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.
Neisseria meningitidis is a human pathogen that causes septicemia and meningitis with high mortality. The disease progression is rapid and much remains unknown about the disease process. The understanding of disease development is crucial for development of novel therapeutic strategies and vaccines against meningococcal disease. The use of bioluminescent imaging combined with a mouse disease model allowed us to investigate the progression of meningococcal sepsis over time. Injection of bacteria in blood demonstrated waves of bacterial clearance and growth, which selected for Opa-expressing bacteria, indicating the importance of this bacterial protein. Further, N. meningitidis accumulated in the thyroid gland, while thyroid hormone T4 levels decreased. Bacteria reached the mucosal surfaces of the upper respiratory tract, which required expression of the meningococcal PilC1 adhesin. Surprisingly, PilC1 was dispensable for meningococcal growth in blood and for crossing of the blood-brain barrier, indicating that the major role of PilC1 is to interact with mucosal surfaces. This in vivo study reveals disease dynamics and organ targeting during meningococcal disease and presents a potent tool for further investigations of meningococcal pathogenesis and vaccines in vivo. This might lead to development of new strategies to improve the outcome of meningococcal disease in human patients.
Summary: PilC is a phase-variable protein associated with pilus-mediated adherence of pathogenic Neisseria to target cells. In this study, 24 strains of Neisseria gonorrhoeae with known epidemiological data were examined for expression of PilC. All strains produced PilC independently of serovar and site of isolation. To investigate whether the PilC protein is conserved or variable among gonococcal strains, the complete nucleotide sequence of pilC in four strains, isolated from either rectum, throat or blood, was determined. The deduced amino acid sequence in these strains differed from each other and from the two PilC proteins of N. gonorrhoeae MS11. These data demonstrate that PilC is commonly expressed, but the PilC sequence may vary among gonococcal strains.
We have previously shown that Lactobacillus gasseri Kx110A1, a human stomach isolate, can colonize mouse stomach and reduce the initial colonization of H. pylori. Here, we investigated the role of the sortase-dependent proteins (SDPs) involved in these functions by the construction of a mutant for srtA, the gene encoding the housekeeping sortase that covalently anchors SDPs to the cell surface. The mutant showed a decrease in hydrophobicity and autoaggregation under acidic conditions, indicating the effect of SDPs on cell surface properties. Correspondingly, the mutant lost the capacity to adhere to gastric epithelial cells, thus resulting in an inability to provide a physical barrier to prevent H. pylori adherence. These results indicate that sortase A is a key determinant of the cell surface properties of L. gasseri Kx110A1 and contributes to Lactobacillus-mediated exclusion of H. pylori. Understanding the molecular mechanisms by which lactobacilli antagonize H. pylori might contribute to the development of novel therapeutic strategies that take advantage of health-promoting bacteria and reduce the burden of antibiotic resistance.
The type IV pili (Tfp) of pathogenic Neisseria (i.e., N. gonorrhoeae and N. meningitidis) are essential for twitching motility. Tfp retraction, which is dependent on the ATPase PilT, generates the forces that move bacteria over surfaces. Neisseria motility has mainly been studied in N. gonorrhoeae whereas the motility of N. meningitidis has not yet been characterized. In this work, we analyzed bacterial motility and monitored Tfp retraction using live-cell imaging of freely moving bacteria. We observed that N. meningitidis moved over surfaces at an approximate speed of 1.6 μm/s, whereas N. gonorrhoeae moved with a lower speed (1.0 μm/s). An alignment of the meningococcal and gonococcal pilT promoters revealed a conserved single base pair variation in the −10 promoter element that influence PilT expression. By tracking mutants with altered pilT expression or pilE sequence, we concluded that the difference in motility speed was independent of both. Live-cell imaging using total internal reflection fluorescence microscopy demonstrated that N. gonorrhoeae more often moved with fewer visible retracting filaments when compared to N. meningitidis. Correspondingly, meningococci also displayed a higher level of piliation in transmission electron microscopy. Nevertheless, motile gonococci that had the same number of filaments as N. meningitidis still moved with a lower speed. These data reveal differences in both speed and piliation between the pathogenic Neisseria species during twitching motility, suggesting a difference in Tfp-dynamics.
In this study, we characterize the interaction between non-piliated (P−) Neisseria gonorrhoeae and human epithelial cells. P− mutants lacking the pilus subunit protein PilE attach at low levels to cells. Although the binding may not lead to heavy inflammatory responses, the interaction between P−Neisseria and host cells most probably play a role in colonization and asymptomatic carriage of the pathogen. Here we show that the adherence of P−N. gonorrhoeae is blocked by GDP-β-S [guanosine 5′-O-(thio)diphosphate], a non-hydrolyzable GTP analogue, and by C3 exotoxin, an inhibitor of the small G-protein Rho. G-protein activators such as cholera toxin, that activates Gs, and fluoroaluminate, a general G-protein activator, induced bacterial adherence. Furthermore, increase of the extracellular free [Ca2+] dramatically enhanced adherence of non-piliated Neisseria. The pharynx and the urogenital tract are natural entry sites of the pathogenic Neisseria species, and at both sites the epithelial cells can be exposed to wide variations in Ca2+ concentration. Taken together, these data show the importance of extracellular Ca2+ in the pathogenic Neisseria-host interaction, and reveal a novel function of cholera toxin, namely induction of bacterial adherence.
Lactobacilli are known to prevent colonization by many pathogens; nevertheless, the mechanisms of their protective effect are largely unknown. In this work, we investigated the role of lactobacilli during infection of epithelial cells with group A streptococci (GAS). GAS cause a variety of illnesses ranging from noninvasive disease to more severe invasive infections, such as necrotizing fasciitis and toxic shock-like syndrome. Invasion of deeper tissues is facilitated by GAS-induced apoptosis and cell death. We found that lactobacilli inhibit GAS-induced host cell cytotoxicity and shedding of the complement regulator CD46. Further, survival assays demonstrated that lactic acid secreted by lactobacilli is highly bactericidal toward GAS. In addition, lactic acid treatment of GAS, but not heat killing, prior to infection abolishes the cytotoxic effects against human cells. Since lipoteichoic acid (LTA) of GAS is heat resistant and cytotoxic, we explored the effects of lactic acid on LTA. By applying such an approach, we demonstrate that lactic acid reduces epithelial cell damage caused by GAS by degrading both secreted and cell-bound LTA. Taken together, our experiments reveal a mechanism by which lactobacilli prevent pathogen-induced host cell damage.
Bacterial auto-aggregation is a critical step during adhesion of N. meningitidis to host cells. The precise mechanisms and functions of bacterial auto-aggregation still remain to be fully elucidated. In this work, we characterize the role of a meningococcal hypothetical protein, NMB0995/NMC0982, and show that this protein, here denoted NafA, acts as an anti-aggregation factor. NafA was confirmed to be surface exposed and was found to be induced at a late stage of bacterial adherence to epithelial cells. A NafA deficient mutant was hyperpiliated and formed bundles of pili. Further, the mutant displayed increased adherence to epithelial cells when compared to the wild-type strain. In the absence of host cells, the NafA deficient mutant was more aggregative than the wild-type strain. The in vivo role of NafA in sepsis was studied in a murine model of meningococcal disease. Challenge with the NafA deficient mutant resulted in lower bacteremia levels and mortality when compared to the wild-type strain. The present study reveals that meningococcal NafA is an anti-aggregation factor with strong impact on the disease outcome. These data also suggest that appropriate bacterial auto-aggregation is controlled by both aggregation and anti-aggregation factors during Neisseria infection in vivo.
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