A role for fibroblast growth factor receptor 1 in the pathogenesis of Neisseria meningitidis.

Neisseria meningitidis (the meningococcus) remains an important cause of human disease, including meningitis and sepsis. Adaptation to the host environment includes many interactions with specific cell surface receptors, resulting in intracellular signalling and cytoskeletal rearrangements that contribute to pathogenesis. Here, we assessed the interactions between meningococci and Fibroblast Growth Factor Receptor 1-IIIc (FGFR1-IIIc): a receptor specific to endothelial cells of the microvasculature, including that of the blood-brain barrier. We show that the meningococcus recruits FGFR1-IIIc onto the surface of human blood microvascular endothelial cells (HBMECs). Furthermore, we demonstrate that expression of FGFR1-IIIc is required for optimal invasion of HBMECs by meningococci. We show that the ability of N. meningitidis to interact with the ligand-binding domain of FGFR1-IIIc is shared with the other pathogenic Neisseria species, N. gonorrhoeae, but not with commensal bacteria including non-pathogenic Neisseria species.

Uptake of Neisserial autotransporter lipoprotein (NalP) promotes an increase in human brain microvascular endothelial cell metabolic activity.

Neisseria meningitidis is normally a human nasopharyngeal commensal but is also capable of causing life-threatening sepsis and meningitis. N. meningitidis secretes several virulence-associated proteins including Neisserial autotransporter lipoprotein (NalP), an immunogenic, type Va autotransporter harboring an S8-family serine endopeptidase domain. NalP has been previously characterized as a cell-surface maturation protease which processes other virulence-associated meningococcal surface proteins, and as a factor contributing to the survival of meningococci in human serum due to its ability to cleave complement factor C3. Here, recombinant NalP (rNalP) fragments were purified and used to investigate the interaction of NalP with host cells. Flow cytometry and confocal microscopy demonstrated binding and uptake of rNalP into different human cell types. High-resolution microscopy confirmed that internalized rNalP predominantly localized to the perinuclear region of cells. Abolition of rNalP protease activity using site-directed mutagenesis did not influence uptake or sub-cellular localization, but inactive rNalP (rNalPS426A) was unable to induce an increase in human brain microvascular endothelial cell metabolic activity provoked by proteolytically-active rNalP. Our data suggests a more complex and multifaceted role for NalP in meningococcal pathogenesis than was previously understood which includes novel intra-host cell functions.

Genomic Analysis of Serogroup Y Neisseria meningitidis Isolates Reveals Extensive Similarities Between Carriage-Associated and Disease-Associated Organisms.

BACKGROUND: Neisseria meningitidis is a frequent colonizer of the human nasopharynx, with asymptomatic carriage providing the reservoir for invasive, disease-causing strains. Serogroup Y (MenY) strains are a major cause of meningococcal disease. High-resolution genetic analyses of carriage and disease isolates can establish epidemiological relationships and identify potential virulence factors. METHODS: Whole-genome sequence data were obtained for 99 MenY carriage isolates recovered in the United Kingdom during 1997-2010. Sequences were compared to those of 73 MenY invasive isolates recovered during 2010-2011, using a gene-by-gene approach. RESULTS: Comparisons across 1605 core genes resolved 91% of isolates into one of 8 clusters containing closely related disease and carriage isolates. Six clusters contained carried meningococci isolated during 1997-2001, suggesting temporal stability. One cluster of isolates, predominately sharing the designation Y: P1.5-1,10-1: F4-1: ST-1655 (cc23), was resolved into one subcluster with 86% carriage isolates and a second with 90% invasive isolates. These subclusters were defined by specific allelic differences in 5 core genes encoding glycerate kinase (glxK), valine-pyruvate transaminase (avtA), superoxide dismutase (sodB), and 2 hypothetical proteins. CONCLUSIONS: High-resolution genetic analyses detected long-term temporal stability and temporally overlapping carriage and disease populations for MenY clones but also evidence of a disease-associated clone.

