme-linked immunosorbent assays, biotype IV strains adhered to 16HBE14o(-) and HEp-2 cells of respiratory origin as well as to ME180 and HeLa cells of genital origin. This adherence was not pilus specific, however, as GM-1, a known pilus receptor analog, did not inhibit binding of biotype IV strains to ME180, HEp-2, or HeLa cells, and GM-1 inhibition of binding to 16HBE14o(-) cells did not correlate with the presence of hifE. While both nonpiliated variants and hifA and hifB (encoding the pilus chaperone) mutants of respiratory strain AAr176 showed reduced binding (64 to 87% of that of piliated AAr176) to 16HBE14o(-) and ME180 cells, hifA and hifB mutants of the biotype IV strains showed minimal reduction in binding to these cell lines (91 to 98% of that of wild-type strains). Thus, although biotype IV H. influenzae isolates of the cryptic genospecies possess the genes that code for HifA- and HifE-containing hemagglutinating pili, epithelial cell adherence exhibited by these strains is not mediated by expression of hemagglutinating pili.

The binding of uropathogenic Escherichia coli to the urothelial surface is a crucial initial event for establishing urinary tract infection because it allows the bacteria to gain a foothold on the urothelial surface, thus preventing them from being removed by micturition. In addition, it triggers bacterial invasion as well as host urothelial defense. This binding is mediated by the FimH adhesin located at the tip of the bacterial type 1-fimbrium, a filamentous attachment apparatus, and its urothelial receptor. We have prepared a biotinylated, recombinant FimH-FimC adhesin:chaperone complex and used it to identify its mouse urothelial receptor. The FimH-FimC complex binds specifically to a single 24 kDa major mouse urothelial plaque protein, which we identified as uroplakin Ia by mass spectrometry, cDNA cloning and immunoreactivity. The terminal mannosyl moieties on Asn-169 of uroplakin Ia are responsible for FimH as well as concanavalin A binding. Although FimH binds to uroplakin Ia with only moderate strength (K(d) approximately 100 nM between pH 4 and 9), the binding between multiple fimbriae of a bacterium and the crystalline array of polymerized uroplakin receptors should achieve high avidity and stable bacterial attachment. The FimH-FimC complex binds preferentially to the mouse urothelial umbrella cells in a pattern similar to uroplakin staining. Our results indicate that the structurally related uroplakins Ia and Ib are glycosylated differently, that uroplakin Ia serves as the urothelial receptor for the type 1-fimbriated E. coli, and that the binding of uropathogenic bacteria to uroplakin Ia may play a key role in mediating the urothelial responses to bacterial attachment.

Salmonella enterica serovar Enteritidis is a leading cause of food poisoning in the USA and Europe. Although Salmonella serovars share many fimbrial operons, a few fimbriae are limited to specific Samonella serovars. SEF14 fimbriae are restricted to group D Salmonella and the genes encoding this virulence factor were acquired relatively recently. Genomic, genetic and gene expression studies have been integrated to investigate the ancestry, regulation and expression of the sef genes. Genomic comparisons of the Salmonella serovars sequenced revealed that the sef operon is inserted in leuX in Salmonella Enteritidis, Salmonella Paratyphi and Salmonella Typhi, and revealed the presence of a previously unidentified 25 kb pathogenicity island in Salmonella Typhimurium at this location. Salmonella Enteritidis contains a region of homology between the Salmonella virulence plasmid and the chromosome downstream of the sef operon. The sef operon itself consists of four co-transcribed genes, sefABCD, and adjacent to sefD there is an AraC-like transcriptional activator that is required for expression of the sef genes. Expression of the sef genes was optimal during growth in late exponential phase and was repressed during stationary phase. The regulation was coordinated by the RpoS sigma factor.

Leukotoxin produced by Mannheimia (Pasteurella) haemolytica is an important virulence factor in shipping fever pneumonia in feedlot cattle and is a critical protective antigen. In this study, the immune response to a chimeric protein generated by combining a gene fragment encoding neutralizing epitopes of M. haemolytica leukotoxin and a fimbrial protein gene (fim N) from Bordetella bronchiseptica was evaluated. The recombinant gene was cloned in a bacterial expression vector under the control of the tac promoter and expressed as a fusion protein with glutathione-S-transferase (GST) in Escherichia coli. Immunization of mice with the recombinant protein, GST-LTXFIM elicited a significantly stronger anti-leukotoxin antibody response than comparable immunizations with GST-LTX fusion proteins lacking FIM N. The GST-LTXFIM was also more stable than GST-LTX during storage at -80 degrees C, thus alleviating a stability problem inherent to leukotoxin. This chimeric protein may be a candidate for inclusion in new generation vaccines against shipping fever pneumonia.

