The effect of mutation on Rhodococcus equi virulence plasmid gene expression and mouse virulence.

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.