Novel Non-Antimicrobial Treatment For Enteric Salmonellosis

In the US, non-typhoidal Salmonellae are the leading cause of human bacterial foodborne gastroenteritis. Adding to this problem is the alarming increase in Salmonella strains that are resistant to multiple antibiotics. In order to address the growing concernabout multi-drug resistant Salmonella infections in humans and production animals, producers need new strategies to decrease the use of antibiotics while preserving the ability to prevent and/or treat infection. Therefore, we propose an innovative antiinfective strategy to combat Salmonella that targets the host response, rather than the pathogen.Our long-term goal is to design novel strategies to inhibit Salmonella shedding in cattle. The purpose of our proposal is to establish the host cell protein MARCKS (Myristoylated Alanine Rich C-Kinase Substrate) as a viable, non-antimicrobial target to combat enteric salmonellosis in cattle. MARCKS is a ubiquitously expressed hostcell protein that regulates the actin cytoskeleton and antimicrobial responses of neutrophils. Actin is targeted by Salmonella for epithelial invasion and intracellular survival. Neutrophils play a key role in Salmonella enteritis, causing inflammation that ultimately helps Salmonella to colonize the gut. Our preliminary data implicate MARCKSin Salmonella-induced neutrophil activation and Salmonella infection in calves. We show that a MARCKS inhibiting peptide alters Salmonella epithelial invasion and survival during infection. Therefore, we hypothesize that MARCKS protein functionis essential for Salmonella-induced neutrophil recruitment and activation, epithelial invasion, and the overall survival of Salmonella in the host intestine. To address this hypothesis, we propose the following objectives:Objective 1 - Determine the effects of MARCKS inhibition on neutrophil antibacterial responses to Salmonella in vitro and during calf infection. (A) We will use a function blocking peptide (MANS) to determine MARCKS role in Salmonella-mediated neutrophil adhesion, migration, phagocytosis, cytokine and reactive oxygen species production, and Salmonella killing in vitro. (B) We will establish the effects of MARCKS inhibition either prior to or after infection on neutrophil recruitment and cytokine production in a Salmonella calf infection model.Objective 2 - Determine the effects of MARCKS inhibition on Salmonella epithelial invasion in vitro and during calf infection. (A) We will use a function blocking peptide (MANS) and protein knockdown (siRNA) to determine MARCKS role in Salmonella epithelial invasion and magnification of the inflammatory response in vitro. (B) We will establish the effects of MARCKS inhibition either prior to or after infection on Salmonella intestinal colonization and mucosal injury in a calf infection model.Objective 3 - Determine the effects of in vivo MARCKS inhibition on development of antimicrobial resistance. We will establish the effects of in vivo MARCKS inhibition on antimicrobial resistance development in Salmonella and the intestinal microflora.