Optimization of Molecular Approaches to Genogroup Neisseria meningitidis Carriage Isolates and Implications for Monitoring the Impact of New Serogroup B Vaccines.

The reservoir for Neisseria meningitidis (Nm) is the human oropharynx. Implementation of Nm serogroup C (NmC) glycoconjugate vaccines directly reduced NmC carriage. Prophylactic vaccines are now available to prevent disease caused by the five major Nm disease causing serogroups (ABCWY). Nm serogroup B (NmB) vaccines are composed of antigens that are conserved across Nm serogroups and therefore have the potential to impact all Nm carriage. To assess the effect of these vaccines on carriage, standardized approaches to identify and group Nm are required. Real-time PCR (rt-PCR) capsule grouping assays that were internally controlled to confirm Nm species were developed for eight serogroups associated with carriage (A, B, C, E, W, X, Y and Z). The grouping scheme was validated using diverse bacterial species associated with carriage and then used to evaluate a collection of diverse Nm carriage isolates (n=234). A scheme that also included porA and ctrA probes was able to speciate the isolates, while ctrA also provided insights on the integrity of the polysaccharide loci. Isolates were typed for the Nm vaccine antigen factor H binding protein (fHbp), and were found to represent the known diversity of this antigen. The porA rt-PCR yielded positive results with all 234 of the Nm carriage isolates. Genogrouping assays classified 76.5% (179/234) of these isolates to a group, categorized 53 as nongenogroupable (NGG) and two as mixed results. Thirty seven NGG isolates evidenced a disrupted capsular polysaccharide operon judged by a ctrA negative result. Only 28.6% (67/234) of the isolates were serogrouped by slide agglutination (SASG), highlighting the reduced capability of carriage strains to express capsular polysaccharide. These rt-PCR assays provide a comprehensive means to identify and genogroup N. meningitidis in carriage studies used to guide vaccination strategies and to assess the impact of novel fHbp containing vaccines on meningococcal carriage.

Nuclear trafficking, histone cleavage and induction of apoptosis by the meningococcal App and MspA autotransporters.

Neisseria meningitidis, a major cause of bacterial meningitis and septicaemia, secretes multiple virulence factors, including the adhesion and penetration protein (App) and meningococcal serine protease A (MspA). Both are conserved, immunogenic, type Va autotransporters harbouring S6-family serine endopeptidase domains. Previous work suggested that both could mediate adherence to human cells, but their precise contribution to meningococcal pathogenesis was unclear. Here, we confirm that App and MspA are in vivo virulence factors since human CD46-expressing transgenic mice infected with meningococcal mutants lacking App, MspA or both had improved survival rates compared with mice infected with wild type. Confocal imaging showed that App and MspA were internalized by human cells and trafficked to the nucleus. Cross-linking and enzyme-linked immuno assay (ELISA) confirmed that mannose receptor (MR), transferrin receptor 1 (TfR1) and histones interact with MspA and App. Dendritic cell (DC) uptake could be blocked using mannan and transferrin, the specific physiological ligands for MR and TfR1, whereas in vitro clipping assays confirmed the ability of both proteins to proteolytically cleave the core histone H3. Finally, we show that App and MspA induce a dose-dependent increase in DC death via caspase-dependent apoptosis. Our data provide novel insights into the roles of App and MspA in meningococcal infection.

Deciphering the complex three-way interaction between the non-integrin laminin receptor, galectin-3 and Neisseria meningitidis.

