The Hydroxamate Siderophore Rhequichelin Is Required for Virulence of the Pathogenic Actinomycete Rhodococcus equi

We previously showed that the facultative intracellular pathogen Rhodococcus equi produces a nondiffusible and catecholate-containing siderophore (rhequibactin) involved in iron acquisition during saprophytic growth. Here, we provide evidence that the rhbABCDE cluster directs the biosynthesis of a hydroxamate siderophore, rhequichelin, that plays a key role in virulence. The rhbC gene encodes a nonribosomal peptide synthetase that is predicted to produce a tetrapeptide consisting of N(5)-formyl-N(5)-hydroxyornithine, serine, N(5)-hydroxyornithine, and N(5)-acyl-N(5)-hydroxyornithine. The other rhb genes encode putative tailoring enzymes mediating modification of ornithine residues incorporated into the hydroxamate product of RhbC. Transcription of rhbC was upregulated during growth in iron-depleted medium, suggesting that it plays a role in iron acquisition. This was confirmed by deletion of rhbCD, rendering the resulting strain R. equi SID2 unable to grow in the presence of the iron chelator 2,2-dipyridyl. Supernatant of the wild-type strain rescued the phenotype of R. equi SID2. The importance of rhequichelin in virulence was highlighted by the rapid increase in transcription levels of rhbC following infection and the inability of R. equi SID2 to grow within macrophages. Unlike the wild-type strain, R. equi SID2 was unable to replicate in vivo and was rapidly cleared from the lungs of infected mice. Rhequichelin is thus a key virulence-associated factor, although nonpathogenic Rhodococcus species also appear to produce rhequichelin or a structurally closely related compound. Rhequichelin biosynthesis may therefore be considered an example of cooption of a core actinobacterial trait in the evolution of R. equi virulence

Characterization of the Role of the Pathogenicity Island and vapG in the Virulence of the Intracellular Actinomycete Pathogen Rhodococcus equi▿

Rhodococcus equi, a facultative intracellular pathogen of macrophages, causes severe, life-threatening pneumonia in young foals and in people with underlying immune deficiencies. R. equi virulence is dependent on the presence of a large virulence plasmid that houses a pathogenicity island (PAI) encoding a novel family of surface-localized and secreted proteins of largely unknown function termed the virulence-associated proteins (VapACDEFGHI). To date, vapA and its positive regulators virR and orf8 are the only experimentally established virulence genes residing on the virulence plasmid. In this study, a PAI deletion mutant was constructed and, as anticipated, was attenuated for growth both in macrophages and in mice due to the absence of vapA expression. Expression of vapA in the PAI mutant from a constitutive promoter, thereby eliminating the requirement for the PAI-encoded vapA regulators, resulted in delayed bacterial clearance in vivo, yet full virulence was not restored, indicating that additional virulence genes are indeed located within the deleted pathogenicity island region. Based on previous reports demonstrating that the PAI-carried gene vapG is highly upregulated in macrophages and in the lungs of R. equi-infected foals, we hypothesized that vapG could be an important virulence factor. However, analysis of a marked vapG deletion mutant determined the gene to be dispensable for growth in macrophages and in vivo in mice

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

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

Protection Elicited by a Double Leucine and Pantothenate Auxotroph of Mycobacterium tuberculosis in Guinea Pigs

We developed a live, fully attenuated Mycobacterium tuberculosis vaccine candidate strain with two independent attenuating auxotrophic mutations in leucine and pantothenate biosynthesis. The ΔleuD ΔpanCD double auxotroph is fully attenuated in the SCID mouse model and highly immunogenic and protective in the extremely sensitive guinea pig tuberculosis model, reducing both bacterial burden and disease pathology