Salmonella infections are a major health problem worldwide. Salmonella causes disease by expressing genes that are located on pathogenicity islands. Pathogenicity islands are large tracts of acquired genes that promote virulence and have a different AT content from the rest of the bacterial chromosome. Genes that reside on Salmonella Pathogenicity Island-1 (SPI-1) enable Salmonella to adhere to and invade epithelial cells, whereas SPI-2 genes are required for systemic infection. Specialized secretory systems termed type three secretion systems are encoded on each pathogenicity island that provide Salmonella with the means to secrete effector molecules into the host that alter host functions and promote pathogenesis. The present proposal focuses on the control of SPI-2 gene expression. It is one of the most critical virulence determinants of Salmonella, yet the complex molecular biology of its transcriptional regulation, is not understood. In particular, the identification of the pathways for gene expression in vivo remains poorly defined. Our research is focused on defining these pathways in molecular terms. SPI-2 gene expression is controlled by a two-component regulatory system SsrA/B, whose expression is in turn controlled by additional regulatory networks, including the EnvZ/OmpR two- component system, the transcriptional activator SlyA and the global repressor H-NS. The complex regulation of SPI-2 requires integration of multiple environmental signals to ensure that these important virulence genes are expressed at the appropriate time within the macrophage phagosome. In this proposal, we will test the hypothesis that EnvZ senses the acidified cytoplasm when Salmonella resides in the macrophage vacuole. Using hydrogen-deuterium exchange mass spectrometry, we will identify the regions of EnvZ that change conformation as a result of acid stress. The levels of OmpR~P produced will be determined using phos-tag reagents and the effect on SsrA/B expression and activation will be determined. Our preliminary studies indicate that the Salmonella cytoplasm is acidified to pH 5.7 in the vacuole and that this process is completely dependent on OmpR. We will identify the OmpR targets involved in intracellular acidification by microarray and examine their effect on enhancing Salmonella survival in the vacuole. Knowing the targets provides us with a strategy to inhibit Salmonella replication in the vacuole, which is required for dissemination and systemic infection. The mechanism of gating that controls the secretion of effector molecules into the host macrophage is controversial and may vary in different host backgrounds. Using the tools we developed in the previous funding period, we will examine type three secretion in HeLa cells, macrophages and 3T3 fibroblasts and identify host factors that alter Salmonella function during infection. As a result of our studies, we will have a enhanced understanding of the molecular events that occur as a result of Salmonella infection and how these processes can be targeted in the host.

Public Health Relevance

Veterans suffer from both acute and chronic bacterial infections. Our studies are mechanistic, aimed at understanding how pathogens respond to environmental stimuli and activate expression of virulence genes. The two-component systems that we study have implications beyond Salmonella, extending to all other pathogens. Drug-resistant Salmonella infections are a problem in all hospitals, including VA hospitals and septicemia due to Salmonella is a problem in immune-compromised HIV positive patients, a high incidence occurs in veterans. Salmonella-associated acute gastroenteritis is a significant cause of morbidity among deployed military personnel and high population density closed communities (e.g. military bases). The number of typhoid fever cases exceeds 20 million per year worldwide and remains a potential concern among military personnel. Reactive arthritis (ReA or Reiter syndrome) is an autoimmune condition that develops in response to Salmonella infection; outbreaks of enteric Reiter syndrome have been reported aboard military vessels.

National Institute of Health (NIH)
Veterans Affairs (VA)
Non-HHS Research Projects (I01)
Project #
Application #
Study Section
Infectious Diseases B (INFB)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Jesse Brown VA Medical Center
United States
Zip Code
Desai, Stuti K; Kenney, Linda J (2017) To ?P or Not to ?P? Non-canonical activation by two-component response regulators. Mol Microbiol 103:203-213
Lin, Meishan; Zhang, Ge; Fahie, Monifa et al. (2017) Engineering a Novel Porin OmpGF Via Strand Replacement from Computational Analysis of Sequence Motif. Biochim Biophys Acta Biomembr 1859:1180-1189
Desai, Stuti K; Winardhi, Ricksen S; Periasamy, Saravanan et al. (2016) The horizontally-acquired response regulator SsrB drives a Salmonella lifestyle switch by relieving biofilm silencing. Elife 5:
Foo, Yong Hwee; Spahn, Christoph; Zhang, Hongfang et al. (2015) Single cell super-resolution imaging of E. coli OmpR during environmental stress. Integr Biol (Camb) 7:1297-308
Chakraborty, Smarajit; Mizusaki, Hideaki; Kenney, Linda J (2015) A FRET-based DNA biosensor tracks OmpR-dependent acidification of Salmonella during macrophage infection. PLoS Biol 13:e1002116
Winardhi, Ricksen S; Yan, Jie; Kenney, Linda J (2015) H-NS Regulates Gene Expression and Compacts the Nucleoid: Insights from Single-Molecule Experiments. Biophys J 109:1321-9
Cheong, Fook Chiong; Wong, Chui Ching; Gao, YunFeng et al. (2015) Rapid, high-throughput tracking of bacterial motility in 3D via phase-contrast holographic video microscopy. Biophys J 108:1248-56
Foo, Yong Hwee; Gao, Yunfeng; Zhang, Hongfang et al. (2015) Cytoplasmic sensing by the inner membrane histidine kinase EnvZ. Prog Biophys Mol Biol 118:119-29
Lim, Ci Ji; Kenney, Linda J; Yan, Jie (2014) Single-molecule studies on the mechanical interplay between DNA supercoiling and H-NS DNA architectural properties. Nucleic Acids Res 42:8369-78
Adediran, Jimmy; Leatham-Jensen, Mary P; Mokszycki, Matthew E et al. (2014) An Escherichia coli Nissle 1917 missense mutant colonizes the streptomycin-treated mouse intestine better than the wild type but is not a better probiotic. Infect Immun 82:670-82

Showing the most recent 10 out of 14 publications