The non-integrin laminin receptor (LAMR1/RPSA) and galectin-3 (Gal-3) are multi-functional host molecules with roles in diverse pathological processes, particularly of infectious or oncogenic origins. Using bimolecular fluorescence complementation and confocal imaging, we demonstrate that the two proteins homo- and heterodimerize, and that each isotype forms a distinct cell surface population. We present evidence that the 37 kDa form of LAMR1 (37LRP) is the precursor of the previously described 67 kDa laminin receptor (67LR), whereas the heterodimer represents an entity that is distinct from this molecule. Site-directed mutagenesis confirmed that the single cysteine (C(173)) of Gal-3 or lysine (K(166)) of LAMR1 are critical for heterodimerization. Recombinant Gal-3, expressed in normally Gal-3-deficient N2a cells, dimerized with endogenous LAMR1 and led to a significantly increased number of internalized bacteria (Neisseria meningitidis), confirming the role of Gal-3 in bacterial invasion. Contact-dependent cross-linking determined that, in common with LAMR1, Gal-3 binds the meningococcal secretin PilQ, in addition to the major pilin PilE. This study adds significant new mechanistic insights into the bacterial-host cell interaction by clarifying the nature, role and bacterial ligands of LAMR1 and Gal-3 isotypes during colonization.

Phase variation mediates reductions in expression of surface proteins during persistent meningococcal carriage.

Asymptomatic and persistent colonization of the upper respiratory tract by Neisseria meningitidis occurs despite elicitation of adaptive immune responses against surface antigens. A putative mechanism for facilitating host persistence of this bacterial commensal and pathogen is alterations in expression of surface antigens by simple sequence repeat (SSR)-mediated phase variation. We investigated how often phase variation occurs during persistent carriage by analyzing the SSRs of eight loci in multiple isolates from 21 carriers representative of 1 to 6 months carriage. Alterations in repeat number were detected by a GeneScan analysis and occurred at 0.06 mutations/gene/month of carriage. The expression states were determined by Western blotting and two genes, fetA and nadA, exhibited trends toward low expression states. A critical finding from our unique examination of combinatorial expression states, "phasotypes," was for significant reductions in expression of multiple phase-variable surface proteins during persistent carriage of some strains. The immune responses in these carriers were examined by measuring variant-specific PorA IgG antibodies, capsular group Y IgG antibodies and serum bactericidal activity in concomitant serum samples. Persistent carriage was associated with high levels of specific IgG antibodies and serum bactericidal activity while recent strain acquisition correlated with a significant induction of antibodies. We conclude that phase-variable genes are driven into lower expression states during long-term persistent meningococcal carriage, in part due to continuous exposure to antibody-mediated selection, suggesting localized hypermutation has evolved to facilitate host persistence.

A novel O-linked glycan modulates Campylobacter jejuni major outer membrane protein-mediated adhesion to human histo-blood group antigens and chicken colonization.

Campylobacter jejuni is an important cause of human foodborne gastroenteritis; strategies to prevent infection are hampered by a poor understanding of the complex interactions between host and pathogen. Previous work showed that C. jejuni could bind human histo-blood group antigens (BgAgs) in vitro and that BgAgs could inhibit the binding of C. jejuni to human intestinal mucosa ex vivo. Here, the major flagella subunit protein (FlaA) and the major outer membrane protein (MOMP) were identified as BgAg-binding adhesins in C. jejuni NCTC11168. Significantly, the MOMP was shown to be O-glycosylated at Thr(268); previously only flagellin proteins were known to be O-glycosylated in C. jejuni. Substitution of MOMP Thr(268) led to significantly reduced binding to BgAgs. The O-glycan moiety was characterized as Gal(ß1-3)-GalNAc(ß1-4)-GalNAc(ß1-4)-GalNAcα1-Thr(268); modelling suggested that O-glycosylation has a notable effect on the conformation of MOMP and this modulates BgAg-binding capacity. Glycosylation of MOMP at Thr(268) promoted cell-to-cell binding, biofilm formation and adhesion to Caco-2 cells, and was required for the optimal colonization of chickens by C. jejuni, confirming the significance of this O-glycosylation in pathogenesis.

Pro-inflammatory cytokines can act as intracellular modulators of commensal bacterial virulence.

