Piliated Strains Research Articles

Animal Protection and Structural Studies of a Consensus Sequence Vaccine Targeting the Receptor Binding Domain of the Type IV Pilus of Pseudomonas aeruginosa

One of the main obstacles in the development of a vaccine against Pseudomonas aeruginosa is the requirement that it is protective against a wide range of virulent strains. We have developed a synthetic-peptide consensus-sequence vaccine (Cs1) that targets the host receptor-binding domain (RBD) of the type IV pilus of P. aeruginosa. Here, we show that this vaccine provides increased protection against challenge by the four piliated strains that we have examined (PAK, PAO, KB7 and P1) in the A.BY/SnJ mouse model of acute P. aeruginosa infection. To further characterize the consensus sequence, we engineered Cs1 into the PAK monomeric pilin protein and determined the crystal structure of the chimeric Cs1 pilin to 1.35 Å resolution. The substitutions (T130K and E135P) used to create Cs1 do not disrupt the conserved backbone conformation of the pilin RBD. In fact, based on the Cs1 pilin structure, we hypothesize that the E135P substitution bolsters the conserved backbone conformation and may partially explain the immunological activity of Cs1. Structural analysis of Cs1, PAK and K122-4 pilins reveal substitutions of non-conserved residues in the RBD are compensated for by complementary changes in the rest of the pilin monomer. Thus, the interactions between the RBD and the rest of the pilin can either be mediated by polar interactions of a hydrogen bond network in some strains or by hydrophobic interactions in others. Both configurations maintain a conserved backbone conformation of the RBD. Thus, the backbone conformation is critical in our consensus-sequence vaccine design and that cross-reactivity of the antibody response may be modulated by the composition of exposed side-chains on the surface of the RBD. This structure will guide our future vaccine design by focusing our investigation on the four variable residue positions that are exposed on the RBD surface.

Novel Strategy to Prevent Otitis Media Caused by Colonizing Streptococcus pneumoniae

Novel Strategy to Prevent Otitis Media Caused by Colonizing Streptococcus pneumoniae

Type IV pili of Neisseria gonorrhoeae influence the activation of human CD4+ T cells.

Neisseria gonorrhoeae is the causative agent of the sexually transmitted disease gonorrhea, and infection with this organism is typically associated with an intense inflammatory response. In many individuals, however, the infection is asymptomatic and can progress to serious secondary complications. The type IV pili of Neisseria gonorrhoeae mediate binding of the bacteria to host cells and are involved in cellular signal transduction. In these studies we have demonstrated that gonococcal pili influence human CD4+ T cells by using isogenic strains of N. gonorrhoeae with piliated and nonpiliated phenotypes. To determine the impact of piliation on the cellular status, we examined the expression of activation markers, cellular proliferation, and the production of cytokines after infection. The activation marker CD69 showed significantly increased expression on cells infected with the piliated strain, and this expression was dependent on costimulation of the T-cell receptor. Infection with piliated gonococci also altered T-cell proliferation and influenced the production of the regulatory cytokine interleukin-10. PilC, the putative pilus adhesin, was also observed to influence cellular activation but had no impact on the proliferation of cells further indicating that pilus-mediated adhesion is important in gonococcal stimulation of CD4+ T cells. These results show that the piliation status of gonococci influences CD4+ T-cell activation and that the adhesion mediated by pilus components aids in the regulation of the T-cell response to N. gonorrhoeae.

Aptamers That Preferentially Bind Type IVB Pili and Inhibit Human Monocytic-Cell Invasion by Salmonella enterica Serovar Typhi

