Identification of genes required for the fitness of Rhodococcus equi during the infection of mice via signature-tagged transposon mutagenesis.

Rhodococcus equi is a Gram-positive facultative intracellular bacterium that causes pyogranulomatous pneumonia in foals and immunocompromised people. In the present study, signature-tagged transposon mutagenesis was applied for the negative selection of R. equi mutants that cannot survive in vivo. Twenty-five distinguishable plasmid-transposon (plasposon) vectors by polymerase chain reaction (PCR), each containing a unique oligonucleotide tag, were constructed and used to select the transposon mutants that have in vivo fitness defects using a mouse systemic infection model. Of the 4,560 transposon mutants, 102 mutants were isolated via a real-time PCR-based screening as the mutants were unable to survive in the mouse model. Finally, 50 single transposon insertion sites were determined via the self-cloning strategy. The insertion of the transposon was seen on the virulence plasmid in 15 of the 50 mutants, whereas the remaining 35 mutants had the insertion of transposon on the chromosome. The chromosomal mutants contained transposon insertions in genes involved in cellular metabolism, DNA repair and recombination, gene regulation, non-ribosomal peptide synthesis, and unknown functions. Additionally, seven of the chromosomal mutants showed a reduced ability to multiply in the macrophages in vitro. In this study, we have identified several biosynthetic pathways as fitness factors associated with the growth within macrophages and survival in mice.

Rescue of an intracellular avirulent Rhodococcus equi replication defect by the extracellular addition of virulence-associated protein A.

Rhodococcus equi is a facultative intracellular bacterium that can escape from bactericidal mechanisms associated with phagocytosis. Virulence-associated protein A (VapA), encoded on a virulence-associated plasmid, is essential for intracellular survival in macrophages, but its function is not known. Here, we show that the extracellular addition of recombinant glutathione S-transferase (GST)-VapA fusion protein rescued the intracellular replication defect of a mutant lacking the vapA gene. Furthermore, the virulence-plasmid-cured strain could also multiply to nearly wild-type levels by the addition of GST-VapA. The present data suggest that VapA can alter the intraphagocytic environment, thereby affecting its suitability for the growth of R. equi.