The long polar fimbria, LpfA, which is part of the H-NS/Ler regulon and is required for cell adherence of EHEC [32, 54, 55], might represent such a factor. Altogether, the cell adherence and A/E lesion phenotypes of the sspA mutant are consistent with the finding that SspA positively regulates the expression of genes encoding
the T3SS including those of the LEE by negatively affecting H-NS levels. Figure 5 SspA is required for cell adherence and A/E lesion formation. HEp-2 cells were ARRY-438162 in vivo infected by wild type EHEC EDL933 (A) and its mutant derivatives of sspA (B), sspA pQEsspA (C), sspA pQEsspA84-86 (D), hns (E) and hns sspA (F). Bacterial adherence was examined by phase-contrast images (left panels) and the actin cytoskeleton of infected HEp-2 cells by buy 4EGI-1 fluorescent microscopic images (right panels). Representative images are shown. Black and white arrowheads indicate bacteria and A/E lesions, respectively. The correlation between the effects of sspA on the transcription of H-NS/Ler-regulated virulence genes and on A/E lesion formation upon infection of HEp-2 cells supports the conclusion that SspA upregulates the expression of LEE and other virulence genes by reducing the accumulation of H-NS in the cell. A reduced cellular selleck kinase inhibitor H-NS level mediated by SspA will derepress the H-NS regulon and thereby allow the expression of transcriptional activators
such as Ler and GrlA. These two activators then form a positive transcriptional regulatory loop partially by preventing H-NS-mediated repression [28]. Accumulation of Ler will in turn antagonize H-NS function and with that enhance the expression of virulence genes controlled by Ler [26]. At present, the molecular mechanism behind SspA-mediated regulation of the H-NS level during stationary phase and in infection to facilitate virulence gene expression in EHEC is unknown. Also, it remains to be determined whether SspA directly affects transcription of virulence genes as is the case for SspA in Francisella tularensis,
where SspA along with two other transcription factors and ppGpp activates transcription Methane monooxygenase to link the nutritional status to virulence gene expression [56, 57]. We observed that SspA positively affects additional H-NS-controlled virulence traits of EHEC such as stationary phase-induced acid tolerance (data not shown), which enables survival of the pathogen during passage through the low pH environment of the human gastrointestinal tract, and thereby contributes to a low infectious dose [58, 59]. Also, sspA positively affects EHEC motility (data not shown), which could influence virulence as motility enables the pathogen to penetrate the intestinal mucus layer during colonization of host cells. This further supports an important role of sspA in EHEC virulence.