Salmonella enterica serovar Typhi is an important pathogen exclusively for humans and causes typhoid or enteric fever. It has been shown that type IVB pili, encoded by the S. enterica serovar Typhi pil operon located in Salmonella pathogenicity island 7, are important in the pathogenic process. In this study, by using both an adhesion-invasion assay and fluorescence quantitative PCR analysis, we demonstrated that the entry of type IVB piliated S. enterica serovar Typhi A21-6 (pil(+) Km(r)) into human THP-1 monocytic cells was greater than that of a nonpiliated S. enterica serovar Typhi pilS::Km(r) (pil mutant) strain. We have applied a systematic evolution of ligands by exponential enrichment approach to select oligonucleotides (aptamers) as ligands that specifically bind to type IVB pili. Using this approach, we identified a high-affinity single-stranded RNA aptamer (S-PS(8.4)) as a type IVB pilus-specific ligand and further found that the selected aptamer (S-PS(8.4)) could significantly inhibit the entry of the piliated strain (but not that of the nonpiliated strain) into human THP-1 cells. The binding affinities between aptamers and pre-PilS (structural protein of type IVB pili) were determined by nitrocellulose filter-binding assays, and the K(d) value was determined to be 8.56 nM for the S-PS(8.4) aptamer alone. As an example of an aptamer against type IVB pili of S. enterica serovar Typhi, the aptamer S-PS(8.4) can serve as a tool for analysis of bacterial type IVB pilus-host cell interactions and may yield information for the development of putative new drugs against S. enterica serovar Typhi bacterial infections, useful both in prevention of infection and in therapeutic treatment.

A Study of Surface Proteins, Other Adhesins and Iron Acquiring Mechanisms in the Pathogenesis of Neisseria gonorrhoea strains Isolated from STD Patients in Ethiopia

Background information: Neisseria gonorrhoea is one of the most world wide prevalent Gram-negative diplococci bacteria causing the disease called Sexually Transmitted Disease on humans. Its stages of infection involve, first, colonization of columnar epithelial cells of mucosal surfaces; then an inflammatory response is elicited; and finally, the phagocytosis of the organisms by PMNs results in the formation of purulent discharges where the PMNs are found accumulated in it. The antigenic composition of N.gonorrhoea is complex with three major classes that are associated with the surface layers, namely, the pilus protein antigens, the polysaccharide component of cell wall and the outer membrane protein constitution. The pathogenicity determinant factors of N.gonorrhoea have been proved to include: Colonial Morphology which indicates that the virulence factors are based on the five major colony forms designated as T1, T2, T3, T4 and T5, indicating differences in surface antigens, virulence and in colony appearances; and, the Pili are the other pathogenesis determinant factors that are functioning by having adherence to the mucosal cell surface and serving as conjugal transfer of genetic material. It has been observed that piliated bacterial strains are resistant to phagocytosis, giving inefficient digestion ability to the macrophages compared to nonpiliated strains. Thus, types T1 and T2 are most virulent to humans because they are the piliated (fimbriated) strains. The Ethiopian situation in relation to gonococcal infections indicates that it is known to be highly prevalent among both sexes of most age groups. However no work has been conducted to assess the prevalence of the different virulence factors of N.gonorrhoeae strains. Objective : The aim of this study was thus to assess the prevalence of all the essential surface antigens as virulence factors of N.gonorrhoeae among the most vulnerable group of individuals for the infection and try to assess and recommend the possibility of getting important surface components to serve as vaccine candidates against gonococcal infections. Patients & methods: a total of over 300 STD male and female patients from three study sites, Addis Ababa, Nazareth, and Awassa, were used as study subjects by collecting urogenital specimen after their clinical examination. The specimens were then used for the isolation of the N.gonorrhoea strains that were identified for their virulence types, tested for their antimicrobial sensitivity patterns and determined their surface antigenic components for the most prevalent virulence factors using haemagglutination and other detection assay methods. Results: It was found that out of the total 335 specimens, N.gonorrhoea strains were isolated in 59% positive rate from males compared to the 43% positive rate was obtained from the females. The sensitivity pattern of these strains indicate that there were 19 different antibiogram patterns showing that only 12.3% were sensitive to all drugs while over 70% of the strains were resistant against more than two combined drugs and above 90% of these multi-drug resistant strains showed to be PPNGs. Out the total N.gonorrhoea isolates, 200 were tested for their haemagglutination and Iron-binding protein production properties and 35% of them were found to be Mannose Resistant Haemagglutinating (MRHA) strains while 20% were Mannose Sensitive Haemaggluintating (MSHA) strains when tested using the human AB blood types of. By the use guinea pig blood cells this was found to be 15% MRHA and 25% MSHA strains. When tested for their iron-binding protein (IBP) production, the MRHAs were positive for IBPs while the MSHA were positive for this property in 10%. Finally, based on the results obtained conclusions and recommendations are given at the end of the paper. [J Ethiopia Med Pract. 2002;4(1):14– 20]

Fimbrial ghf gene cluster of genital strains of Haemophilus spp.

