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The effect of mutation on Rhodococcus equi virulence plasmid gene expression and mouse virulence.
Abstract: An 81 kb virulence plasmid containing a pathogenicity island (PI) plays a crucial role in the pathogenesis of Rhodococcus equi pneumonia in foals but its specific function in virulence and regulation of plasmid-encoded virulence genes is unclear. Using a LacZ selection marker developed for R. equi in this study, in combination with an apramycin resistance gene, an efficient two-stage homologous recombination targeted gene mutation procedure was used to mutate three virulence plasmid genes, a LysR regulatory gene homologue (ORF4), a ResD-like two-component response regulator homologue (ORF8), and a gene (ORF10) of unknown function that is highly expressed by R. equi inside macrophages, as well as the chromosomal gene operon, phoPR. Virulence testing by liver clearance after intravenous injection in mice showed that the ORF4 and ORF8 mutants were fully attenuated, that the phoPR mutant was hypervirulent, and that virulence of the ORF10 mutant remained unchanged. A virulence plasmid DNA microarray was used to compare the plasmid gene expression profile of each of the four gene-targeted mutants against the parental R. equi strain. Changes were limited to PI genes and gene induction was observed for all mutants, suggesting that expression of virulence plasmid genes is dominated by a negative regulatory network. The finding of attenuation of ORF4 and ORF8 mutants despite enhanced transcription of vapA suggests that factors other than VapA are important for full expression of virulence. ORF1, a putative Lsr antigen gene, was strongly and similarly induced in all mutants, implying a common regulatory pathway affecting this gene for all four mutated genes. ORF8 is apparently the centre of this common pathway. Two distinct highly correlated gene induction patterns were observed, that of the ORF4 and ORF8 mutants, and that of the ORF10 and phoPR mutants. The gene induction pattern distinguishing these two groups paralleled their virulence in mice.
Publication Date: 2004-10-27 PubMed ID: 15504593DOI: 10.1016/j.vetmic.2004.08.005Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
- Research Support
- Non-U.S. Gov't
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.
The research investigates the effects of mutations in specific genes of Rhodococcus equi virulence plasmid on the organism’s virulence and the gene expression. The study revealed a negative regulatory network dominating the expression of virulence plasmid genes, and established that VapA is not the only factor affecting virulence – other yet unidentified elements also play a significant role.
Objective of the research
- This study aimed to understand the function of a virulence plasmid in Rhodococcus equi pneumonia’s pathogenesis and regulation of plasmid-encoded virulence genes.
- The study used specific mutations to modify the virulence genes to better understand their role.
Methods and procedures
- A two-stage homologous recombination targeted gene mutation procedure, combined with a LacZ selection marker and an apramycin resistance gene, was used to mutate three virulence plasmid genes and a chromosomal gene operon.
- The targeted genes included a regulatory gene, a two-component response regulator, a gene highly expressed within macrophages, and the chromosomal gene operon, phoPR.
- The virulence of these mutants was analysed by liver clearance tests in mice after an intravenous injection.
- A virulence plasmid DNA microarray was utilised to compare the plasmid gene expression profile of each of the four gene-targeted mutants against the parental strain of R. equi.
Key findings
- The study shows that the regulatory gene and the response regulator gene mutants were fully attenuated, the phoPR mutant was hypervirulent, and the virulence of the highly-expressed gene mutant remained unchanged.
- The research discovers that expression of virulence plasmid genes is controlled by a negative regulatory network, with the response regulator gene appearing to be central.
- The findings suggest that factors other than VapA, an established virulence-associated protein, are significant for full expression of virulence, challenging the previous assumption.
- Interestingly, a putative Lsr antigen gene was strongly induced across all mutants, implying a shared regulatory pathway affecting this gene for all the four mutated genes.
- Two distinct highly correlated gene induction patterns were observed, which coincided with their virulence in mice, providing insights into the complex interplay between gene expression and virulence.
Cite This Article
APA
Ren J, Prescott JF.
(2004).
The effect of mutation on Rhodococcus equi virulence plasmid gene expression and mouse virulence.
Vet Microbiol, 103(3-4), 219-230.
https://doi.org/10.1016/j.vetmic.2004.08.005 Publication
Researcher Affiliations
- Department of Pathobiology, University of Guelph, Guelph, Ont., Canada N1G 2W1.
