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.

Figure 1

Schematic diagram of the 1-step strategy for single-crossover mutation. The homologous region is located in the middle of the target gene, so that the entire recombinant vector is integrated in the middle of the gene after a single crossover event. An aacC4-specific primer (right-pointing arrow at bottom) and a primer specific for part of the gene outside the homologous target region (left-pointing arrow at bottom) were used to confirm the single-crossover mutation.

Figure 2

Growth curves for the htrA, narG, and pepD mutant strains and the parent strain (103⁺) of Rhodococcus equi at 37°C. Results are presented as optical density at 540 nm (OD₅₄₀) at intervals after inoculation into growth medium.