We analyzed the LKP fimbrial gene clusters of six piliated strains of a cryptic genospecies of Haemophilus isolated from the genital tracts of adult patients (five strains) and from an infected neonate. In a group of 19 genital strains, LKP-like genes have been found in only these 6 strains. In addition to the ghfA, ghfD, and ghfE genes previously described, we characterized two genes, designated ghfB and ghfC, encoding the putative chaperone and assembly platform proteins. All six strains had a complete and unique LKP-like gene cluster consisting of the five genes ghfA to ghfE, homologous to genes hifA to hifE of Haemophilus influenzae. The sequences of the coding and intergenic regions of the ghf clusters of the six strains were remarkably homologous. Unlike hif clusters, which are inserted between purE and pepN, the ghf cluster was inserted between purK and pepN on the chromosome. Analysis of the flanking regions of the ghf cluster identified a large deletion, identical in the 5' end regions of all strains, including the whole purE gene and much of the purK gene. Ultrastructural observations, an attempt at enriching LKP fimbriae, and hemagglutination experiments demonstrated that none of the strains had LKP-type fimbriae. Nevertheless, reverse transcription (RT)-PCR showed that ghf genes were transcribed in four of the six strains. Sequencing of the intergenic ghfA-ghfB regions, including the ghf gene promoters, showed that the absence of transcripts in the remaining two strains was due to a decrease in the number of TA repeats (4 or 9 repeats rather than 10) between the -10 and -35 boxes of the two overlapping and divergent promoters. The other four strains, which had ghf transcripts, had the optimal 10 TA repeats (one strain) or 5 repeats associated with putative alternative -35 boxes (three strains). The absence of 10 repeated palindromic sequences of 44 or 45 nucleotides upstream of ghfB induces an increased instability of mRNA, as quantified by real-time RT-PCR, and may explain why the LKP fimbrial gene cluster is not expressed in these strains.

A heat-stable component of Bartonella henselae upregulates intercellular adhesion molecule-1 expression on vascular endothelial cells.

Bartonella henselae upregulated the expression of intercellular adhesion molecule-1 (ICAM-1) on human umbilical vein endothelial cells (HUVECs). The induction level of ICAM-1 depended on the inoculation bacterial dose. ICAM-1 expression began increasing 4 h after infection and reached a sustained peak beginning at 12 h after B. henselae infection; this time course was similar to that of lipopolysaccharide (LPS) of Escherichia coli. The stimulatory effect was abolished when live B. henselae were separated from HUVECs by a filter membrane. The nonpiliated strain, which is unable to invade endothelial cells, induced ICAM-1 expression to the same extent as the piliated strain. Inactivation of B. henselae by ultraviolet (UV) irradiation, heat (56 degrees C, 30 min), or sonication did not alter its stimulatory activity. Polymyxin B, which strongly inhibited the effect of LPS, did not exert any influence on the stimulatory activity of B. henselae. Furthermore, the effect of sonicated B. henselae was not inhibited even by boiling, which was also the case with LPS. Our data suggest that some heat-stable component of B. henselae binds to the endothelial cell surface, inducing ICAM-1 expression. Though the participation of LPS could not be completely ruled out, we suppose that some unidentified heat-stable proteins, lipids, or polysaccharides may be the stimulatory factor(s). The ability of B. henselae to enhance the expression of adhesion molecules on endothelial cells may be an important mechanism in the pathogenesis of B. henselae infection.