Interactions between commensal pathogens and hosts are critical for disease development but the underlying mechanisms for switching between the commensal and virulent states are unknown. We show that the human pathogen Neisseria meningitidis, the leading cause of pyogenic meningitis, can modulate gene expression via uptake of host pro-inflammatory cytokines leading to increased virulence. This uptake is mediated by type IV pili (Tfp) and reliant on the PilT ATPase activity. Two Tfp subunits, PilE and PilQ, are identified as the ligands for TNF-α and IL-8 in a glycan-dependent manner, and their deletion results in decreased virulence and increased survival in a mouse model. We propose a novel mechanism by which pathogens use the twitching motility mode of the Tfp machinery for sensing and importing host elicitors, aligning with the inflamed environment and switching to the virulent state.

Prevalence and phase variable expression status of two autotransporters, NalP and MspA, in carriage and disease isolates of Neisseria meningitidis.

Neisseria meningitidis is a human nasopharyngeal commensal capable of causing life-threatening septicemia and meningitis. Many meningococcal surface structures, including the autotransporter proteins NalP and MspA, are subject to phase variation (PV) due to the presence of homopolymeric tracts within their coding sequences. The functions of MspA are unknown. NalP proteolytically cleaves several surface-located virulence factors including the 4CMenB antigen NhbA. Therefore, NalP is a phase-variable regulator of the meningococcal outer membrane and secretome whose expression may reduce isolate susceptibility to 4CMenB-induced immune responses. To improve our understanding of the contributions of MspA and NalP to meningococcal-host interactions, their distribution and phase-variable expression status was studied in epidemiologically relevant samples, including 127 carriage and 514 invasive isolates representative of multiple clonal complexes and serogroups. Prevalence estimates of >98% and >88% were obtained for mspA and nalP, respectively, with no significant differences in their frequencies in disease versus carriage isolates. 16% of serogroup B (MenB) invasive isolates, predominately from clonal complexes ST-269 and ST-461, lacked nalP. Deletion of nalP often resulted from recombination events between flanking repetitive elements. PolyC tract lengths ranged from 6-15 bp in nalP and 6-14 bp in mspA. In an examination of PV status, 58.8% of carriage, and 40.1% of invasive nalP-positive MenB isolates were nalP phase ON. The frequency of this phenotype was not significantly different in serogroup Y (MenY) carriage strains, but was significantly higher in invasive MenY strains (86.3%; p<0.0001). Approximately 90% of MenB carriage and invasive isolates were mspA phase ON; significantly more than MenY carriage (32.7%) or invasive (13.7%) isolates. This differential expression resulted from different mode mspA tract lengths between the serogroups. Our data indicates a differential requirement for NalP and MspA expression in MenB and MenY strains and is a step towards understanding the contributions of phase-variable loci to meningococcal biology.

Naturally occurring IgG antibody levels to the Staphylococcus aureus protein IsdB in humans.

Staphylococcus aureus is a well-recognized, clinically important cause of nosocomial infections, and as such, a vaccine to prevent S. aureus infections would be an important achievement. A Phase IIB/III study of V710, a vaccine containing iron-regulated surface determinant B (IsdB), demonstrated significant sero-conversion rates in cardiovascular surgery patients following a single pre-surgery immunization. However, the vaccine was not efficacious in preventing bacteremia or deep sternal wound infection post-surgery, thus raising the possibility that IsdB might not be available for immune recognition during infection. The purpose of the work described herein was to evaluate and quantify the naturally occurring anti-IsdB levels at baseline and over time during infection, to understand whether IsdB is expressed during a S. aureus infection in hospitalized non-vaccinated patients. We evaluated baseline and follow-up titers in 3 populations: (1) healthy subjects, (2) hospitalized patients with non-S. aureus infections, and (3) hospitalized patients with S. aureus infections. Baseline anti-IsdB levels generally overlapped between the 3 groups, but were highly variable within each group. In healthy subjects, baseline and follow-up levels were highly correlated (Spearman's rho = 0.93), and the geometric mean fold-rise (GMFR) in anti-IsdB levels between study entry and last value was 0.9-fold (95% confidence interval (CI): 0.8 to 1.0 ; p = 0.09), showing no trend over time. The convalescent GMFR in anti-IsdB levels from baseline was 1.7-fold (95% CI: 1.3 to 2.2, p = 0.0008) during S. aureus infection, significantly different from the 1.0-fold GMFR (95% CI: 0.9-1.2, p = 0.60) in non-S. aureus infection, p = 0.005. Additionally, S. aureus isolates (51) obtained from the hospitalized patient group expressed the IsdB protein in vitro. Collectively, these data suggest that IsdB expression levels rise substantially following infection with S. aureus, but not with other pathogens, and IsdB is likely well-conserved across S. aureus strains.