MeSH Terms
- Actinomycetales Infections / microbiology
- Actinomycetales Infections / veterinary
- Amino Acid Sequence
- Animals
- Bacterial Proteins / chemistry
- Bacterial Proteins / genetics
- Base Sequence
- DNA, Bacterial / analysis
- Gene Expression Regulation, Bacterial
- Genetic Markers
- Horse Diseases / microbiology
- Horses
- Macrophages / microbiology
- Mice
- Molecular Sequence Data
- Mutation
- Oligonucleotide Array Sequence Analysis / veterinary
- Operon / genetics
- Plasmids / genetics
- Polymerase Chain Reaction / methods
- Polymerase Chain Reaction / veterinary
- Rhodococcus equi / genetics
- Rhodococcus equi / pathogenicity
- Transcriptional Activation
- Virulence / genetics
Citations
This article has been cited 13 times.- da Silveira BP, Cohen ND, Lawhon SD, Watson RO, Bordin AI. Protective immune response against Rhodococcus equi: An innate immunity-focused review. Equine Vet J 2025 May;57(3):563-586.
- Hansen P, von Bargen K, Jünger-Leif A, Haas A. Laboratory Plasticware Induces Expression of a Bacterial Virulence Factor. mSphere 2022 Oct 26;7(5):e0031122.
- Rofe AP, Davis LJ, Whittingham JL, Latimer-Bowman EC, Wilkinson AJ, Pryor PR. The Rhodococcus equi virulence protein VapA disrupts endolysosome function and stimulates lysosome biogenesis. Microbiologyopen 2017 Apr;6(2).
- Coulson GB, Miranda-CasoLuengo AA, Miranda-CasoLuengo R, Wang X, Oliver J, Willingham-Lane JM, Meijer WG, Hondalus MK. Transcriptome reprogramming by plasmid-encoded transcriptional regulators is required for host niche adaption of a macrophage pathogen. Infect Immun 2015 Aug;83(8):3137-45.
- Valero-Rello A, Hapeshi A, Anastasi E, Alvarez S, Scortti M, Meijer WG, MacArthur I, Vázquez-Boland JA. An Invertron-Like Linear Plasmid Mediates Intracellular Survival and Virulence in Bovine Isolates of Rhodococcus equi. Infect Immun 2015 Jul;83(7):2725-37.
- Wang X, Coulson GB, Miranda-Casoluengo AA, Miranda-Casoluengo R, Hondalus MK, Meijer WG. IcgA is a virulence factor of Rhodococcus equi that modulates intracellular growth. Infect Immun 2014 May;82(5):1793-800.
- Tripathi VN, Harding WC, Willingham-Lane JM, Hondalus MK. Conjugal transfer of a virulence plasmid in the opportunistic intracellular actinomycete Rhodococcus equi. J Bacteriol 2012 Dec;194(24):6790-801.
- Letek M, González P, Macarthur I, Rodríguez H, Freeman TC, Valero-Rello A, Blanco M, Buckley T, Cherevach I, Fahey R, Hapeshi A, Holdstock J, Leadon D, Navas J, Ocampo A, Quail MA, Sanders M, Scortti MM, Prescott JF, Fogarty U, Meijer WG, Parkhill J, Bentley SD, Vázquez-Boland JA. The genome of a pathogenic rhodococcus: cooptive virulence underpinned by key gene acquisitions. PLoS Genet 2010 Sep 30;6(9):e1001145.
- Coulson GB, Agarwal S, Hondalus MK. Characterization of the role of the pathogenicity island and vapG in the virulence of the intracellular actinomycete pathogen Rhodococcus equi. Infect Immun 2010 Aug;78(8):3323-34.
- von Bargen K, Polidori M, Becken U, Huth G, Prescott JF, Haas A. Rhodococcus equi virulence-associated protein A is required for diversion of phagosome biogenesis but not for cytotoxicity. Infect Immun 2009 Dec;77(12):5676-81.
- Letek M, Ocampo-Sosa AA, Sanders M, Fogarty U, Buckley T, Leadon DP, González P, Scortti M, Meijer WG, Parkhill J, Bentley S, Vázquez-Boland JA. Evolution of the Rhodococcus equi vap pathogenicity island seen through comparison of host-associated vapA and vapB virulence plasmids. J Bacteriol 2008 Sep;190(17):5797-805.
- Byrne GA, Russell DA, Chen X, Meijer WG. Transcriptional regulation of the virR operon of the intracellular pathogen Rhodococcus equi. J Bacteriol 2007 Jul;189(14):5082-9.
- Pei Y, Parreira V, Nicholson VM, Prescott JF. Mutation and virulence assessment of chromosomal genes of Rhodococcus equi 103. Can J Vet Res 2007 Jan;71(1):1-7.
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