Adherence of Burkholderia cepacia to respiratory tract epithelial cells and inhibition with dextrans

Adherence of Burkholderia cepacia to cells of the respiratory tract of patients with cystic fibrosis (CF) appears to be a necessary precondition for colonization and infection. To date, no effective anti-adhesive strategy has been devised for preventing B. cepacia infection in CF patients. It was found in this study that B. cepacia adhered to respiratory epithelial cells both in vitro and in vivo. However, strains with cable-like pili (Cbl) exhibited the typical clump formation on pneumocytes, whereas non-cable piliated strains predominantly showed single-cell adherence. Dextrans (nominally 4000-10000 Da) significantly inhibited adhesion of B. cepacia to A549 pneumocytes. When compared on an equal weight basis, the nominally 10000 Da dextran was most inhibitory. A dose-dependent inhibitory effect (up to 80 mg ml(-1)) was observed for most strains. Dextran exerted less of an anti-adhesive effect on the two Cbl+ strains than on the others which were Cbl-. Dextrans appeared to block the adherence in a non-specific fashion, as shown by the observations that the inhibitory effect was readily reversible and oligosaccharides composed of 2-4 glucose units with the same alpha-1,6 linkage were not inhibitory. The mean molecular masses of dextrans used in this study, as determined by gel filtration and MS, were approximately 10-fold lower than those indicated by the manufacturers. Our data suggest that dextran of nominal molecular mass 4000 Da at a concentration of 40 mg ml(-1) (10 mM according to manufacturer's quoted molecular mass) or more may be useful in patients with CF to prevent colonization and infection with B. cepacia.

Live Bartonella henselae enhances endothelial cell proliferation without direct contact

The proliferation of human umbilical vein endothelial cells (HUVECs) cocultivated with live B. henselae was enhanced in a bacterial dose-dependent manner, and the stimulatory effect was specific to vascular endothelial cells. The inactivation of B. henselae by UV or heat treatment abolished its stimulatory activity, suggesting that live bacteria is necessary for the growth stimulation effect. To investigate the role of direct contact, live B. henselae were separated from HUVECs by a filter membrane (Millicell-CM insert). Even under this condition, an enhanced proliferation of HUVECs was observed. However, no morphological changes in the HUVECs were apparent compared to the B. henselae -infected cells. Furthermore, we isolated a nonpiliated strain of B. henselae that is unable to attach to and enter into endothelial cells. The nonpiliated strain possessed the ability to stimulate the proliferation of cocultivated HUVECs the same as the piliated strain. Moreover, the culture supernatants of B. henselae were also able to induce HUVEC proliferation. Our results indicate that the stimulation of HUVEC proliferation by B. henselae is mediated by soluble factor(s) secreted from the bacteria.

Fibronectin and α5β1 integrin mediate binding of Pseudomonas aeruginosa to repairing airway epithelium

Initial infection of the airway by Pseudomonas aeruginosa may occur through a variety of bacterial strategies including binding to epithelial receptors present at the surface of the respiratory epithelium. In order to characterize the adherence sites for P. aeruginosa in damaged and repairing bronchial tissue, an ex vivo model of airway epithelial injury and repair was developed using primary cell cultures of nasal cells from 14 subjects with polyposis. P. aeruginosa strongly adhered to flattened dedifferentiated (FD) bronchial and nasal cytokeratin 13-positive epithelial cells in the process of migration for repair. In in vitro experiments, competitive binding inhibition assays demonstrated that alpha5beta1 integrins and cellular fibronectin, in particular the RGD sequence, are receptors involved in P. aeruginosa adherence to FD nasal epithelial cells. Fluorescent cell sorting analysis and immunofluorescence techniques revealed that the alpha5beta1 integrins are overexpressed and apically exposed in FD nasal epithelial cells. One 50 kDa outer membrane protein was identified in piliated and nonpiliated strains of P. aeruginosa that was involved in binding to cellular fibronectin and alpha5beta1 epithelial integrins. These results demonstrate that Pseudomonas aeruginosa adherence is related to the dedifferentiation of airway epithelium during the repair process which unmasks and upregulates the alpha5beta1 integrin expression and induces active synthesis of cellular fibronectin. These epithelial receptors are then used by a Pseudomonas aeruginosa 50 kDa outer membrane protein as sites of bacterial adherence.

