T-cell response during Rhodococcus equi infection in a murine experimental model.

Rhodococcus equi is a facultative intracellular bacterium that can cause pneumonia in both young horses and immunocompromised humans. In this study, we have tried to determine the T-cell populations that recognize this pathogen during murine infection, as well as the bacterial antigens recognized by these cells. When BALB/c mice were hyperimmunized with a virulent R. equi strain, we did not observe preferential expansion of a particular T-cell subset in their spleens. However, when the splenic T lymphocytes of the hyperimmunized BALB/c mice were cultured in the presence of killed bacteria, we found that alpha/beta CD4+ T cells proliferated and exhibited increased levels of the interleukin-2 receptor (IL2R). In order to ensure antigen specificity, two different controls were included in these experiments: (i) T-cell proliferation and expression of the IL2R in the presence of the major membrane constituent of Bacillus megaterium were studied comparatively with the presence of the R. equi bacterial antigen, and (ii) T-cell proliferation and expression of the IL2R from naive, non-infected mice in the presence of bacterial antigens were compared to those observed in hyperimmunized mice. In our study, the T cells from hyperimmunized mice did not significantly proliferate nor were they activated in the presence of non-related bacterial antigens, and T cells from naive mice were not found to significantly recognize R. equi antigens. When we studied the localization of R. equi antigens that could stimulate the in vitro proliferation and activation of T cells, we found that they were constituents of the bacterial cell wall and the cytoplasm, but they were not excreted in the culture medium. For these experiments, T-cell recognition of the bacterial antigens in hyperimmunized mice was compared to that of naive mice. With T-cell immunoblotting, we found that T-cell proliferation and activation were obtained with proteins having molecular masses of approximately 65, 43, 30, 22-27 and 15-17 kDa. It is noteworthy that among the recognized bacterial antigens, some have been described as being associated with virulence.

Interaction of virulent and non-virulent Rhodococcus equi human isolates with phagocytes, fibroblast- and epithelial-derived cells.

Rhodococcus equi is a facultative, intracellular, Gram-positive coccobacillus, increasingly reported in pneumonia of AIDS-infected patients. We investigated killing resistance properties of human R. equi virulent and avirulent human strains. Avirulent beta-lactam-susceptible strains had lower intracellular colony forming units after 45 min incubation in murine macrophages J774 and human monocyte-macrophage TPH-1 than those of virulent strains. Only virulent beta-lactam-resistant strains persisted within macrophages for at least 18 min only. A beta-lactam-resistant mutant was obtained from a beta-lactam-susceptible strain after selection in a penicillin G-containing culture medium. This mutant strain, like the natural virulent strains, persisted within macrophages, harboured cell-associated appendages, produced phage-like particles and induced, after its intravenous inoculation, a chronic infection in BALB/c nude mice. Supernatant culture of virulent strains transferred partial macrophage-killing resistance properties to avirulent strains. The same supernatant was toxic for L-929, HeLa and Vero cell cultures. These supernatant effects were heat-inactivated, trypsin-inactivated and did not seem to be linked to phage-like particle presence. These data argue that virulence, beta-lactam-resistance, and macrophage-killing resistance are associated in human R. equi isolates. Moreover, only virulent strains produced uncharacterized toxic factors.