Influence of type IV pilus retraction on the architecture of the Neisseria gonorrhoeae-infected cell cortex

Abstract

Early in infection, Neisseria gonorrhoeae can be observed to attach to the epithelial cell surface as microcolonies and induce dramatic changes to the host cell cortex. We tested the hypothesis that type IV pili (Tfp) retraction plays a role in the ultrastructure of both the host cell cortex and the bacterial microcolony. Using serial ultrathin sectioning, transmission electron microscopy and 3D reconstruction of serial 2D images, we have obtained what we believe to be the first 3D reconstructions of the N. gonorrhoeae-host cell interface, and determined the architecture of infected cell microvilli as well as the attached microcolony. Tfp connect both wild-type (wt) and Tfp retraction-deficient bacteria with each other, and with the host cell membrane. Tfp fibres and microvilli form a lattice in the wt microcolony and at its periphery. Wt microcolonies induce microvilli formation and increases of surface area, leading to an approximately ninefold increase in the surface area of the host cell membrane at the site of attachment. In contrast, Tfp retractiondeficient microcolonies do not affect these parameters. Wt microcolonies had a symmetrical, dome-shaped structure with a circular 'footprint', while Tfp retraction-deficient microcolonies were notably less symmetrical. These findings support a major role for Tfp retraction in microvilli and microcolony architecture. They are consistent with the biophysical attributes of Tfp and the effects of Tfp retraction on epithelial cell signalling. INTRODUCTIONNeisseria gonorrhoeae (the gonococcus, or GC) remains a significant public health concern. The Gram-negative diplococcus causes a million cases of gonorrhoea annually in Western Europe and North America, and over 62 million cases worldwide (World Health Organization). Humans are the only known reservoir for GC. The exquisite tropism of GC for man and the ability of GC to establish a carrier state (Turner et al., 2002) reflect a highly evolved relationship between pathogen and host.Gonococcal attachment is promoted by type IV pili (Tfp), peritrichous fibres that are 6 nm in width and up to several micrometres in length. The Tfp filament is composed of helical polymers of pilin subunits (Craig et al., 2006;Parge et al., 1995) and minor proteins (Carbonnelle et al., 2005;Winther-Larsen et al., 2005). Tfp participate in important biological processes such as DNA uptake/genetic exchange, twitching motility, attachment, and host cell signalling (Mattick, 2002;. These activities require, or are enhanced by, the retraction of Tfp fibres (Wolfgang et al., 2000).Gonococcal Tfp retraction is an activity that requires PilT, the Tfp retraction motor subunit (Wolfgang et al., 1998). Retracting Tfp generate strong 'pull' forces, ranging from 50 picoNewtons (pN) to 1 nanoNewton (nN) (Biais et al., 2008;Opitz et al., 2009). Repeated cycles of Tfp extension, substrate attachment and Tfp retraction allow GC to crawl over surfaces and aggregate into microcolonies. Tfp retraction from bacteria in a microcolony allows the community to craw...

Seminal Plasma Promotes Neisseria gonorrhoeae Aggregation and Biofilm Formation

“…The presence of nonfunctional pili may then interfere with the ability of gonococci to achieve close cell-to-cell contact, in effect by holding neighboring bacteria at a distance. Indeed, lack of PilT function has previously been demonstrated to alter aggregation of bacteria (35) and microcolony morphology (37). The presence or absence of Opa proteins did not alter SP-mediated loss of surface adherence (Fig.…”

Dynamics of brush border remodeling induced by enteropathogenicE. coli

“…22,23 In A431 epidermoid carcinoma cells, retraction of the N. gonnorrhoeae type IV pilus induces protrusion formation and elongation. 24 To determine if BFP retraction plays a role in the elongation of microvilli observed on the surface of differentiated Caco-2 BBE cells, we utilized EPEC- bfpF +BfpF ara , a strain in which an arabinose-inducible copy of BfpF compensates for a mutated variant of BfpF that cannot retract pili. 8 We scored attachment events and binned them into the following categories: (1) microvilli that did not respond to presence of bacteria (Fig.…”

Construction and Characterization of a Derivative of Neisseria gonorrhoeae Strain MS11 Devoid of All opa Genes

“…Gonococci appear to enter HEC-1-B cells as individual bacteria and then replicate in the cells to produce intracellular inclusions of bacteria (39). Although gonococcal microcolonies are able to interact with and induce membrane ruffling on epithelial cells (14), the bulk of the vacuoles containing intracellular gonococci rarely contain more than a few bacteria (19,30). This suggests that the clumping of gonococci is actually a deterrent to cellular invasion.…”