Mapping the laminin receptor binding domains of Neisseria meningitidis PorA and Haemophilus influenzae OmpP2.

Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae are major bacterial agents of meningitis. They each bind the 37/67-kDa laminin receptor (LamR) via the surface protein adhesins: meningococcal PilQ and PorA, H. influenzae OmpP2 and pneumococcal CbpA. We have previously reported that a surface-exposed loop of the R2 domain of CbpA mediates LamR-binding. Here we have identified the LamR-binding regions of PorA and OmpP2. Using truncated recombinant proteins we show that binding is dependent on amino acids 171-240 and 91-99 of PorA and OmpP2, respectively, which are predicted to localize to the fourth and second surface-exposed loops, respectively, of these proteins. Synthetic peptides corresponding to the loops bound LamR and could block LamR-binding to bacterial ligands in a dose dependant manner. Meningococci expressing PorA lacking the apex of loop 4 and H. influenzae expressing OmpP2 lacking the apex of loop 2 showed significantly reduced LamR binding. Since both loops are hyper-variable, our data may suggest a molecular basis for the range of LamR-binding capabilities previously reported among different meningococcal and H. influenzae strains.

Influence of the combination and phase variation status of the haemoglobin receptors HmbR and HpuAB on meningococcal virulence.

Neisseria meningitidis can utilize haem, haemoglobin and haemoglobin-haptoglobin complexes as sources of iron via two TonB-dependent phase variable haemoglobin receptors, HmbR and HpuAB. HmbR is over-represented in disease isolates, suggesting a link between haemoglobin acquisition and meningococcal disease. This study compared the distribution of HpuAB and phase variation (PV) status of both receptors in disease and carriage isolates. Meningococcal disease (n = 214) and carriage (n = 305) isolates representative of multiple clonal complexes (CCs) were investigated for the distribution, polyG tract lengths and ON/OFF status of both haemoglobin receptors, and for the deletion mechanism for HpuAB. Strains with both receptors or only hmbR were present at similar frequencies among meningococcal disease isolates as compared with carriage isolates. However, >90 % of isolates from the three CCs CC5, CC8 and CC11 with the highest disease to carriage ratios contained both receptors. Strains with an hpuAB-only phenotype were under-represented among disease isolates, suggesting selection against this receptor during systemic disease, possibly due to the receptor having a high level of immunogenicity or being inefficient in acquisition of iron during systemic spread. Absence of hpuAB resulted from either complete deletion or replacement by an insertion element. In an examination of PV status, one or both receptors were found in an ON state in 91 % of disease and 71 % of carriage isolates. We suggest that expression of a haemoglobin receptor, either HmbR or HpuAB, is of major importance for systemic spread of meningococci, and that the presence of both receptors contributes to virulence in some strains.

Persistence, replacement, and rapid clonal expansion of meningococcal carriage isolates in a 2008 university student cohort.