In this study, a total of 118 Escherichia coli strains isolated from dogs (93) and cats (25) with urinary tract infection (UTI) were tested in a multiplex polymerase chain reaction for the presence of adhesin-encoding genes (pap, sfa, and afa), hemolysin encoding genes (hly), cytotoxic necrotizing factor 1 (cnf1) and aerobactin (aer) genes. Virulence gene frequencies detected in those isolates which had been randomly collected (68 canine strains) were: 43% pap, 57% sfa, 1% afa, 44% hly, 41% cnf1 and 34% aer. These frequencies were much higher in the remaining 50 hemolytic strains of either cat or dog origin. Virulence factor associations in the 80 hemolytic strains studied revealed that 50/80 simultaneously had two adhesin genes (pap and sfa) and two cytotoxin genes (hly and cnf1), and 15/80 in addition had the aer gene. The major structural subunit and antigenic determinant of P fimbriae of uropathogenic E. coli is PapA. Polymorphism in this subunit was studied by an F antigen-specific papA allele polymerase chain reaction in 51 canine and 22 feline pap positive E. coli strains. The most prevalent canine papA alleles were F10 (39%), F15 (37%) and F12 (35%). In feline strains F15 (50%) was more frequent, other allele frequencies were F12 (45%), F14 and F10 (27%) and F16 (23%). Only nine canine and two feline strains were negative for one of the 11 serologically defined F types of P fimbriae. Three copies of the pap operon were found in 16/51 canine and 9/22 feline UTI E. coli pap positive strains. In this study, we show that a particular combination of virulence genes appears with high frequency in dog and cat urinary tract E. coli strains (pap, sfa, hly, and cnf1). In spite of the more frequent presence of F10, F12 and F15 papA alleles in this virulence gene combination, the occurrence of different papA alleles in strains where up to three copies of the pap operon are present accounts for the observed P fimbriae diversity.

The phylogenetic distributions of multiple putative virulence factors (VFs) and papA (P fimbrial structural subunit) alleles among 182 Escherichia coli blood isolates from patients with diverse-source bacteremia were defined. Phylogenetic correspondence among these strains, the E. coli Reference (ECOR) collection, and other collections of extraintestinal pathogenic E. coli (ExPEC) was assessed. Although among the 182 bacteremia isolates phylogenetic group B2 predominated, exhibited the greatest concentration of individual VFs, and contained the largest number of familiar virulent clones, other phylogenetic groups exhibited greater concentrations of certain VFs than did group B2 and included several additional virulent clones. Certain of the newly detected VF genes, e.g., fyuA (yersiniabactin; 76%) and focG (F1C fimbriae; 25%), were as prevalent or more prevalent than their more familiar traditional counterparts, e.g., iut (aerobactin; 57%) and sfaS (S fimbriae; 14%), thus possibly offering additional useful targets for preventive interventions. Considerable diversity of VF profiles was observed at every level within the phylogenetic tree, including even within individual lineages. This suggested that many different pathways can lead to extraintestinal virulence in E. coli and that the evolution of ExPEC, which involves extensive horizontal transmission of VFs and continuous remodeling of pathogenicity-associated islands, is a highly active, ongoing process.

The assembly of pilus colonization factor antigen III (CFA/III) of enterotoxigenic Escherichia coli (ETEC) requires the processing of CFA/III major pilin (CofA) by a prepilin peptidase (CofP), similar to other type IV pilus formation systems. CofA is produced initially as a 26.5-kDa preform pilin (prepilin) and then processed to a 20.5-kDa mature pilin by CofP which is predicted to be localized in the inner membrane. In the present experiment, we determined the nucleotide sequence of the whole region for CFA/III formation and identified a cluster of 14 genes, including cofA and cofP. Several proteins encoded by cof genes were similar to previously described proteins, such as the toxin-coregulated pili of Vibrio cholerae and the bundle-forming pili of enteropathogenic E. coli. The G+C content of the cof gene cluster was 37%, which was significantly lower than the average for the E. coli genome (50%). The introduction of a recombinant plasmid containing the cof gene cluster into the E. coli K-12 strain conferred CFA/III biogenesis and the ability of adhesion to the human colon carcinoma cell line Caco-2. This is the first report of a complete nucleotide sequence of the type IV pili found in human ETEC, and our results provide a useful model for studying the molecular mechanism of CFA/III biogenesis and the role of CFA/III in ETEC infection.