SummaryThe attachment of pathogenic Neisseria species to human cells, in which type IV pili (Tfp) play a key but incompletely defined role, depends on the ability of these bacteria to establish contacts with the target cells but also interbacterial interactions. In an effort to improve our understanding of the molecular mechanisms of N. meningitidis adherence to human cells, we screened a collection of defined mutants for those presenting reduced attachment to a human cell line. Besides underscoring the central role of Tfp in this process, this analysis led to the identification of mutants interrupted in a novel gene termed pilX , that displayed an adherence as impaired as that of a nonpiliated mutant but quantitatively and qualitatively unaltered fibres. Moreover, the pilX gene, which encodes a pilin-like protein that copurifies with Tfp fibres, was also found to be essential for bacterial aggregation. We provide here several piece of evidence suggesting that PilX has intrinsic aggregative but no adhesive properties and that the reduced numbers of adherent bacteria seen with a pilX mutant result from the absence of interbacterial interactions. These data extend the current model for Tfp-facilitated adherence of N. meningitidis to human cells by suggesting that the pili lead to an increase in net initial adherence primarily by mediating a cooperation between the bacteria, which is supported by the finding that a major effect on initial adherence could be observed in a wild-type (WT) genetic background after a mechanical removal of the bacterial aggregates.
SummaryType IV pili (Tfp) play a critical role in the pathogenic lifestyle of Neisseria meningitidis and N. gonorrhoeae , notably by facilitating bacterial attachment to human cells, but our understanding of their biogenesis, during which the fibres are assembled in the periplasm, then emerge onto the cell surface and are stabilized, remains fragmentary. We therefore sought to identify the genes required for Tfp formation in N. meningitidis by screening a genome-wide collection of mutants for those that were unable to form aggregates, another phenotype mediated by these organelles. Fifteen proteins, of which only seven were previously characterized, were found to be essential for Tfp biogenesis. One novel component, named PilW, was studied in more detail. We found that PilW is an outer-membrane protein necessary for the stabilization of the fibres but not for their assembly or surface localization, because Tfp could be restored on the surface in a pilW mutant by a mutation in the twitching motility gene pilT . However, Tfp-linked properties, including adherence to human cells, were not restored in a pilW/T mutant, which suggests that PilW is also essential for the functionality of the fibres. Together with the finding that PilW is important for the stability of PilQ multimers, our results extend the current model for Tfp biogenesis by suggesting that a multiprotein machinery in the outer-membrane is involved in the terminal stage of Tfp biogenesis during which growing fibres are not only stabilized, but also become perfectly functional.
Multiple regulated neutrophil cell death programs contribute to host defense against infections. However, despite expressing all necessary inflammasome components, neutrophils are thought to be generally defective in Caspase-1-dependent pyroptosis. By screening different bacterial species, we found that several Pseudomonas aeruginosa (P. aeruginosa) strains trigger Caspase-1-dependent pyroptosis in human and murine neutrophils. Notably, deletion of Exotoxins U or S in P. aeruginosa enhanced neutrophil death to Caspase-1-dependent pyroptosis, suggesting that these exotoxins interfere with this pathway. Mechanistically, P. aeruginosa Flagellin activates the NLRC4 inflammasome, which supports Caspase-1-driven interleukin (IL)-1β secretion and Gasdermin D (GSDMD)-dependent neutrophil pyroptosis. Furthermore, P. aeruginosa-induced GSDMD activation triggers Calcium-dependent and Peptidyl Arginine Deaminase-4-driven histone citrullination and translocation of neutrophil DNA into the cell cytosol without inducing extracellular Neutrophil Extracellular Traps. Finally, we show that neutrophil Caspase-1 contributes to IL-1β production and susceptibility to pyroptosis-inducing P. aeruginosa strains in vivo. Overall, we demonstrate that neutrophils are not universally resistant for Caspase-1-dependent pyroptosis.
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