The effect of subinhibitory concentrations of antibiotics on adherence of Pseudomonas aeruginosa to cystic fibrosis (CF) and non-CF-affected tracheal epithelial cells

this project investigated the proposition that Pseudomonas aeruginosa binds preferentially to cystic fibrosis (CF) respiratory cells. In addition, disadherence of P. aeruginosa by subinhibitory concentrations of antibiotics was examined as a possible explanation for clinical improvement seen in chronically colonized patients when treated with anti-pseudomonal agents. we used a distinctive HPV-transformed respiratory cell line to compare adherence of two strains of P. aeruginosa to CF and non-CF respiratory cells. The effect of subinhibitory concentrations of antipseudomonal antibiotics on adherence of P. aeruginosa to cell monolayers was measured. piliated P. aeruginosa bound significantly better to CF than non-CF-affected cells (P=0.003). Adherence was significantly reduced when organisms were preincubated in subinhibitory concentrations of antibiotics overnight (P<0.001). these results support the presence of an altered receptor present on CF-affected cells that binds piliated strains of P. aeruginosa. Bacterial disadherence due to subinhibitory concentrations of antibiotics may partially explain why clinical improvement is observed in CF patients with acute respiratory exacerbations.

Influence of urinary constituents on the adherence of Escherichia coli to human uroepithelial cells in vitro

To study the influence of some urinary constituents on the adhesion ability of Escherichia coli to uroepithelial cells, a standard piliated strain (E. coli 31-B), its mutant lacking in type I pili (E. coli BH-5), and a wild-type laboratory E. coli isolate of serotype 04 possessing both MR and MS pili were used. The average number of bacteria adhering per uroepithelial cell was greater when grown in tryptone soya broth (TSB), compared with Fe-CDM grown strains. A similar effect was observed when strains were grown under iron-deficient conditions. Urinary constituents such as glucose, lactose, urea, and creatinine singly, or in combination, at different concentrations, when added to Fe-CDM medium, altered the adhesion potential of the growing organism. This effect, however, was strain-dependent. Its implication in the clinical set-up is discussed.

Pseudomonas aeruginosa strains possess specific adhesins for laminin.

Pseudomonas aeruginosa is a major human pathogen known to infect tissues that have been previously damaged in some way. In wounded human respiratory tissues, P. aeruginosa cells were found attached to exposed basement membranes following epithelial denudation, suggesting that the affinity for extracellular matrix proteins may account for the bacterium's opportunistic character. By using microtiter wells coated with different P. aeruginosa strains, we demonstrated that laminin binds to both colonizing bacterial strains, isolated from asymptomatic carriers, and strains isolated from infected patients. Binding of soluble laminin to piliated P. aeruginosa PAK and to the nonpiliated isogenic mutant PAK/p--was shown to be saturable. Binding of laminin to the piliated PAK strain was not different from binding to th nonpiliated PAK/p--strain but was significantly higher than binding to the avirulent, nonpiliated PAK-N1 rpoN mutant. By transmission electron microscopy, we localized the laminin-binding sites on a loose material in the outermost layer of the bacteria. Western immunoblotting results suggested that 57- and 59-kDa nonpilus adhesins from the microbial outer membranes account for the binding of P. aeruginosa to laminin. We speculate that bacterial affinity for laminin may be of biological significance in the pathogenesis of P. aeruginosa infection of injured tissues.

