Hfq is an RNA chaperone that functions as a pleiotropic regulator for RNA metabolism in bacteria. In several pathogenic bacteria, Hfq contributes indirectly to virulence by binding to riboregulators that modulate the stability or translation efficiency of RNA transcripts. To characterize the role of Hfq in the pathogenicity of Neisseria gonorrhoeae, we generated an N. gonorrhoeae hfq mutant. Infectivity and global changes in gene expression caused by the hfq mutation in N. gonorrhoeae strain MS11 were analyzed. Transcriptional analysis using a custom‐made N. gonorrhoeae microarray revealed that 369 ORFs were differentially regulated in the hfq mutant, MS11hfq, in comparison with the wild‐type strain (202 were upregulated, and 167 were downregulated). The loss‐of‐function mutation in hfq led to pleiotropic phenotypic effects, including an altered bacterial growth rate and reduced adherence to epithelial cells. Twitching motility and microcolony formation were not affected. Hfq also appears to play a minor role in inducing the inflammatory response of infected human epithelial cells. Interleukin‐8 production was slightly decreased, and activation of c‐Jun N‐terminal kinase, a mitogen‐activated protein kinase, was reduced in MS11hfq‐infected epithelial cells in comparison with wild type‐infected cells. However, activation of nuclear factor kappa B, extracellular signal‐regulated kinase 1/2 and p38 remained unchanged. The data presented suggest that Hfq plays an important role as a post‐transcriptional regulator in N. gonorrhoeae strain MS11 but does not contribute significantly to its virulence in cell culture models.

The ATPase protein PilT mediates retraction of type IV pili (Tfp). Tfp retraction of Neisseria gonorrhoeae causes many signal transduction events and changes in gene expression in infected epithelial cells. To find out whether a pilT mutation and lack of Tfp retraction, respectively, lead also to gene regulation in bacteria we performed microarrays comparing the transcriptional profiles of the N. gonorrhoeae parent strain MS11 and its isogenic pilT mutant during growth in vitro. A loss-of-function-mutation in pilT led to altered transcript levels of 63 open reading frames. Levels of pilE transcripts and its deduced protein the major Tfp subunit pilin, were increased most markedly by a mutation in pilT. Further studies revealed that pilE expression was also controlled by two other genes encoding Tfp biogenesis proteins, pilD and pilF. Our studies strongly suggest that pilE expression is a finely-tuned process.

SummaryThe early stage of infection with Neisseria gonorrhoeae (Ngo), the causative agent of gonorrhoea, is marked by type IV pilus (Tfp)-mediated attachment and the formation of bacterial microcolonies on epithelial cells. Retraction of the Ngo Tfp generates substantial force on its substrate which can elicit host cell signalling. Here, we observed that this retraction force could also activate nuclear factor (NF)-kB, the central signalling cascade of innate immunity. Using a p65-GFP-expressing epithelial cell line, we show that piliated Ngo induce asynchronous NF-kB activation in infected cells, which is temporally associated with the formation of gonococcal microcolonies. A mutant lacking PilT, an ATPase necessary for Tfp retraction, induced markedly reduced NF-kB activation. This was accompanied by decreased NF-kB target gene transcription and cytokine release. The impaired ability of the pilT mutant to activate NF-kB was compensated by applying mechanical shear stress to the infected host cells, indicating that the mechanical forces generated by retractile pili are involved in the retraction-dependent activation of NF-kB elicited by gonococcal microcolonies. Thus, our work provides evidence for an intriguing relationship between microcolony growth, pilus retraction and host cell signalling, with likely implications with regard to the course of symptomatic versus asymptomatic gonococcal infections.

In this report, we show that bacterial plasmids that harbor the Tn10 transposon (i.e., the IncHI1 plasmid R27) modify expression of different Salmonella regulons responding to the presence of tetracycline (Tc) in the medium. By using as a model the Tc-dependent upregulation of the ibpAB operon (which belongs to the heat shock regulon), we have identified Tn10-tetA (coding for a Tc efflux pump) and adjacent tetC sequences as required for ibpAB upregulation. Characterization of transcripts in the tetAC region showed that tetA transcription can continue into tetC sequences, generating a long 3'UTR sequence, which can protect transcripts from RNA processing, thus increasing the expression of TetA protein. In the presence of Tc, the DnaK and IbpA chaperones are overexpressed and translocated to the periplasm and to the membrane fraction respectively. DnaK targeting unfolded proteins is known to induce heat shock by avoiding RpoH proteolysis. We correlate expression levels of Tn10-encoded TetA protein with heat shock induction in Salmonella, likely because TetA activity compromises protein secretion.

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