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Home / Equine Research Bank / Can J Vet Res / Mutation and virulence assessment of chromosomal genes of Rh...
Canadian journal of veterinary research = Revue canadienne de recherche veterinaire2006; 71(1); 1-7;

Mutation and virulence assessment of chromosomal genes of Rhodococcus equi 103.

Abstract: 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. Rhodococcus equi peut causer une pneumonie sévère ou fatale chez les poulains aussi bien que chez les animaux ou humains immunocompromis. Sa capacité à persister dans les macrophages est fondamentale à sa pathogénie, mais la base de ce phénomène est mal connue. Afin d’examiner plus en détails l’application générale d’un nouveau système de mutation génétique dirigée et d’évaluer l’importance de différents gènes dans la résistance à la réponse immunitaire innée, un dérèglement des gènes codant pour la protéase htrA (htrA), la nitrate réductase (narG), la peptidase D (pepD), la phosphoribosylaminoimidazole-succinocarboxamide synthétase (purC) et la superoxide dismutase (sodC) de la souche 103 de R. equi a été produit à l’aide d’une approche utilisant une recombinaison homologue à double croisement. Une évaluation de la virulence par évaluation de la clairance après injection intraveineuse chez la souris a permis de démontrer que les mutants htrA et narG étaient complètement atténués, les mutants purC et sodC étaient inchangés et le mutant pepD était légèrement atténué. Une complémentation avec le plasmide vecteur pREM a rétabli la virulence des mutants htrA et pepD mais pas celle du mutant narG. Une approche de mutation par croisement unique était plus simple et rapide que la technique de recombinaison homologue à double croisement et a été utilisée afin d’obtenir des mutations dans six autres gènes potentiellement impliqués dans la virulence (clpB, fadD8, fbpB, glnA1, regX3 et sigF). Ces mutants n’étaient pas atténués dans l’épreuve de clairance chez la souris. Il a été impossible d’obtenir des mutants pour les gènes furA, galE et sigE en utilisant l’approche de mutation par croisement unique. En résumé, le système à mutation dirigée a une application globale mais le résultat désiré n’est pas toujours obtenu, peut-être parce que certains gènes sont essentiels dans les conditions de culture utilisées ou parce que le succès de la mutation dépend des gènes ciblés. (Traduit par Docteur Serge Messier)
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Publication Date: 2006-12-30 PubMed ID: 17193875PubMed Central: PMC1636002
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This study explores the mutation and virulence of specific chromosomal genes in Rhodococcus equi 103, a bacteria that can cause severe pneumonia. Key genes contributing to the survival of the bacteria within immune cells were identified, and some were found to be critical for infection. However, the effectiveness of the gene mutation approach varied depending on the specific genes targeted.

Objective of the Study

  • The aim of the study was to understand the role of different genes in the resistance of Rhodococcus equi to the immune system and to assess the ability of a targeted gene mutation system in disrupting the function of these genes.

Methodology

  • The researchers used a double-crossover homologous recombination method to disrupt genes that they hypothesized to be important for the bacteria’s survival and virulence. These include high-temperature requirement A (htrA), nitrate reductase (narG), peptidase D (pepD), phosphoribosylaminoimidazole-succinocarboxamide synthase (purC), and superoxide dismutase (sodC).
  • The modified bacteria strains were tested for their virulence by observing their clearance after injecting them into mice.
  • To achieve further mutations in other potential virulence genes, the researchers also used a simpler single-crossover mutation approach.

Results

  • The study found that the htrA and narG mutants were completely attenuated, meaning they lost their ability to cause disease.
  • The purC and sodC mutants were unchanged, and the pepD mutant was slightly attenuated.
  • Using an external plasmid (pREM), the researchers were able to restore virulence in the htrA and pepD mutants, but not in the narG mutant.
  • For six additional genes targeted using the single-crossover approach, no attenuation was observed.
  • The researchers were unable to obtain mutants for three genes (furA, galE, and sigE) using the single-crossover mutation approach.

Conclusions

  • The targeted gene mutation system was useful but was not consistently successful across all the targeted genes. This could be because certain genes are essential under the growth conditions used, or the success of mutation varies depending on the specific genes targeted.

Cite This Article

APA
Pei Y, Parreira V, Nicholson VM, Prescott JF. (2006). Mutation and virulence assessment of chromosomal genes of Rhodococcus equi 103. Can J Vet Res, 71(1), 1-7.

Publication

Canadian journal of veterinary research = Revue canadienne de recherche veterinaire
ISSN: 0830-9000
NlmUniqueID: 8607793
Country: Canada
Language: English
Volume: 71
Issue: 1
Pages: 1-7

Researcher Affiliations

Pei, Yanlong
  • Department of Pathobiology, University of Guelph, Guelph, Ontario N1G 2W1.
Parreira, Valeria
    Nicholson, Vivian M
      Prescott, John F

        MeSH Terms

        • Actinomycetales Infections / microbiology
        • Actinomycetales Infections / veterinary
        • Amino Acid Sequence
        • Animals
        • Base Sequence
        • Biological Assay
        • DNA, Bacterial / chemistry
        • DNA, Bacterial / genetics
        • Gene Expression Regulation, Bacterial
        • Horse Diseases / microbiology
        • Horses
        • Mice
        • Molecular Sequence Data
        • Mutation
        • Plasmids / genetics
        • Rhodococcus equi / genetics
        • Rhodococcus equi / pathogenicity
        • Virulence / genetics

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        This article has been cited 10 times.
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