A study of meningococcal carriage dynamics was performed with a cohort of 190 first-year students recruited from six residential halls at Nottingham University, United Kingdom. Pharyngeal swabs were obtained on four occasions between November 2008 and May 2009. Direct plating and culture on selective media were succeeded by identification and characterization of meningococci using PCR-based methodologies. Three serogroup Y clones and one serogroup 29E clone were highly prevalent in particular residential halls in November 2008, which is indicative of rapid clonal expansion since the start of the academic year. Persistent carriage of the same meningococcal strain for at least 5 to 6 months was observed in 45% of carriers, with infrequent evidence of antigenic variation in PorA. Sequential carriage of heterologous meningococcal strains occurred in 36% of carriers and involved strains with different capsules and antigenic variants of PorA and FetA in 83% of the cases. These clonal replacement strains also exhibited frequent differences in the presence and antigenic structures of two other surface proteins, NadA and HmbR. This study highlights the low level of antigenic variation associated with persistent carriage but, conversely, the importance of alterations in the repertoire of antigenic variants for sequential carriage of meningococcal strains. Rapid clonal expansion of potentially pathogenic strains in residential halls has implications for the implementation of public health interventions in university populations.

The role of glyceraldehyde 3-phosphate dehydrogenase (GapA-1) in Neisseria meningitidis adherence to human cells.

BACKGROUND: Glyceraldehyde 3-phosphate dehydrogenases (GAPDHs) are cytoplasmic glycolytic enzymes, which although lacking identifiable secretion signals, have also been found localized to the surface of several bacteria (and some eukaryotic organisms); where in some cases they have been shown to contribute to the colonization and invasion of host tissues. Neisseria meningitidis is an obligate human nasopharyngeal commensal which can cause life-threatening infections including septicaemia and meningitis. N. meningitidis has two genes, gapA-1 and gapA-2, encoding GAPDH enzymes. GapA-1 has previously been shown to be up-regulated on bacterial contact with host epithelial cells and is accessible to antibodies on the surface of capsule-permeabilized meningococcal cells. The aims of this study were: 1) to determine whether GapA-1 was expressed across different strains of N. meningitidis; 2) to determine whether GapA-1 surface accessibility to antibodies was dependent on the presence of capsule; 3) to determine whether GapA-1 can influence the interaction of meningococci and host cells, particularly in the key stages of adhesion and invasion. RESULTS: In this study, expression of GapA-1 was shown to be well conserved across diverse isolates of Neisseria species. Flow cytometry confirmed that GapA-1 could be detected on the cell surface, but only in a siaD-knockout (capsule-deficient) background, suggesting that GapA-1 is inaccessible to antibody in in vitro-grown encapsulated meningococci. The role of GapA-1 in meningococcal pathogenesis was addressed by mutational analysis and functional complementation. Loss of GapA-1 did not affect the growth of the bacterium in vitro. However, a GapA-1 deficient mutant showed a significant reduction in adhesion to human epithelial and endothelial cells compared to the wild-type and complemented mutant. A similar reduction in adhesion levels was also apparent between a siaD-deficient meningococcal strain and an isogenic siaD gapA-1 double mutant. CONCLUSIONS: Our data demonstrates that meningococcal GapA-1 is a constitutively-expressed, highly-conserved surface-exposed protein which is antibody-accessible only in the absence of capsule. Mutation of GapA-1 does not affect the in vitro growth rate of N. meningitidis, but significantly affects the ability of the organism to adhere to human epithelial and endothelial cells in a capsule-independent process suggesting a role in the pathogenesis of meningococcal infection.

Human antibody responses to the meningococcal factor H binding protein (LP2086) during invasive disease, colonization and carriage.

