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Identification of a VapA virulence factor functional homolog in Rhodococcus equi isolates housing the pVAPB plasmid.

ABSTRACT: 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 ?vapA 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 ?vapA 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.

SUBMITTER: Willingham-Lane JM

PROVIDER: S-EPMC6171844 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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VapA of Rhodococcus equi binds phosphatidic acid.

Project description:Rhodococcus equi is a multihost, facultative intracellular bacterial pathogen that primarily causes pneumonia in foals less than six months in age and immunocompromised people. Previous studies determined that the major virulence determinant of R. equi is the surface bound virulence associated protein A (VapA). The presence of VapA inhibits the maturation of R. equi-containing phagosomes and promotes intracellular bacterial survival, as determined by the inability of vapA deletion mutants to replicate in host macrophages. While the mechanism of action of VapA remains elusive, we show that soluble recombinant VapA32-189 both rescues the intramacrophage replication defect of a wild type R. equi strain lacking the vapA gene and enhances the persistence of nonpathogenic Escherichia coli in macrophages. During macrophage infection, VapA was observed at both the bacterial surface and at the membrane of the host-derived R. equi containing vacuole, thus providing an opportunity for VapA to interact with host constituents and promote alterations in phagolysosomal function. In support of the observed host membrane binding activity of VapA, we also found that rVapA32-189 interacted specifically with liposomes containing phosphatidic acid in vitro. Collectively, these data demonstrate a lipid binding property of VapA, which may be required for its function during intracellular infection.

| S-EPMC5777868 | biostudies-literature

The Rhodococcus equi virulence protein VapA disrupts endolysosome function and stimulates lysosome biogenesis.

Project description:Rhodococcus equi (R. equi) is an important pulmonary pathogen in foals that often leads to the death of the horse. The bacterium harbors a virulence plasmid that encodes numerous virulence-associated proteins (Vaps) including VapA that is essential for intracellular survival inside macrophages. However, little is known about the precise function of VapA. Here, we demonstrate that VapA causes perturbation to late endocytic organelles with swollen endolysosome organelles having reduced Cathepsin B activity and an accumulation of LBPA, LC3 and Rab7. The data are indicative of a loss of endolysosomal function, which leads cells to upregulate lysosome biogenesis to compensate for the loss of functional endolysosomes. Although there is a high degree of homology of the core region of VapA to other Vap proteins, only the highly conserved core region of VapA, and not VapD of VapG, gives the observed effects on endolysosomes. This is the first demonstration of how VapA works and implies that VapA aids R. equi survival by reducing the impact of lysosomes on phagocytosed bacteria.

| S-EPMC5387311 | biostudies-literature

An Invertron-Like Linear Plasmid Mediates Intracellular Survival and Virulence in Bovine Isolates of Rhodococcus equi.

Project description:We report a novel host-associated virulence plasmid in Rhodococcus equi, pVAPN, carried by bovine isolates of this facultative intracellular pathogenic actinomycete. Surprisingly, pVAPN is a 120-kb invertron-like linear replicon unrelated to the circular virulence plasmids associated with equine (pVAPA) and porcine (pVAPB variant) R. equi isolates. pVAPN is similar to the linear plasmid pNSL1 from Rhodococcus sp. NS1 and harbors six new vap multigene family members (vapN to vapS) in a vap pathogenicity locus presumably acquired via en bloc mobilization from a direct predecessor of equine pVAPA. Loss of pVAPN rendered R. equi avirulent in macrophages and mice. Mating experiments using an in vivo transconjugant selection strategy demonstrated that pVAPN transfer is sufficient to confer virulence to a plasmid-cured R. equi recipient. Phylogenetic analyses assigned the vap multigene family complement from pVAPN, pVAPA, and pVAPB to seven monophyletic clades, each containing plasmid type-specific allelic variants of a precursor vap gene carried by the nearest vap island ancestor. Deletion of vapN, the predicted "bovine-type" allelic counterpart of vapA, essential for virulence in pVAPA, abrogated pVAPN-mediated intramacrophage proliferation and virulence in mice. Our findings support a model in which R. equi virulence is conferred by host-adapted plasmids. Their central role is mediating intracellular proliferation in macrophages, promoted by a key vap determinant present in the common ancestor of the plasmid-specific vap islands, with host tropism as a secondary trait selected during coevolution with specific animal species.

| S-EPMC4468562 | biostudies-literature

Virulence plasmid of Rhodococcus equi contains inducible gene family encoding secreted proteins.

