The meningococcal PilT protein is required for induction of intimate attachment to epithelial cells following pilus-mediated adhesion

Abstract

The ability of Neisseria meningitidis (MC) to interact with cellular barriers is essential to its pathogenesis. With epithelial cells, this process has been modeled in two steps. The initial stage of localized adherence is mediated by bacterial pili. After this phase, MC disperse and lose piliation, thus leading to a diffuse adherence. At this stage, microvilli have disappeared, and MC interact intimately with cells and are, in places, located on pedestals of actin, thus realizing attaching and effacing (AE) lesions. The bacterial attributes responsible for these latter phenotypes remain unidentified. Considering that bacteria are nonpiliated at this stage, pili cannot be directly responsible for this effect. However, the initial phase of pilus-mediated localized adherence is required for the occurrence of diffuse adherence, loss of microvilli, and intimate attachment, because nonpiliated bacteria are not capable of such a cellular interaction. In this work, we engineered a mutation in the cytoplasmic nucleotide-binding protein PilT and showed that this mutation increased piliation and abolished the dispersal phase of bacterial clumps as well as the loss of piliation. Furthermore, no intimate attachment nor AE lesions were observed. On the other hand, PilT ؊ MC remained adherent as piliated clumps at all times. Taken together these data demonstrate that the induction of diffuse adherence, intimate attachment, and AE lesions after pilusmediated adhesion requires the cytoplasmic PilT protein.Neisseria meningitidis (MC) is a pathogen responsible for septicemia and meningitis. To reach the meninges, MC must interact with two cellular barriers, one in the nasopharynx and one in the brain, the blood-brain barrier. A multistep model has been proposed for MC interaction with cells (1). The first step corresponds to pilus-mediated adhesion. Pili emanate from the bacterial surface and are assembled from protein subunits called pilin. They initiate adherence by serving as a long-range adhesin and recruit other bacteria into a growing microcolony. This pilus-mediated adhesion phase leads to an initial phase of localized adherence. After this first step MC disperse from these microcolonies and spread at the apical surface of the cells to form a single monolayer of bacteria covering the surface. At this stage of diffuse adherence, MC have lost their pili and are involved in an intimate attachment with disappearance of microvilli and formation of pedestals with actin polymerization, thus realizing attaching and effacing (AE) lesions resembling those observed with enteropathogenic Escherichia coli (2, 3). The loss of piliation is not caused by phase variation because bacteria recovered as cellassociated colony-forming units (cfu) are piliated. The bacterial attributes responsible for the diffuse adherence phenotype, including intimate attachment and AE lesions, have not been identified yet; however, the loss of piliation suggests that pili per se are not involved in this step. Furthermore, an inoculum of nonpiliated bac...

3D structure/function analysis of PilX reveals how minor pilins can modulate the virulence properties of type IV pili

“…6). It is possible to envision that PilX senses and modulates the tension along filaments, thereby controlling the switch between pilus extension and retraction (32) and finetuning the regular ordered aggregates of Neisseria on human cells (28). In this context, the slight unraveling of ␣2 observed in two monomers leads us to speculate that PilX could accommodate some of the retraction force through conformational changes (Fig.…”

“…Whatever aspect of Tfp biology is to be studied, pilus retraction must be taken into account because it generates remarkable mechanical forces (25), likely promoted by domain movements within PilT (26). PilT-powered retraction is indispensable for intimate adhesion (27,28), twitching motility (7,8), competence for DNA transformation (29) and signaling to host cells (30), yet it must be counteracted to achieve efficient biogenesis of pilus filaments. Accordingly, almost half of the 15 proteins essential for Tfp biogenesis in N. meningitidis are required to stabilize the fibers by counterbalancing the action of PilT (31).…”

Neisseria gonorrhoeae PilV, a type IV pilus-associated protein essential to human epithelial cell adherence

“…Pilus retraction accounts for the absence of pilus fibers in some classes of biogenesis mutants (4, 13) and has been proposed to be responsible for the dramatic reduction in piliation seen after prolonged interaction of N. meningitidis with a human epithelial cell line (25). It appears then that, under certain circumstances, the equilibrium between pilus growth and retraction can be shifted from one in which extrusion is favored to one in which retraction predominates.…”

Down‐regulation of pili and capsule of Neisseria meningitidis upon contact with epithelial cells is mediated by CrgA regulatory protein

“…The persistence of pili may hinder intimate adhesion, but pili seem to disappear during intimate adhesion (Pujol et al, 1999). The mechanism by which the pili disappear is unknown.…”