Identification of genes required for the fitness of <i>Rhodococcus equi</i> during the infection of mice via signature-tagged transposon mutagenesis

Abstract

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 4560 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.

Evaluation of vaccine candidates against Rhodococcus equi in BALB/c mice infection model: cellular and humoral immune responses

“…The results showed that all five vaccine candidate-immunized mice produced strong Th1 and Th2 immune responses and strong antibody responses. Cellular immunity plays a key role in the clearance and immunity of intracellular pathogens [ 34 , 35 ]. In mice, while both CD4 + and CD8 + T lymphocytes contribute to host defence against R. equi , CD4 + helper T lymphocytes play a major role in the clearance of virulent R. equi [ 23 , 36 – 39 ].…”

“…Immunocompetent mice can clear infected R. equi and usually do not experience severe pneumonia, but the organ bacterial load and pathology correlate with the degree of virulence of R. equi [ 34 , 35 ]. Therefore, to evaluate the immunoprotective effect of vaccine candidates, we selected the lungs and livers as the organs of interest, and these organs were found to be reliable indicators for assessing the infection status of mice following R. equi challenge [ 52 , 53 ].…”

“…In addition, we used a BALB/c mouse infection model to evaluate the immunogenicity and immunoprotection of the vaccine candidates. Although BALB/c mice are the most widely used species in R. equi infection studies [ 34 , 35 ], they cannot model the immunological complexity of foals and fully imitate foal R. equi pneumonia.…”