Virulence of Rhodococcus equi isolated from cats and dogs.
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ABSTRACT:
SUBMITTER: Takai S
PROVIDER: S-EPMC193802 | biostudies-literature | 2003 Sep
REPOSITORIES: biostudies-literature
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Virulence of Rhodococcus equi isolated from cats and dogs.
Takai Shinji S Martens Ronald J RJ Julian Alan A Garcia Ribeiro Márcio M Rodrigues de Farias Marconi M Sasaki Yukako Y Inuzuka Kazuho K Kakuda Tsutomu T Tsubaki Shiro S Prescott John F JF
Journal of clinical microbiology 20030901 9
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. ...[more]
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Project description:Rhodococcus equi can cause severe or fatal pneumonia in foals as well as in immunocompromised animals and humans. Its ability to persist in macrophages is fundamental to how it causes disease, but the basis of this is poorly understood. To examine further the general application of a recently developed system of targeted gene mutation and to assess the importance of different genes in resistance to innate immune defenses, we disrupted the genes encoding high-temperature requirement A (htrA), nitrate reductase (narG), peptidase D (pepD), phosphoribosylaminoimidazole-succinocarboxamide synthase (purC), and superoxide dismutase (sodC) in strain 103 of R. equi using a double-crossover homologous recombination approach. Virulence testing by clearance after intravenous injection in mice showed that the htrA and narG mutants were fully attenuated, the purC and sodC mutants were unchanged, and the pepD mutant was slightly attenuated. Complementation with the pREM shuttle plasmid restored the virulence of the htrA and pepD mutants but not that of the narG mutant. A single-crossover mutation approach was simpler and faster than the double-crossover homologous recombination technique and was used to obtain mutations in 6 other genes potentially involved in virulence (clpB, fadD8, fbpB, glnA1, regX3, and sigF). These mutants were not attenuated in the mouse clearance assay. We were not able to obtain mutants for genesfurA, galE, and sigE using the single-crossover mutation approach. In summary, the targeted-mutation system had general applicability but was not always completely successful, perhaps because some genes are essential under the growth conditions used or because the success of mutation depends on the target genes.
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Project description:Rhodococcus equi is a facultative intracellular pathogen of macrophages and the causative agent of foal pneumonia. R. equi virulence is usually assessed by analyzing intracellular growth in macrophages by enumeration of bacteria following cell lysis, which is time consuming and does not allow for a high throughput analysis. This paper describes the use of an impedance based real-time method to characterize proliferation of R. equi in macrophages, using virulent and attenuated strains lacking the vapA gene or virulence plasmid. Image analysis suggested that the time-dependent cell response profile (TCRP) is governed by cell size and roundness as well as cytoxicity of infecting R. equi strains. The amplitude and inflection point of the resulting TCRP were dependent on the multiplicity of infection as well as virulence of the infecting strain, thus distinguishing between virulent and attenuated strains.
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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.
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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.
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