Discovery of a newNeisseria gonorrhoeaeType IV pilus assembly factor, TfpC

Abstract

Neisseria gonorrhoeae rely 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, non-retracted 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 there are orthologues in numerous bacteria species but not all Type IV pilin expressing bacteria contain orthologous genes. Co-evolution and NMR analysis indicates that TfpC contains an N-terminal transmembrane helix, a substantial extended/unstructured region and a highly charge C-terminal coiled-coil domain.ImportanceMost 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 the Type IV pili on the bacterial cell surface. There are similar proteins found in the other members of the Neisseria genus and many other bacterial species important for human health.

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

“…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 (Table 1, Fig.…”

“…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.…”

“…Since different segments in the single stranded region of TfpR1 are predicted to interact with the 5′-UTR of dinD (nt. [43][44][45][46][47][48][49][50][51][52] and tfpC (nt. 51-63) (Fig.…”