Project description:Rhodococcus equi causes severe pyogranulomatous pneumonia in foals. This facultative intracellular pathogen produces similar lesions in immunocompromised humans, particularly in AIDS patients. Virulent strains of R. equi bear a large plasmid that is required for intracellular survival within macrophages and for virulence in foals and mice. Only two plasmid-encoded proteins have been described previously; a 15- to 17-kDa surface protein designated virulence-associated protein A (VapA) and an antigenically related 20-kDa protein (herein designated VapB). These two proteins are not expressed by the same R. equi isolate. We describe here the substantial similarity between VapA and VapB. Moreover, we identify three additional genes carried on the virulence plasmid, vapC, -D, and -E, that are tandemly arranged downstream of vapA. These new genes are members of a gene family and encode proteins that are approximately 50% homologous to VapA, VapB, and each other. vapC, -D, and -E are found only in R. equi strains that express VapA and are highly conserved in VapA-positive isolates from both horses and humans. VapC, -D, and -E are secreted proteins coordinately regulated by temperature with VapA; the proteins are expressed when R. equi is cultured at 37 degrees C but not at 30 degrees C, a finding that is compatible with a role in virulence. As secreted proteins, VapC, -D, and -E may represent targets for the prevention of rhodococcal pneumonia. An immunologic study using VapA-specific antibodies and recombinant Vap proteins revealed no evidence of cross-reactivity despite extensive sequence similarity over the carboxy terminus of all four proteins.

| S-EPMC97935 | biostudies-literature

Conjugal transfer of a virulence plasmid in the opportunistic intracellular actinomycete Rhodococcus equi.

Project description:Rhodococcus equi is a facultative intracellular, Gram-positive, soilborne actinomycete which can cause severe pyogranulomatous pneumonia with abscessation in young horses (foals) and in immunocompromised people, such as persons with AIDS. All strains of R. equi isolated from foals and approximately a third isolated from humans contain a large, ~81-kb plasmid which is essential for the intramacrophage growth of the organism and for virulence in foals and murine in vivo model systems. We found that the entire virulence plasmid could be transferred from plasmid-containing strains of R. equi (donor) to plasmid-free R. equi strains (recipient) at a high frequency and that plasmid transmission reestablished the capacity for intracellular growth in macrophages. Plasmid transfer required living cells and cell-to-cell contact and was unaffected by the presence of DNase, factors pointing to conjugation as the major means of genetic transfer. Deletion of a putative relaxase-encoding gene, traA, located in the proposed conjugative region of the plasmid, abolished plasmid transfer. Reversion of the traA mutation restored plasmid transmissibility. Finally, plasmid transmission to other Rhodococcus species and some additional related organisms was demonstrated. This is the first study showing a virulence plasmid transfer in R. equi, and it establishes a mechanism by which the virulence plasmid can move among bacteria in the soil.

| S-EPMC3510604 | biostudies-literature

Evolution of the Rhodococcus equi vap pathogenicity island seen through comparison of host-associated vapA and vapB virulence plasmids.

Project description:The pathogenic actinomycete Rhodococcus equi harbors different types of virulence plasmids associated with specific nonhuman hosts. We determined the complete DNA sequence of a vapB(+) plasmid, typically associated with pig isolates, and compared it with that of the horse-specific vapA(+) plasmid type. pVAPB1593, a circular 79,251-bp element, had the same housekeeping backbone as the vapA(+) plasmid but differed over an approximately 22-kb region. This variable region encompassed the vap pathogenicity island (PAI), was clearly subject to selective pressures different from those affecting the backbone, and showed major genetic rearrangements involving the vap genes. The pVAPB1593 PAI harbored five different vap genes (vapB and vapJ to -M, with vapK present in two copies), which encoded products differing by 24 to 84% in amino acid sequence from the six full-length vapA(+) plasmid-encoded Vap proteins, consistent with a role for the specific vap gene complement in R. equi host tropism. Sequence analyses, including interpolated variable-order motifs for detection of alien DNA and reconstruction of Vap family phylogenetic relationships, suggested that the vap PAI was acquired by an ancestor plasmid via lateral gene transfer, subsequently evolving by vap gene duplication and sequence diversification to give different (host-adapted) plasmids. The R. equi virulence plasmids belong to a new family of actinobacterial circular replicons characterized by an ancient conjugative backbone and a horizontally acquired niche-adaptive plasticity region.

| S-EPMC2519538 | biostudies-literature

The LysR-type transcriptional regulator VirR is required for expression of the virulence gene vapA of Rhodococcus equi ATCC 33701.

