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Diversion of phagosome trafficking by pathogenic Rhodococcus equi depends on mycolic acid chain length.

ABSTRACT: Rhodococcus equi is a close relative of Mycobacterium spp. and a facultative intracellular pathogen which arrests phagosome maturation in macrophages before the late endocytic stage. We have screened a transposon mutant library of R. equi for mutants with decreased capability to prevent phagolysosome formation. This screen yielded a mutant in the gene for β-ketoacyl-(acyl carrier protein)-synthase A (KasA), a key enzyme of the long-chain mycolic acid synthesizing FAS-II system. The longest kasA mutant mycolic acid chains were 10 carbon units shorter than those of wild-type bacteria. Coating of non-pathogenic E. coli with purified wild-type trehalose dimycolate reduced phagolysosome formation substantially which was not the case with shorter kasA mutant-derived trehalose dimycolate. The mutant was moderately attenuated in macrophages and in a mouse infection model, but was fully cytotoxic.Whereas loss of KasA is lethal in mycobacteria, R. equi kasA mutant multiplication in broth was normal proving that long-chain mycolic acid compounds are not necessarily required for cellular integrity and viability of the bacteria that typically produce them. This study demonstrates a central role of mycolic acid chain length in diversion of trafficking by R. equi.

SUBMITTER: Sydor T

PROVIDER: S-EPMC3864644 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Rhodococcus equi virulence-associated protein A is required for diversion of phagosome biogenesis but not for cytotoxicity.

Project description:Rhodococcus equi is a gram-positive facultative intracellular pathogen that can cause severe bronchopneumonia in foals and AIDS patients. Virulence is plasmid regulated and is accompanied by phagosome maturation arrest and host cell necrosis. A replacement mutant in the gene for VapA (virulence-associated protein A), a major virulence factor of R. equi, was tested for its activities during macrophage infection. Early in infection, phagosomes containing the vapA mutant did not fuse with lysosomes and did not stain with the acidotropic fluor LysoTracker similar to those containing virulent wild-type R. equi. However, vapA mutant phagosomes had a lower average pH. Late in infection, phagosomes containing the vapA mutant were as frequently positive for LysoTracker as phagosomes containing plasmid-cured, avirulent bacteria, whereas those with virulent wild-type R. equi were still negative for the fluor. Macrophage necrosis after prolonged infection with virulent bacteria was accompanied by a loss of organelle staining with LysoTracker, suggesting that lysosome proton gradients had collapsed. The vapA mutant still killed the macrophages and yet did not affect the pH of host cell lysosomes. Hence, VapA is not required for host cell necrosis but is required for neutralization of phagosomes and lysosomes or their disruption. This is the first report of an R. equi mutant with altered phagosome biogenesis.

| S-EPMC2786453 | biostudies-literature

Endocytic trafficking of sphingomyelin depends on its acyl chain length.

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Characterization of mycolic acids from the pathogen Rhodococcus equi by tandem mass spectrometry with electrospray ionization.

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| S-EPMC3006067 | biostudies-literature

Pangenome and Phylogenomic Analysis of the Pathogenic Actinobacterium Rhodococcus equi.

Project description:We report a comparative study of 29 representative genomes of the animal pathogen Rhodococcus equi The analyses showed that R. equi is genetically homogeneous and clonal, with a large core genome accounting for ?80% of an isolates' gene content. An open pangenome, even distribution of accessory genes among the isolates, and absence of significant core-genome recombination, indicated that gene gain/loss is a main driver of R. equi genome evolution. Traits previously predicted to be important in R. equi physiology, virulence and niche adaptation were part of the core genome. This included the lack of a phosphoenolpyruvate:carbohydrate transport system (PTS), unique among the rhodococci except for the closely related Rhodococcus defluvii, reflecting selective PTS gene loss in the R. equi-R. defluvii sublineage. Thought to be asaccharolytic, rbsCB and glcP non-PTS sugar permease homologues were identified in the core genome and, albeit inefficiently, R. equi utilized their putative substrates, ribose and (irregularly) glucose. There was no correlation between R. equi whole-genome phylogeny and host or geographical source, with evidence of global spread of genomovars. The distribution of host-associated virulence plasmid types was consistent with the exchange of the plasmids (and corresponding host shifts) across the R. equi population, and human infection being zoonotically acquired. Phylogenomic analyses demonstrated that R. equi occupies a central position in the Rhodococcus phylogeny, not supporting the recently proposed transfer of the species to a new genus.

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The hydroxamate siderophore rhequichelin is required for virulence of the pathogenic actinomycete Rhodococcus equi.

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Fatal Infection in an Alpaca (<i>Vicugna pacos</i>) Caused by Pathogenic <i>Rhodococcus equi</i>.

Project description:Rhodococcus (R.) equi is a pathogen primarily known for infections in equine foals, but is also present in numerous livestock species including New World camelids. Moreover, R. equi is considered an emerging zoonotic pathogen. In this report, we describe in detail a fatal rhodococcal infection in an alpaca (Vicugna pacos), to our best knowledge, for the first time. The alpaca died due to a septicemic course of an R. equi infection resulting in emaciation and severe lesions including pyogranulomas in the lungs and pericardial effusion. The onset of the infection was presumably caused by aspiration pneumonia. R. equi could be isolated from the pyogranulomas in the lung and unequivocally identified by MALDI-TOF MS analysis and partial sequencing of the 16S rRNA gene, the 16S-23S internal transcribed spacer (ITS) region and the rpoB gene. The isolate proved to possess the vapA gene in accordance with tested isolates originating from the lungs of infected horses. The R. equi isolates revealed low minimal inhibitory concentrations (MIC values) for doxycycline, erythromycin, gentamycin, neomycin, rifampicin, trimethoprim/sulfamethoxazole, tetracycline and vancomycin in antibiotic susceptibility testing. Investigations on the cause of bacterial, especially fatal, septicemic infections in alpacas are essential for adequately addressing the requirements for health and welfare issues of this New World camelid species. Furthermore, the zoonotic potential of R. equi has to be considered with regard to the One Health approach.

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International Spread of Multidrug-Resistant Rhodococcus equi.

Project description:A multidrug-resistant clone of the animal and human pathogen Rhodococcus equi, MDR-RE 2287, has been circulating among equine farms in the United States since the 2000s. We report the detection of MDR-RE 2287 outside the United States. Our finding highlights the risk for MDR-RE spreading internationally with horse movements.

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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.

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Spread of Multidrug-Resistant Rhodococcus equi, United States.

Project description:Multidrug resistance has been detected in the animal and zoonotic human pathogen Rhodococcus equi after mass macrolide/rifampin antibioprophylaxis in endemically affected equine farms in the United States. Multidrug-resistant (MDR) R. equi emerged upon acquisition of pRERm46, a conjugative plasmid conferring resistance to macrolides, lincosamides, streptogramins, and, as we describe, tetracycline. Phylogenomic analyses indicate that the increasing prevalence of MDR R. equi since it was first documented in 2002 is caused by a clone, R. equi 2287, attributable to coselection of pRErm46 with a chromosomal rpoBS531F mutation driven by macrolide/rifampin therapy. pRErm46 spillover to other R. equi genotypes has given rise to a novel MDR clone, G2016, associated with a distinct rpoBS531Y mutation. Our findings illustrate that overuse of antimicrobial prophylaxis in animals can generate MDR pathogens with zoonotic potential. MDR R. equi and pRErm46-mediated resistance are currently disseminating in the United States and are likely to spread internationally through horse movements.

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