SummarySalmonella enterica serovar Typhimurium invades intestinal epithelial cells using a type three secretion system (TTSS) encoded on Salmonella Pathogenicity Island 1 (SPI1). The SPI1 TTSS injects effector proteins into the cytosol of host cells where they promote actin rearrangement and engulfment of the bacteria. We previously identified RtsA, an AraC-like protein similar to the known HilC and HilD regulatory proteins. Like HilC and HilD, RtsA activates expression of SPI1 genes by binding upstream of the master regulatory gene hilA to induce its expression. HilA activates the SPI1 TTSS structural genes. Here we present evidence that hilA expression, and hence the SPI1 TTSS, is controlled by a feedforward regulatory loop. We demonstrate that HilC, HilD and RtsA are each capable of independently inducing expression of the hilC , hilD and rtsA genes, and that each can independently activate hilA . Using competition assays in vivo , we show that each of the hilA regulators contribute to SPI1 induction in the intestine. Of the three, HilD has a predominant role, but apparently does not act alone either in vivo or in vitro to sufficiently activate SPI1. The two-component regulatory systems, SirA/BarA and OmpR/EnvZ, function through HilD, thus inducing hilC, rtsA and hilA . However, the two-component systems are not responsible for environmental regulation of SPI1. Rather, we show that 'SPI1 inducing conditions' cause independent activation of the rtsA , hilC and hilD genes in the absence of known regulators. Our model of SPI1 regulation provides a framework for future studies aimed at understanding this complicated regulatory network.
The impact of bacterial morphology on virulence and transmission attributes of pathogens is poorly understood. The prevalent enteric pathogen
Campylobacter jejuni
displays a helical shape postulated as important for colonization and host interactions. However, this had not previously been demonstrated experimentally.
C. jejuni
is thus a good organism for exploring the role of factors modulating helical morphology on pathogenesis. We identified an uncharacterized gene, designated
pgp1
(peptidoglycan peptidase 1), in a calcofluor white-based screen to explore cell envelope properties important for
C. jejuni
virulence and stress survival. Bioinformatics showed that Pgp1 is conserved primarily in curved and helical bacteria. Deletion of
pgp1
resulted in a striking, rod-shaped morphology, making
pgp1
the first
C. jejuni
gene shown to be involved in maintenance of
C. jejuni
cell shape. Pgp1 contributes to key pathogenic and cell envelope phenotypes. In comparison to wild type, the rod-shaped
pgp1
mutant was deficient in chick colonization by over three orders of magnitude and elicited enhanced secretion of the chemokine IL-8 in epithelial cell infections. Both the
pgp1
mutant and a
pgp1
overexpressing strain – which similarly produced straight or kinked cells – exhibited biofilm and motility defects. Detailed peptidoglycan analyses via HPLC and mass spectrometry, as well as Pgp1 enzyme assays, confirmed Pgp1 as a novel peptidoglycan DL-carboxypeptidase cleaving monomeric tripeptides to dipeptides. Peptidoglycan from the
pgp1
mutant activated the host cell receptor Nod1 to a greater extent than did that of wild type. This work provides the first link between a
C. jejuni
gene and morphology, peptidoglycan biosynthesis, and key host- and transmission-related characteristics.
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