Salmonella infect their animal hosts by entering into and traversing the intestinal epithelial barrier. Penetration of the epithelium is primarily achieved by direct invasion of host epithelial cells using a panel of effector molecules secreted through a Type III secretion apparatus. SipC, which forms part of the tip of this apparatus, has long cytoplasmic N- and C-termini, which nucleate the assembly of actin filament networks that are required for bacterial engulfment. Using a yeast two-hybrid screen, we identified a number of host proteins that interact selectively with the C-terminus of SipC. These include proteins that regulate actin assembly and dynamics, as well as proteins that mediate vesicular transport. We hypothesize that Salmonella uses SipC to nucleate the assembly of signaling and cytoskeletal machinery at sites of bacterial attachment to coordinate actin remodeling with the delivery of new membrane to form the nascent phagosome. This hypothesis will be explored in Specific Aim 1. Salmonella infection triggers the recruitment of inflammatory monocytes to the intestine, which have an important role in clearance of the infection. However, Salmonella can also use phagocytes as vehicles for their systemic dissemination, and the success or failure of the host response lies in the balance between these two processes. We are examining the role of Focal Adhesion Kinase (FAK) in the migration and function of monocytic cells. Using mice that conditionally lack FAK in cells of the myeloid lineage, we found that infiltration of inflammatory macrophages into the Peyer's patches and mesenteric lymph nodes is impaired in the absence of FAK, and that this surprisingly correlates with reduced bacterial colonization of all tissues examined. Conversely, neutrophil infiltration into the same tissues is enhanced, suggesting a differential regulation of macrophage and neutrophil recruitment to infected tissues. We hypothesize that inflammatory macrophages are necessary for bacterial survival in the tissue microenvironment, by providing a protective niche for bacterial replication and dissemination, and that in the absence of this niche bacteria are more efficiently killed by infiltrating neutrophils. This hypothesis will be tested in Specific Aim 2. Recent evidence indicates the existence of a novel population of lysozyme-expressing dendritic cells unique to the Peyer's patch, that are the first cells targeted by Salmonella upon transiting the intestinal epithelium. However nothing is known about the role of these cells in Salmonella infection. Characterization of these cells forms the basis for Specific Aim 3. Together, the results of these studies will provide significant new insight into the mechanisms used by Salmonella to infect their animal hosts, and the role of the innate immune response in combating the infection. The molecular details that emerge from these studies may provide new targets for next generation antibiotics or therapies for the prevention of Salmonellosis.

Public Health Relevance

Infection with pathogenic Salmonella strains causes symptoms ranging from gastritis to potentially fatal systemic disease such as Typhoid Fever. The goals of this research are to define the host cell machinery that is subverted by Salmonella to enter the intestinal epithelium and spread systemically, and to characterize the innate immune responses to Salmonella infection.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Gastrointestinal Mucosal Pathobiology Study Section (GMPB)
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Grey, Michael J
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University of Virginia
Anatomy/Cell Biology
Schools of Medicine
United States
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Casanova, James E (2017) Bacterial Autophagy: Offense and Defense at the Host-Pathogen Interface. Cell Mol Gastroenterol Hepatol 4:237-243
Owen, Katherine A; Anderson, C J; Casanova, James E (2016) Salmonella Suppresses the TRIF-Dependent Type I Interferon Response in Macrophages. MBio 7:e02051-15
Mingo, Rebecca M; Simmons, James A; Shoemaker, Charles J et al. (2015) Ebola virus and severe acute respiratory syndrome coronavirus display late cell entry kinetics: evidence that transport to NPC1+ endolysosomes is a rate-defining step. J Virol 89:2931-43
Owen, Katherine A; Meyer, Corey B; Bouton, Amy H et al. (2014) Activation of focal adhesion kinase by Salmonella suppresses autophagy via an Akt/mTOR signaling pathway and promotes bacterial survival in macrophages. PLoS Pathog 10:e1004159
Owen, Katherine A; Abshire, Michelle Y; Tilghman, Robert W et al. (2011) FAK regulates intestinal epithelial cell survival and proliferation during mucosal wound healing. PLoS One 6:e23123
Das, Soumita; Owen, Katherine A; Ly, Kim T et al. (2011) Brain angiogenesis inhibitor 1 (BAI1) is a pattern recognition receptor that mediates macrophage binding and engulfment of Gram-negative bacteria. Proc Natl Acad Sci U S A 108:2136-41
Nichols, Christina D; Casanova, James E (2010) Salmonella-directed recruitment of new membrane to invasion foci via the host exocyst complex. Curr Biol 20:1316-20
Ly, Kim Thien; Casanova, James E (2009) Abelson tyrosine kinase facilitates Salmonella enterica serovar Typhimurium entry into epithelial cells. Infect Immun 77:60-9
Shi, Jing; Casanova, James E (2006) Invasion of host cells by Salmonella typhimurium requires focal adhesion kinase and p130Cas. Mol Biol Cell 17:4698-708
Dunphy, Jillian L; Moravec, Radim; Ly, Kim et al. (2006) The Arf6 GEF GEP100/BRAG2 regulates cell adhesion by controlling endocytosis of beta1 integrins. Curr Biol 16:315-20

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