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Abstract
Rhodococcus equi is a ubiquitous organism that causes pyogranulomatous pneumonia in foals. Previous work on R. equi has ascertained that the organism carries an ~80 kb virulence plasmid known as pVapA; within this plasmid, is a region that contains features consistent with a pathogenicity island. This pathogenicity island encompasses a family of virulence associated proteins, or Vaps, that do not share sequence homology with any other proteins nor have an identified function. Investigation of these Vap proteins has determined that VapA is able to allow intramacrophage replication of R. equi, wherein none of the remaining Vap proteins on pVapA share this capacity. Further studies with a vapA deletion mutant of R. equi have determined the importance of this protein during inhibition of phagosomal maturation; however, it has yet to be defined how VapA functions to promote bacterial virulence. Experimentation within has shown the capacity of recombinant VapA (rVapA) to rescue the vapA deletion mutant of R. equi during macrophage infection. Subsequently, this protein was assessed for its ability to bind host phospholipids to better understand VapAs mechanism of action. It was seen that rVapA was able to interact with phosphatidic acid. Furthermore, staining for VapA during intramacrophage replication or in vitro expression of the protein in the model organism Saccharomyces cerevisiae revealed the presence of VapA at the surface of eukaryotic membranes. Consequently, the basic amino acid residues within VapA were mutated in an attempt to hinder VapA membrane localization and phosphatidic acid binding, as it is hypothesized that VapAs interaction with phosphatidic acid is at least partially responsible for the ability of VapA to alter the outcome of R. equi intramacrophage replication. Mutating residues within the conserved domain of VapA prohibited bacterial replication within the macrophage and disrupted the membrane localization during yeast expression. It remains to be proven whether these mutations alter the ability of the protein to bind phosphatidic acid. Together, these findings suggest that VapA is able to bind phosphatidic acid and that amino acid residues located within the conserved domain of the protein are important for VapAs ability to alter macrophage infection with R. equi.