Doctor of Philosophy, University of Illinois at Urbana Champaign (2009)
Holden Maecker, Postdoctoral Faculty Sponsor
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 FliZ-mediated 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.
View details for DOI 10.1128/JB.00635-10
View details for Web of Science ID 000283994300017
View details for PubMedID 20889744
Disulfide bond formation in periplasmic proteins is catalysed by the DsbA/DsbB system in most Gram-negative bacteria. Salmonella enterica serovar Typhimurium also encodes a paralogous pair of proteins to DsbA and DsbB, DsbL and DsbI, respectively, downstream of a periplasmic arylsulfate sulfotransferase (ASST). We show that DsbL and DsbI function as a redox pair contributing to periplasmic disulfide bond formation and, as such, affect transcription of the Salmonella pathogenicity island 1 (SPI1) type three secretion system genes and activation of the RcsCDB system, as well as ASST activity. In contrast to DsbA/DsbB, however, the DsbL/DsbI system cannot catalyse the disulfide bond formation required for flagellar assembly. Phylogenic analysis suggests that the assT dsbL dsbI genes are ancestral in the Enterobacteriaceae, but have been lost in many lineages. Deletion of assT confers no virulence defect during acute Salmonella infection of mice.
View details for DOI 10.1099/mic.0.032904-0
View details for Web of Science ID 000272918200022
View details for PubMedID 19797361
Upon contact with intestinal epithelial cells, Salmonella enterica serovar Typhimurium injects a set of effector proteins into the host cell cytoplasm via the Salmonella pathogenicity island 1 (SPI1) type III secretion system (T3SS) to induce inflammatory diarrhea and bacterial uptake. The master SPI1 regulatory gene hilA is controlled directly by three AraC-like regulators: HilD, HilC, and RtsA. Previous work suggested a role for DsbA, a periplasmic disulfide bond oxidase, in SPI1 T3SS function. RtsA directly activates dsbA, and deletion of dsbA leads to loss of SPI1-dependent secretion. We have studied the dsbA phenotypes by monitoring expression of SPI1 regulatory, structural, and effector genes. Here we present evidence that loss of DsbA independently affects SPI1 regulation and SPI1 function. The dsbA-mediated feedback inhibition of SPI1 transcription is not due to defects in the SPI1 T3SS apparatus. Rather, the transcriptional response is dependent on both the flagellar protein FliZ and the RcsCDB system, which also affects fliZ transcription. Thus, the status of disulfide bonds in the periplasm affects expression of the SPI1 system indirectly via the flagellar apparatus. RcsCDB can also affect SPI1 independently of FliZ. All regulation is through HilD, consistent with our current model for SPI1 regulation.
View details for DOI 10.1128/JB.01323-07
View details for Web of Science ID 000252080400008
View details for PubMedID 17951383