FliZ Regulates Expression of theSalmonellaPathogenicity Island 1 Invasion Locus by Controlling HilD Protein Activity inSalmonella entericaSerovar Typhimurium

Abstract

A prerequisite for Salmonella enterica to cause both intestinal and systemic disease is the direct injection of effector proteins into host intestinal epithelial cells via a type three secretion system (T3SS); the T3SS genes are carried on Salmonella pathogenicity island 1 (SPI1). These effector proteins induce inflammatory diarrhea and bacterial invasion. Expression of the SPI1 T3SS is tightly regulated in response to environmental signals through a variety of global regulatory systems. We have previously shown that three AraC-like regulators, HilD, HilC, and RtsA, act in a complex feed-forward regulatory loop to control the expression of the hilA gene, which encodes the direct regulator of the SPI1 structural genes. In this work, we characterize a major positive regulator of this system, the flagellar protein FliZ. Through genetic and biochemical analyses, we show that FliZ posttranslationally controls HilD to positively regulate hilA expression. This mechanism is independent of other flagellar components and is not mediated through the negative regulator HilE or through FliZmediated RpoS regulation. We demonstrate that FliZ controls HilD protein activity and not stability. FliZ regulates HilD in the absence of Lon protease, previously shown to degrade HilD. Indeed, it appears that FliZ, rather than HilD, is the most relevant target of Lon as it relates to SPI1 expression. Mutants lacking FliZ are significantly attenuated in their ability to colonize the intestine but are unaffected during systemic infection. The intestinal attenuation is partially dependent on SPI1, but FliZ has additional pleiotropic effects.During the course of infection, Salmonella enterica serovar Typhimurium induces inflammatory diarrhea and invades intestinal epithelial cells using a type three secretion system (T3SS); the T3SS genes are carried on Salmonella pathogenicity island 1 (SPI1) (20,51,53,55). The SPI1 locus is a 40-kb island and carries all of the genes for a functional T3SS apparatus, a number of secreted effectors, and the regulatory proteins HilA, HilC, and HilD (34). RtsA, homologous to HilD and HilC, is encoded on a 15-kb island inserted in the tRNA PheU gene (15). HilA is the master SPI1 regulator and directly binds to the promoters and activates expression of the prg-org and inv-spa operons, encoding the components of the apparatus (2,8,10,11,32). The expression of hilA is controlled by a complex feed-forward regulatory loop consisting of HilD, HilC, and RtsA, each of which can independently activate hilA expression (13) (Fig. 1). Of these three, HilD has the predominant role, but apparently, it alone cannot activate SPI1 sufficiently in vivo (13). HilC and RtsA act as amplifiers of the inducing signals. A number of additional regulatory systems also affect SPI1 regulation (1,17). Most of these systems seem to act through HilD (18), suggesting that HilD is the integration point for signal transduction into the SPI1 system. RtsA, HilD, and HilC directly activate dsbA, which encodes a periplasmic disulfide bond oxidoredu...

Mathematical model of flagella gene expression dynamics in Salmonella enterica serovar typhimurium

“…Another insight from the model is the prediction that FliZ is the secretiondependent link between flagella and the SPI1 regulatory network. FliZ has been previously shown to be activator of SPI1 gene expression, and our model predicts that this happens post-secretion (Chubiz et al 2010;Iyoda et al 2001;Lucas et al 2000). Previous modeling efforts of the flagellar regulon have been limited to the FliA-FlgM interaction only (Kalir et al 2005;Saini et al 2011).…”

“…This likely implies that post-secretion, FliZ made from the class 3 transcript is responsible for activation of SPI1 and SPI4 gene expression. The role of FliZ in activating SPI1 and SPI4 gene expression has been previously reported (Chubiz et al 2010;Kage et al 2008;Saini and Rao 2010;Saini et al 2010b).…”

“…Second, unlike E. coli, in Salmonella, the flagellar regulon is never truly in the OFF state. And last, in Salmonella, motility is a virulence factor, and proteins from the flagellar network have been shown to regulate genes on Pathogenicity Island of the organism (Chubiz et al 2010;Iyoda et al 2001;Kage et al 2008). To study these questions, we develop a model taking into account all major feedback loops in the network, and study the impact of different interactions on the dynamics of gene expression.…”

“…YdiV controls the amounts of FlhD 4 C 2 amounts in the cell by controlling its exposure to ClpXP protease (Takaya et al 2012). In addition, FliZ is also known to activate expression of the genes located on the Salmonella Pathogenicity Island-1 (SPI1) (Chubiz et al 2010).…”

A type 6 secretion system (T6SS) encoded gene within Salmonella enterica serovar Enteritidis contributes to virulence

“…The authors focused on SEN1005 for the remainder of their studies. Motility is associated with invasion of host cells and uptake by phagocytic cells [14][15][16][17][18][19][20][21]; therefore, the authors tested the mobility of the DSEN1005 strain. Under the tested conditions, the mutant displays reduced motility and expression of the flagellin structural gene, fliC.…”