Project description:The virulence of the intracellular pathogen Rhodococcus equi in foals is dependent on the presence of an 81-kb virulence plasmid encoding the virulence protein VapA. Expression of this protein is induced by exposure to oxidative stress, high temperatures, and low pHs, which reflect the conditions encountered by R. equi when it enters the host environment. The aim of this study was to determine whether the LysR-type transcriptional regulator VirR, which is encoded by the virulence plasmid, is required for the expression of vapA. It was shown that the virR gene is cotranscribed with four downstream genes, one of which encodes a two-component response regulator. The expression of VapA, as monitored by Western blotting, was completely dependent on the presence of virR. Maximal expression was observed when vapA was present together with the complete virR operon, suggesting that at least one of the virR operon genes, in addition to virR, is required for the expression of vapA to wild-type levels. The transcriptional start site of vapA was determined to be a cytidine located 226 bp upstream from the vapA initiation codon. His-tagged VirR protein was expressed in Escherichia coli and purified by nickel affinity chromatography. DNA binding studies showed that purified VirR binds to a DNA fragment containing the vapA promoter. We therefore conclude that VirR is required for the activation of vapA transcription.

| S-EPMC516814 | biostudies-other

Virulence Plasmids of <i>Rhodococcus equi</i> Isolates From Cuban Patients With AIDS.

Project description:Rhodococcus equi is an animal pathogen and zoonotic human opportunistic pathogen associated with immunosuppressive conditions. The pathogenicity of R. equi is linked to three animal host-associated virulence plasmids encoding a family of "Virulence Associated Proteins" (VAPs). Here, the PCR-based TRAVAP molecular typing system for the R. equi virulence plasmids was applied to 26 R. equi strains isolated between 2010 and 2016 at the Institute of Tropical Medicine "Pedro Kourí," Cuba, from individuals living with HIV/AIDS. TRAVAP detects 4 gene markers, traA common to the three virulence plasmids, and vapA, vapB, and vapN specific to each of the host-associated plasmid types (equine pVAPA, porcine pVAPB, and ruminant pVAPN). Of the 26 isolates, six were positive to the vapB (porcine-type) marker, 4 (15.4%) to the vapA (equine-type) marker, and 1 (3.8%) to the vapN (ruminant-type) marker. Most of the isolates 14 (53.8%) were negative to all TRAVAP markers, suggesting they lacked a virulence plasmid. To our knowledge, this work is the first to report the molecular characterization of R. equi isolates from Cuba. Our findings provide insight into the zoonotic origin of R. equi infections in people and the potential dispensability of the virulence plasmid in immunosuppressed patients.

| S-EPMC7947234 | biostudies-literature

B-Cell epitope mapping of the VapA protein of Rhodococcus equi: implications for early detection of R. equi disease in foals.

Project description:Linear B-cell epitopes of the Rhodococcus equi virulence-associated protein (VapA) were mapped using a synthetic peptide bank in this study. The peptides were screened in an enzyme-linked immunosorbent assay (ELISA) with a total of 70 sera from foals with current R. equi disease (51 sera), as well as from foals that had either recovered from R. equi infection 10 months previously (3 sera) or that had no known history of R. equi disease (16 sera). An epitope with the sequence NLQKDEPNGRA was identified and was universally recognized by all 51 sera from foals with R. equi disease and was not recognized by any of the other sera. There was poor reactivity between all sera and peptides relating to other areas of the VapA protein. It is proposed that an ELISA based upon a defined peptide epitope may be used in an improved serological diagnostic test for R. equi infection in foals.

| S-EPMC87987 | biostudies-literature

Virulence of Rhodococcus equi isolated from cats and dogs.

Project description:Nine cat isolates and nine dog isolates of Rhodococcus equi from clinical material were investigated for the presence of the virulence-associated antigens (VapA and VapB) and virulence plasmids. Five of the cat isolates and one dog isolate were VapA positive and contained an 85-kb type I or an 87-kb type I plasmid. The remaining 12 isolates were avirulent R. equi strains and contained no virulence plasmids.

| S-EPMC193802 | biostudies-literature

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