Date Published: October 4, 2018
Publisher: Public Library of Science
Author(s): Jennifer M. Willingham-Lane, Garry B. Coulson, Mary K. Hondalus, Enrico Baruffini.
Rhodococcus equi is a facultative intracellular bacterium of macrophages and is an important pathogen of animals and immunocompromised people wherein disease results in abcessation of the lungs and other sites. Prior work has shown that the presence of the major virulence determinant, VapA, encoded on the pVAPA-type plasmid, disrupts normal phagosome development and is essential for bacterial replication within macrophages. pVAPA- type plasmids are typical of R. equi strains derived from foals while strains from pigs carry plasmids of the pVAPB-type, lacking vapA, and those from humans harbor various types of plasmids including pVAPA and pVAPB. Through the creation and analysis of a series of gene deletion mutants, we found that vapK1 or vapK2 is required for optimal intracellular replication of an R. equi isolate carrying a pVAPB plasmid type. Complementation analysis of a ΔvapA R. equi strain with vapK1 or vapK2 showed the VapK proteins of the pVAPB-type plasmid could restore replication capacity to the macrophage growth-attenuated ΔvapA strain. Additionally, in contrast to the intracellular growth capabilities displayed by an equine R. equi transconjugant strain carrying a pVAPB-type plasmid, a transconjugant strain carrying a pVAPB-type plasmid deleted of vapK1 and vapK2 proved incapable of replication in equine macrophages. Cumulatively, these data indicate that VapK1 and K2 are functionally equivalent to VapA.
The Gram-positive aerobic bacterium, Rhodococcus equi, is found in the soil worldwide and in the fecal matter of herbivores [1, 2]. In addition to being an environmental saprophyte, R. equi is a facultative intracellular pathogen of macrophages, capable of causing disease in a variety of susceptible hosts. The most well-studied disease presentation is in foals, but other hosts include pigs, cattle, and humans. In foals and people, bacterial exposure is generally through inhalation and disease results in pneumonia with the formation of pyogranulomatous lesions within the lungs [2–4]. Disease in swine and cattle typically presents as submandibular lymphadenitis and abscessation of the respiratory lymph nodes, respectively [5–8]. All R. equi isolates from diseased foals and most from affected swine and humans carry a large circular virulence-associated plasmid, whereas, cattle R. equi isolates possess a linear plasmid [9–11]. The R. equi isolates obtained from equine, swine, and cattle carry distinct plasmid types referred to as pVAPA, pVAPB, and pVAPN respectively whereas, R. equi strains infecting humans may carry any of these plasmid types. Located on all of these plasmids is a pathogenicity island (PAI) acquired from a historical horizontal gene transfer event . Though the genetic composition the PAI regions of these plasmid types vary, all contain a novel family of genes known as the virulenceassociatedproteins or vap family [9, 11].
Although the pVAPA-, pVAPB- and pVAPN-type plasmids carried by disease causing strains of R. equi isolates differ greatly in their PAI regions [9, 10], there are conserved components, the most intriguing of which is the presence of multiple members of the unique and R. equi specific vap gene family, all of which share considerable homology at their C-termini [9, 10]. Amplification of vap genes is evident across all plasmid types perhaps indicative of selective pressures driving the maintenance of the vap genes in these plasmids. However, it is clear from this work and our previously published research [14, 24], that the vap genes are not equivalent in function.