Recombinant forms of Neisseria meningitidis factor H binding protein (fHBP) are undergoing clinical trials in candidate vaccines against serogroup B meningococcal disease. Little is known, however, about the host response to fHBP during natural carriage and disease. Here we report a longitudinal study of the antibody response to fHBP in healthy meningococcal carriers and non-carriers, and in patients with invasive meningococcal disease. Using a highly sensitive quantitative immunoassay, anti-fHBP antibodies were detected in sera from all healthy carriers and non-carriers. Carriers had significantly higher anti-fHBP antibody concentrations than non-carriers. Antibody responses similar to those seen in non-carrier subjects were detected in the sera of patients with invasive disease upon their admission to the hospital. The serum anti-fHBP antibody concentrations in these patients generally rose to reach levels similar to those seen in carriers. No correlation between levels of surface fHBP expressed in vitro by the infecting N. meningitidis strain and the magnitude of antibody responses was observed. These data suggest that fHBP is expressed in vivo during both carriage and invasive disease at levels high enough to elicit a robust antibody response.

The moonlighting protein fructose-1, 6-bisphosphate aldolase of Neisseria meningitidis: surface localization and role in host cell adhesion.

Fructose-1, 6-bisphosphate aldolases (FBA) are cytoplasmic glycolytic enzymes, which despite lacking identifiable secretion signals, have also been found localized to the surface of several bacteria where they bind host molecules and exhibit non-glycolytic functions. Neisseria meningitidis is an obligate human nasopharyngeal commensal, which has the capacity to cause life-threatening meningitis and septicemia. Recombinant native N. meningitidis FBA was purified and used in a coupled enzymic assay confirming that it has fructose bisphosphate aldolase activity. Cell fractionation experiments showed that meningococcal FBA is localized both to the cytoplasm and the outer membrane. Flow cytometry demonstrated that outer membrane-localized FBA was surface-accessible to FBA-specific antibodies. Mutational analysis and functional complementation was used to identify additional functions of FBA. An FBA-deficient mutant was not affected in its ability to grow in vitro, but showed a significant reduction in adhesion to human brain microvascular endothelial and HEp-2 cells compared to its isogenic parent and its complemented derivative. In summary, FBA is a highly conserved, surface exposed protein that is required for optimal adhesion of meningococci to human cells.

AasP autotransporter protein of Actinobacillus pleuropneumoniae does not protect pigs against homologous challenge.

Actinobacillus pleuropneumoniae is a major respiratory pathogen of pigs; current vaccines provide only limited protection. AasP, a subtilisin-like serine protease, is a conserved outer membrane-localised autotransporter protein. We hypothesized that AasP would induce protective immunity and may thus constitute a useful component of a vaccine against A. pleuropneumoniae infection. Here we confirm experimentally that AasP is an antigenic in vivo-expressed protein. In pig protection studies, a detectable specific antibody response was induced in response to recombinant AasP. However, the vaccine was not capable of protecting pigs from colonization, infection or severe clinical disease resulting from challenge with the homologous A. pleuropneumoniae strain.

Laminin receptor initiates bacterial contact with the blood brain barrier in experimental meningitis models.

A diverse array of infectious agents, including prions and certain neurotropic viruses, bind to the laminin receptor (LR), and this determines tropism to the CNS. Bacterial meningitis in childhood is almost exclusively caused by the respiratory tract pathogens Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae, but the mechanism by which they initiate contact with the vascular endothelium of the blood brain barrier (BBB) is unknown. We hypothesized that an interaction with LR might underlie their CNS tropism. Using affinity chromatography, coimmunoprecipitation, retagging, and in vivo imaging approaches, we identified 37/67-kDa LR as a common receptor for all 3 bacteria on the surface of rodent and human brain microvascular endothelial cells. Mutagenesis studies indicated that the corresponding bacterial LR-binding adhesins were pneumococcal CbpA, meningococcal PilQ and PorA, and OmpP2 of H. influenzae. The results of competitive binding experiments suggest that a common adhesin recognition site is present in the carboxyl terminus of LR. Together, these findings suggest that disruption or modulation of the interaction of bacterial adhesins with LR might engender unexpectedly broad protection against bacterial meningitis and may provide a therapeutic target for the prevention and treatment of disease.