Protein phosphorylation and Campylobacter jejuni pathogenesis

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ABSTRACT Campylobacter jejuni is a primary bacterial cause of gastroenteritis in the US and throughout the world, with 140 million cases worldwide and 1.3 million cases of C. jejuni gastroenteritis in the US each year. This infection leads to >30,000 deaths annually, primarily in children <5 years old in underdeveloped countries. Most cases of C. jejuni disease are sporadic and result from contaminated food (especially poultry) and exposure to environmental waters. Some C. jejuni infections (~1/1,000) lead to the development of Guillain-Barré Syndrome, the leading cause of acute paralysis in the world, as well as reactive arthritis and other sequelae. Despite the high prevalence of Campylobacter disease and some 40 years of research, the mechanisms by which C. jejuni causes disease remain incompletely understood and severely understudied. Phosphorylation of aspartate residues of bacterial regulatory proteins is a well-known mechanism for mediating changes in gene expression. However, it is increasingly recognized that phosphorylation of proteins at serine/threonine (S/T) residues also modifies protein function. The S/T phosphorylation system uses specific kinases to phosphorylate proteins, and phosphatases to remove the phosphates from S/T residues, in response to environmental signals. Importantly, this occurs in bacterial pathogens, modifying many virulence-associated characteristics. For the first time, our preliminary studies indicate provide new evidence that C. jejuni S/T phosphorylates eight proteins related to the critical pathogenic determinants of motility/chemotaxis, suggesting that S/T phosphorylation modulates C. jejuni virulence. We will define the S/T phosphorylation system in C. jejuni, according to the following hypothesis: Overall Hypothesis: C. jejuni phosphorylates proteins at S/T residues to modulate motility/chemotaxis and related pathogenesis characteristics, by means of novel S/T kinases/phosphatases. We propose a detailed study of protein S/T phosphorylation in C. jejuni, focusing on the mechanism by which the phosphorylation of motility/chemotaxis-related proteins affects their functions, as well as determining the proteins that affect the levels of S/T phosphorylation. We will use a combination of genetic, biochemical, and advanced phosphoproteomics approaches to achieve the goals outlined in these two specific aims: Aim 1. Determine the role of S/T phosphorylation for the function of motility/chemotaxis-related proteins, and Aim 2. Determine the roles of putative S/T kinase/phosphatase proteins in modifying C. jejuni proteins. Together, these will lay the groundwork for our long-term goal of identifying druggable targets to interrupt C. jejuni S/T phosphorylation and interrupting inter-host transmission and pathogenesis in humans.