Functional implications of the expression of PilC proteins in meningococci

Multiple forms of PilC were found in Neisseria meningitidis (Nm) strains isolated from the oropharynx, blood or cerebrospinal fluid expressing either Class I or Class II pili. PilC expression was observed less frequently in case as opposed to carrier isolates. Moreover, PilC and pili were not always co-expressed. Several heavily piliated strains had no detectable PilC protein as determined by Western blotting using an antiserum previously used to detect such proteins in adhesive variants (Nassif et al., 1994). Serogroup B strain MC58 produced large numbers of pili, but expressed barely detectable amounts of PilC. A clonal variant of this strain with increased expression of PilC concurrently exhibited increased adherence to Chang conjunctival epithelial cells and human umbilical vein endothelial cells (Huvecs), but with more rapid binding to the former. No alteration in pilin sequence occurred in this variant, suggesting the involvement of PilC in increased adhesion. A Pil- backswitcher isolated from the hyper-adherent variant was PilC+ but was non-adherent, indicating that any PilC adherence function requires pilus expression. Parental variant (low PilC) produced pili in bundles that were easily detached from the bacterial surface and were frequently associated with Huvec surfaces after bacteria had been sheared off, but pili infrequently replaced bacteria during infection with the PilC-expressing variant. The hyper-adherent variant, which appeared to produce morphologically distinct pilus bundles, was able to withstand considerable shearing force and remained firmly attached to Huvecs. This raises the possibility that the observed hyper-adherence may arise from better anchorage of pili to the bacterial surface in addition to increased adhesion to some host cell surfaces.

Role of Pseudomonas aeruginosa pili in acute pulmonary infection.

The role of piliation in the development and course of acute pulmonary infection was examined using infant BALB/cByJ mice inoculated by intranasal instillation of isogenic Pil+ and Pil- mutants of Pseudomonas aeruginosa PA1244, PAK, and PAO1. The piliated strains caused more cases of pneumonia, bacteremia, and mortality than the nonpiliated strains (chi-square analysis, alpha = 0.001). The piliated strains were more often associated with severe diffuse pneumonias, while the nonpiliated organisms resulted in less severe, focal pneumonias, although these differences did not achieve statistical significance. Purified pilin protein used to inoculate the mice resulted in local inflammatory changes. The nonpiliated strain PA1244-NP was as virulent as the piliated strain PAO1, suggesting that expression of other virulence factors are also important in the development of acute pneumonia. This infant mouse model of pulmonary infection appears to be a useful system for the analysis of P. aeruginosa virulence factors involved in the pathogenesis of pneumonia.

The effect of variations in the expression of pili on the interaction of Neisseria meningitidis with human nasopharyngeal epithelium.

Bacterial adherence is essential to colonization of the nasopharynx by Neisseria meningitidis, and pili may facilitate adherence. Scanning electron microscopy and immunogold labeling were used to study the interaction of 2 piliated and 1 nonpiliated variant of N. meningitidis with nasopharyngeal mucosa in an adenoid organ culture system with an air-mucosal interface. Meningococcal infection caused epithelial damage, loss of ciliated epithelium, and ciliary disorganization, which progressed with time and were greater with the piliated strains. Pili increased adherence of meningococci to the mucosa, and there was tropism of piliated strains for nonciliated cells containing microvilli, damaged epithelium, and sites of cell separation. Bacterial adherence was associated with a change in the appearance of the microvilli.

Pseudomonas aeruginosa selective adherence to and entry into human endothelial cells.

The pathogenesis of Pseudomonas aeruginosa disseminated infections depends on bacterial interaction with blood vessels. We have hypothesized that in order to traverse the endothelial barrier, bacteria would have to adhere to and damage endothelial cells. To test this hypothesis, we studied the adherence to human endothelial cells in primary culture of the piliated P. aeruginosa strain PAK and of two isogenic nonpiliated strains: PAK/p-, which carries a mutation in the pilin structural gene, and PAK-N1, a mutant defective in the regulatory rpoN gene. PAK adhered significantly more than did the pilus-lacking strains. P. aeruginosa was also taken up by endothelial cells, as determined by quantitative bacteriologic assays and by transmission electron microscopy. This internalization of P. aeruginosa seems to be a selective process, since the piliated strain was taken up significantly more than the nonpiliated bacteria and the avirulent Escherichia coli DH5 alpha, even following bacterial centrifugation onto the cell monolayers. A significant fraction of the internalized P. aeruginosa PAK was recovered in a viable form after 6 h of residence within endothelial cells. Progressive endothelial cell damage resulted from PAK intracellular harboring, as indicated by the release of lactate dehydrogenase. An increasing concentration of PAK cells was recovered from the extracellular medium with time, suggesting that ingested bacteria were released from endothelial cells and multiplied freely. We speculate that in vivo the ability of some P. aeruginosa strains to resist intracellular residence would afford protection from host defenses and antibiotics and that the release of viable bacteria into bloodstream may represent a central feature of the pathogenesis of bacteremia in compromised patients.

