INVESTIGATOR: Anne Reid, Salve Reginal University
MENTOR: David Nelson, University of Rhode Island
THEME: Environmental Health Science

ABSTRACT: Over 1 million cases of salmonellosis occur annually in the US, with outbreaks increasingly linked to fruits and vegetables. The role of flagellin methylation in S. enterica is unknown, but hydrophobic interactions, which would be favored by lysine methylation, are known to mediate adhesion to biotic and abiotic surfaces. An understanding of the molecular mechanisms used by S. enterica to interact with plant surfaces will inform the development of strategies to disrupt these interactions, ultimately reducing the burden of disease. Flagellin methylation may also mediate interactions between S. enterica and its human host, and this knowledge could lead to an improved understanding of this pathogen’s host range and disease progression. 
Specific Aim 1: Characterization of flagellin methylation. Flagellin methylation has only to date been described in S. Typhimurium. It is hypothesized that while all non-typhoidal serovars methylate their flagellins, serovars associated with leafy green outbreaks will display increased methylation relative to those that are not linked to this commodity. Lysine residues modified by methylation are predicted to occur in the outward-facing variable domains of the flagellin proteins. Methylation sites are expected to be conserved in serovars whose flagellins display high levels of sequence identity. Flagella will be purified from a subset of S. enterica serovars and probed with an anti-methylated lysine antibody to determine presence and relative levels of methylation. Deletion mutants will be generated to allow for the independent analysis of phase I and II flagellins in serovars with biphasic flagella. Flagellin bands will be excised from SDS-PAGE gels and subjected to methylation mapping using mass spectrometry to identify methylation sites and relative abundance of methylated peptides. Specific Aim 2: Determination of the role of flagellin methylation in cell-cell interactions. The increased hydrophobicity conferred by methylation is predicted to improve attachment to leafy greens and host cells. The N-lysine methylase gene will be deleted in S. enterica serovars, and the attachment of wild-type and methylation-deficient mutants to plant surfaces (leafy greens and a plant cell wall model) will be compared. The mutants and their parent strains will also be subjected to attachment and uptake assays in cultured epithelial and macrophage cells to determine the role of methylation in host cell interactions.

RELEVANCE: The contamination of foods by S. enterica is an important environmental health issue, as their consumption can lead to costly and devastating multi-state outbreaks. Understanding how flagellin methylation contributes to this pathogen’s interaction with plant surfaces could lead to the development of strategies to interfere with these interactions and decrease the burden of salmonellosis in the US. These findings could have broader implications as flagellin methylation occurs in additional human pathogens (e.g., Yersina).