The pathogenesis of experimental Emergomycosis in mice.

Emergomyces africanus is a recently identified thermally-dimorphic fungal pathogen that causes disseminated infection in people living with advanced HIV disease. Known as emergomycosis, this disseminated disease is associated with very high case fatality rates. Over the last decade, improved diagnostics and fungal identification in South Africa resulted in a dramatic increase in the number of reported cases. Although the true burden of disease is still unknown, emergomycosis is among the most frequently diagnosed dimorphic fungal infections in Southern Africa; and additional species in the genus have been identified on four continents. Little is known about the pathogenesis and the host's immune response to this emerging pathogen. Therefore, we established a murine model of pulmonary infection using a clinical isolate, E. africanus (CBS 136260). Both conidia and yeast forms caused pulmonary and disseminated infection in mice with organisms isolated in culture from lung, spleen, liver, and kidney. Wild-type C57BL/6 mice demonstrated a drop in body weight at two weeks post-infection, corresponding to a peak in fungal burden in the lung, spleen, liver, and kidney. An increase in pro-inflammatory cytokine production was detected in homogenized lung supernatants including IFN-γ, IL-1ß, IL-6, IL12-p40 and IL-17 at three- and four-weeks post-infection. No significant differences in TNF, IL-12p70 and IL-10 were observed in wild-type mice between one and four-weeks post-infection. Rag-1-deficient mice, lacking mature T-and B-cells, had an increased fungal burden associated with reduced IFN-γ production. Together our data support a protective T-helper type-1 immune response to E. africanus infection. This may provide a possible explanation for the susceptibility of only a subset of people living with advanced HIV disease despite hypothesized widespread environmental exposure. In summary, we have established a novel murine model of E. africanus disease providing critical insights into the host immune components required for eliminating the infection.

In vitro and in vivo toxicity evaluation of non-neuroleptic phenothiazines, antitubercular drug candidates.

The phenothiazine-derived antipsychotic drugs, such as chlorpromazine and thioridazine, are bactericidal against drug-sensitive and drug-resistant strains of Mycobacterium tuberculosis, but produce undesirable side effects at clinically relevant doses. We have previously modified four novel phenothiazines and maintained their antimycobacterial activity. This study evaluated the pharmacological and toxicity profiles of these novel non-neuroleptic phenothiazines, PTZ3, PTZ4, PTZ31 and PTZ32, for their metabolic stability, kinetic solubility and potential cytotoxic effects in vitro. To further support the safet use of these drug candidates, the in vivo pharmacological and toxicity profiles were assessed in C57BL/6 mice via single or repeated oral gavage. In acute toxicity studies, all four modified phenothiazines showed favourable safety in mice. When treated daily with 100 mg/kg of PTZ3 and PTZ4 for 2 weeks, mice displayed no signs of toxicity. Alternatively, treatment with PTZ31 resulted in 20% mortality with no toxicity evident in biochemical or histological analysis, while exposure to PTZ32 resulted in a 45% survival with increased serum concentrations of uric acid and alkaline phosphatase. The combined non-neuroleptic and antimycobacterial effects of the novel phenothiazines PTZ3, PTZ4, PTZ31 and PTZ32 demonstrated favourable pharmacological and toxicity profiles in this study, highlight the potential of these compounds as suitable anti-tuberculosis drug candidates.

Neurons are host cells for Mycobacterium tuberculosis.

Mycobacterium tuberculosis infection of the central nervous system is thought to be initiated once the bacilli have breached the blood brain barrier and are phagocytosed, primarily by microglial cells. In this study, the interactions of M. tuberculosis with neurons in vitro and in vivo were investigated. The data obtained demonstrate that neurons can act as host cells for M. tuberculosis. M. tuberculosis bacilli were internalized by murine neuronal cultured cells in a time-dependent manner after exposure, with superior uptake by HT22 cells compared to Neuro-2a cells (17.7% versus 9.8%). Internalization of M. tuberculosis bacilli by human SK-N-SH cultured neurons suggested the clinical relevance of the findings. Moreover, primary murine hippocampus-derived neuronal cultures could similarly internalize M. tuberculosis. Internalized M. tuberculosis bacilli represented a productive infection with retention of bacterial viability and replicative potential, increasing 2- to 4-fold within 48 h. M. tuberculosis bacillus infection of neurons was confirmed in vivo in the brains of C57BL/6 mice after intracerebral challenge. This study, therefore, demonstrates neurons as potential new target cells for M. tuberculosis within the central nervous system.

Novel non-neuroleptic phenothiazines inhibit Mycobacterium tuberculosis replication.

OBJECTIVES: Phenothiazines are a commercially available class of psychotropic drugs known to show antituberculosis activity. At clinically relevant bactericidal doses, however, the psychotropic drugs produce undesirable side effects in addition to their neuroleptic properties. This study aimed to evaluate rationally designed novel phenothiazines as antimycobacterial drug candidates. METHODS: Remodelling of psychotropic drugs by substitution of characteristic N-alkylamine side chains, important for CNS activity, with N-alkylsulphonates gave novel drug candidates, which were then tested for post-synaptic receptor binding affinity in a radioligand displacement assay. The bactericidal activities were screened using green fluorescent protein (GFP) microplate assays, and the efficacy of intracellular bacillus killing was evaluated by cfu enumeration. RESULTS: Of the four selected phenothiazine derivatives (PTZ3, PTZ4, PTZ31 and PTZ32) tested, PTZ31 displayed marginal serotonergic activity. The remaining three derivatives did not exhibit dopamine or serotonin receptor binding activity. In vitro results showed significant growth inhibition of virulent Mycobacterium tuberculosis with MICs of 12.5-25 mg/L. None of the phenothiazine derivatives displayed cytotoxicity in infected primary bone marrow-derived macrophages. Moreover, the phenothiazines showed significant antimycobacterial activity of between 40% and 60% against intracellular (ex vivo) M. tuberculosis. CONCLUSIONS: We demonstrate that structural modification of the phenothiazine core is possible in a manner that does not affect the ability of the phenothiazine derivatives to inhibit M. tuberculosis, but that abolishes undesirable dopamine and serotonin receptor binding.