Sticky and Sweet: The Role of Post-Translational Modifications on Neisserial Pili

Neisseria gonorrhoeae and N. meningitidis are obligate human pathogens of medical importance which are highly related at the genomic level and have a conserved array of pathogenicity determinants (Virji, 2009). N. meningitidis resides in the human nasopharynx and is the causative agent of transmissible meningitis and septic shock (Stephens et al., 2007). N. gonorrhoeae is a resident of the urogenital tract, is sexually transmitted and is the causative agent of gonorrhoeae and pelvic inflammatory disease in women (Edwards and Apicella, 2004). Both organisms colonize the mucosal epithelial cell surface via Type IV pili (Tfp), which engages the I-domain containing integrins (Edwards and Apicella, 2004) triggering the recruitment of factors ultimately responsible for the formation and extension of pseudopodia from the epithelial cell (Virji, 2009). During this period, the bacteria proliferate on the cell surface forming microcolonies which eventually disperse on the surface of the epithelial cells. Intimate association between the bacterium and host cell is mediated by the bacterial opacity proteins (Opa) with the epithelial host cell receptor carcinoembryonic antigen-related cell adhesion molecule (CEACAM, CD66) which triggers a cascade of signals resulting in internalization of the bacteria into the host cell (Virji, 2009). Not only is Tfp required for the initial attachment events to the host cell surface, it is also involved in twitching motility (Merz et al., 2000). Tfp is extruded through an outer membrane secretin, PilQ, and is retracted by the motor protein PilT into the periplasm where the fiber is depolymerized. occupancy) of PilE from N. meningitidis strain 8013 (Chamot-Rooke et al., 2011). The equivalent positions in the gonococcal PilE from gonococcal strain MS11 are occupied with phosphoserine (serine 68) and PG (serine 94; Forest et al., 1999). Why there is a difference in the occupancy of serine 68/69 with PG has so far not been explored. However, both PilE structures are glycosylated with different glycans at serine 63 and therefore these attachments may influence the availability of serine 68/69 to PptB. Chamot-Rooke et al. (2011) have shown that the occupancy of PG on PilE controls the ability to form bundled pili. To demonstrate this, they used N. meningitidis strain 8013 which has pilin decorated with GATDH and two PG residues. They showed that PptB is upregulated during attachment to host cells, leading to increased substitution of PilE with PG, ultimately resulting in the separation of the bundled pili into single strands. This observation was used to suggest that the bacterial cells without bundled pili were being released from the microcolony for further dissemination across the host cell surface. Depending on the host strain, glycosylation at serine 63 can consist of the addition of an O-linked trisaccharide (Oac) Gal(β1-4)Gal(α1-3)2,4-diacetamido-2,4,6trideoxyhexose (DATDH) or truncated versions thereof (Power et al., 2003). The genes encoding the enzymes required for the synthesis and transfer of the glycan to PilE have been named pilin glycosylation (pgl) genes. Truncated glycan variants, in which the terminal O-acetyl group (OAc) is missing on the trisaccharide or in which either one or both galactosyl groups are absent, are generated by the phase variable expression of the O-acetyl transferase, PglI (Warren et al., 2004), and the galactosyl transferases, PglA ,and PglE (Power et al., 2003). DATDH is synthesized by the concerted action of three enzymes PglBCD. In a subset of meningococcal isolates, a variant glycosyl group, glyceroamido acetamido trideoxyhexose Cycles of Tfp extension, surface binding, and subsequent retraction result in the ability of the bacterium to crawl across a solid surface. Retraction of a single Tfp fiber generates forces of 50–100 pN, however, this is greatly enhanced when the fibers form bundles, typically containing 8–10 strands, which are co-operatively retracted resulting in the generation of forces in the nanonewton range (Biais et al., 2008). Thus the Tfp motor of Neisseria sp. is one of the most powerful biological motors currently known. Recent work has now shown that post-translational modifications to the pilin subunit, PilE, are the major contributors to the formation and disaggregation of bundled pili. PilE is expressed as a prepilin which undergoes processing by a prepilin peptidase, PilD, during export into the periplasm where subunits are arranged in a helix for extrusion through PilQ to form the shaft of the pilus. During this process of transport, cleavage and polymerization, PilE also undergoes multisite hierarchical post-translational modification (Aas et al., 2006). These O-linked additions are zwitterionic polar residues such as phosphocholine (PC), phosphoethanolamine (PE), and phosphoglycerol (PG), in addition to a separate glycan subunit of variable composition (Aas et al., 2006). The addition of PC and PE to PilE is catalyzed by the pilin phosphoethanolamine transferase (PptA; Aas et al., 2006) which can add up to five residues (four PE and one PC) to serine 68 and serine 156 of PilE from N. gonorrhoeae strain MS11 (Aas et al., 2006). The appearance of PC is due to the conversion of PE to PC by an unknown mechanism after attachment to PilE (Naessan et al., 2008). Variable substitution patterns can arise through the phase variable expression of PptA and the presence of the pilin-like protein, PilV, in the shaft (Aas et al., 2006). The addition of phosphoglycerol (PG) is catalyzed by PptB which adds a single residue to either serine 69 (100% occupancy) or serine 93 (15%