Discovery of a New Neisseria gonorrhoeae Type IV Pilus Assembly Factor, TfpC

Abstract

Neisseria gonorrhoeae relies on type IV pili (T4p) to promote colonization of their human host and to cause the sexually transmitted infection gonorrhea. This organelle cycles through a process of extension and retraction back into the bacterial cell. Through a genetic screen, we identified the NGO0783 locus of N. gonorrhoeae strain FA1090 as containing a gene encoding a protein required to stabilize the type IV pilus in its extended, nonretracted conformation. We have named the gene tfpC and the protein TfpC. Deletion of tfpC produces a nonpiliated colony morphology, and immuno-transmission electron microscopy confirms that the pili are lost in the ΔtfpC mutant, although there is some pilin detected near the bacterial cell surface. A copy of the tfpC gene expressed from a lac promoter restores pilus expression and related phenotypes. A ΔtfpC mutant shows reduced levels of pilin protein, but complementation with a tfpC gene restored pilin to normal levels. Bioinformatic searches show that there are orthologues in numerous bacterial species, but not all type IV pilin-expressing bacteria contain orthologous genes. Coevolution and nuclear magnetic resonance (NMR) analysis indicates that TfpC contains an N-terminal transmembrane helix, a substantial extended/unstructured region, and a highly charged C-terminal coiled-coil domain. IMPORTANCE Most bacterial species express one or more extracellular organelles called pili/fimbriae that are required for many properties of each bacterial cell. The Neisseria gonorrhoeae type IV pilus is a major virulence and colonization factor for the sexually transmitted infection gonorrhea. We have discovered a new protein of Neisseria gonorrhoeae called TfpC that is required to maintain type IV pili on the bacterial cell surface. There are similar proteins found in other members of the Neisseria genus and many other bacterial species important for human health.

The Neisseria gonorrhoeae type IV pilus promotes resistance to hydrogen peroxide- and LL-37-mediated killing by modulating the availability of intracellular, labile iron

“…gonorrhoeae strain FA1090 pilin variant 1-81-S2 (confirmed using primers pilRBS and SP3A ( Table 3 ) [ 67 ]). N-1-60 carries multiple point mutations in the pilE guanine quadruplex sequence upstream of pilE [ 68 ] (confirmed with primers USS2 and pilAREV [ 66 ] ( Table 3 )) and the phase-locked “on” pilC2 (NGO0055) sequence (confirmed using primers pilCfor and pilCdownstream ( Table 3 )), not pilC1 as previously indicated [ 66 ]. For all strains, the 1-81-S2 pilE sequence was confirmed in PCR products using primers pilRBS and SP3A ( Table 3 ) in addition to the non-variable guanine quadruplex and the phase-locked pilC 2.…”

“…Strains used in this study are listed in Table 2 . The parental strain N-1-60 [ 66 ] is N . gonorrhoeae strain FA1090 pilin variant 1-81-S2 (confirmed using primers pilRBS and SP3A ( Table 3 ) [ 67 ]).…”

Identification and initial characterization of a new pair of sibling sRNAs of Neisseria gonorrhoeae involved in type IV pilus biogenesis

“…For initial validation experiments, hits showing complementarity between the predicted sRNA single stranded region and the mRNA region comprising RBS and start codon were taken into consideration (Tables S3 and S4). NGFG_01006 encoding the type IV pilus biogenesis factor TfpC [52] and NGFG_00693 encoding an aminotransferase were the only hits meeting these criteria which scored in the outcome of both in silico target prediction tools. However, no significant differences in the transcript amounts of tfpC and NGFG_00693 were observed in qRT-PCR experiments performed on RNA extracted from MS11 wild-type and the sRNA deletion mutant MS11 ΔtfpR1.…”

“…TfpC, which was only recently identified as a new type IV pilus biogenesis factor, is located in the periplasm and harbours an N-terminal transmembrane domain anchoring the protein to the inner membrane. Deletion of tfpC resulted in nonpiliated colony morphology and reduced transformation competence and it was hypothesized that TfpC stabilizes the pilus in its extended conformation [52]. mRNA quantification indicated that NGFG_01479, encoding the minor pilin PilV [72], is controlled by TfpR1 as well (, ).…”

Structure Determination of Microtubules and Pili: Past, Present, and Future Directions

“…Although cryo-ET has not been used to study the CU pathway, it has been successfully implemented to elucidate the low-resolution features of T4P and T2SS pseudo-pilus assembly devices (Gold et al, 2015;Chang et al, 2016;Ghosal et al, 2019). Work on the T4P systems has revealed pilus structures emanating into the extracellular space (Gold et al, 2015;Chang et al, 2016) and with the expected future increases in cryo-ET resolution, as discussed above, in the future cryo-ET may be able to provide greater detail of how these pili form at the membrane and the role of pilus-specific assembly factors in their biogenesis (Hu et al, 2020). Cryo-EM has had a major impact on the of study filamentous systems over the past decade and it will be interesting to see how EM and its incorporation with other approaches (e.g.…”