Experimental vaccination of young chickens with a live, non‐pathogenic strain ofEscherichia coli

A non-pathogenic, piliated strain of Escherichia coli (BT-7; Frommer et al., 1990), isolated from a meat-type chicken flock, was studied as a candidate for a live vaccine to protect chickens from E. coli infection. Active immunization provided substantial protection of chicks vaccinated at 14 or 21 days of age, resulting in better resistance to challenge than in those vaccinated at 1 or 7 days. Chicks vaccinated at 21 days of age and challenged 1 week later with pathogenic E. coli strains 01-.K1, 02:K1 or 078:K80, exhibited good protection for at least 2 weeks against all strains. Three vaccination routes were found to give the highest resistance to challenge with pathogenic E. coli strain 078:K80. Intramuscular (i.m.) at 7 and 21 days of age, i.m. at 21 days of age and spray at 7, followed by per os at 21 days of age. Vaccination per os once at 7 or twice at 7 and 21 days resulted in good protection. Chicks exhibiting high antibody titres by ELISA were well-protected against challenge.

Adherence to human cells of a cryptic Haemophilus genospecies responsible for genital and neonatal infections.

Haemophilus strains usually identified as Haemophilus influenzae biotype IV belonging to a cryptic genospecies are responsible for genital and neonatal infections. As a first approach to identifying the bacterial factors involved in the pathogenesis of these unusual diseases, we studied the piliation, adherence, and invasion properties of 17 strains assigned to this cryptic genospecies. Twelve strains spontaneously displayed abundant peritrichous piliation, and two strains expressed peritrichous pili after enrichment procedures. For virtually all strains, piliation correlated with adhesion to cultured HeLa cells of genital origin and to a lesser extent with adhesion to HEp-2 cells of laryngeal origin. A variation in the adherence properties of the various strains was observed: all piliated strains except one adhered to 50 to 100% of HeLa cells, with a mean number of bacteria per cell varying from 4 to 50. Adherence was not dependent on the state of growth for most strains, was more pronounced with HeLa cells than with HEp-2 cells for 10 of the 12 highly adherent strains, was time and inoculum dependent, and was not followed by significant invasion of cells. Most of the strains belonging to this unusual Haemophilus clone possess adhesins that do not recognize erythrocyte receptors, since agglutination of human erythrocytes was observed with only 3 of the 14 piliated strains.

Pilus-mediated adsorption of Pseudomonas syringae to the surface of host and non-host plant leaves

SUMMARY: Adsorption of cells of a variety of Pseudomonas syringae pathovars to the leaf surface of host and non-host plants was measured. The strains used were sensitive to the pilus-specific bacteriophage ø6. Phage-resistant non-piliated mutants were isolated and found to have reduced ability to adsorb to plant surfaces. The pilus-mediated adsorption was not host-specific. Piliated strains adsorbed well to both host and non-host plants. Scanning electron microscopy of the P. syringae pathovar syringae strain R32 and the pathovar phaseolicola strain HB10Y revealed a difference between these strains in the distribution of the bacterial cells over the lower bean leaf surface. P. syringae pv. syringae spread evenly over the leaf surface whereas P. syringae pv. phaseolicola adsorbed preferentially to the stomata. No such localization was observed on chloroform-treated leaves, where the cells of both pathovars were evenly distributed. Adsorption of bacteria to leaf disks was independent of divalent cations, and no specific ionic conditions were required.