Host species tropism and the genetic requirements for intracellular replication of Rhodococcus equi
Willingham, Jennifer Michelle
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Rhodococcus equi is an environmental Gram-positive bacterium found worldwide. In addition to being a soil-dwelling organism, it is a pathogen of young foals and pigs who may develop severe pyogranulomatous pneumonia or abscessation of the mandibular lymph nodes, respectively. All R. equi isolates from foals contain a pVAPA-type plasmid, while the majority of swine isolates carry a pVAPB-type plasmid. pVAPA-type plasmid carriage has been demonstrated to enable intracellular replication of the bacteria in host macrophages, as well as, being critical for disease development in foals and in in vivo mouse models. The pathogenicity island (PAI) regions of pVAPA-type and pVAPB-type plasmids house a novel gene family known as the virulence associated proteins or vap family. Of interest is that vap gene composition is distinct amongst these plasmid types, with plasmids from equine isolates possessing six vap genes including the crucial, intracellular replication essential virulence determinant vapA, while plasmids from swine isolates contain vapB and six other unique vaps. The precise mechanism of action of VapA is yet to be determined, however, it has been shown to interfere with acidification of the R. equi-containing phagosome thereby promoting a niche suitable for intracellular growth. Prior to this work, the intracellular growth capabilities of pVAPB-carrying pig isolates had not been evaluated. Therefore, we first sought to analyze the intramacrophage growth potential of several swine isolates and secondly to establish whether plasmid type, pVAPA vs pVAPB, dictates host species-specific intracellular replication capabilities. Examination of various R. equi isolates containing either a pVAPA-type or pVAPB-type plasmid in macrophages of murine, equine, and swine origin revealed that while plasmid possession was critical for intracellular replication, plasmid type was not associated with species-specific intramacrophage growth. Having determined that R. equi isolates possessing pVAPB-type plasmids are capable of intramacrophage replication in the absence of vapA, the component(s) of the pVAPB-type plasmid enabling intracellular growth were investigated. Through analysis of the intramacrophage growth phenotypes of a series of deletion mutants and complementation analysis, it was determined that the pVAPB-type plasmid encoded vapK1 and vapK2 genes are required for replication in macrophages and are functionally equivalent to VapA.