Probing host pathogen cross-talk by transcriptional profiling of both Mycobacterium tuberculosis and infected human dendritic cells and macrophages

This study provides the proof of principle that probing the host and the microbe transcriptomes simultaneously is a valuable means to accessing unique information on host pathogen interactions. Our results also underline the extraordinary plasticity of host cell and pathogen responses to infection, and provide a solid framework to further understand the complex mechanisms involved in immunity to M. tuberculosis and in mycobacterial adaptation to different intracellular environments

Nitazoxanide Stimulates Autophagy and Inhibits mTORC1 Signaling and Intracellular Proliferation of Mycobacterium tuberculosis

Tuberculosis, caused by Mycobacterium tuberculosis infection, is a major cause of morbidity and mortality in the world today. M. tuberculosis hijacks the phagosome-lysosome trafficking pathway to escape clearance from infected macrophages. There is increasing evidence that manipulation of autophagy, a regulated catabolic trafficking pathway, can enhance killing of M. tuberculosis. Therefore, pharmacological agents that induce autophagy could be important in combating tuberculosis. We report that the antiprotozoal drug nitazoxanide and its active metabolite tizoxanide strongly stimulate autophagy and inhibit signaling by mTORC1, a major negative regulator of autophagy. Analysis of 16 nitazoxanide analogues reveals similar strict structural requirements for activity in autophagosome induction, EGFP-LC3 processing and mTORC1 inhibition. Nitazoxanide can inhibit M. tuberculosis proliferation in vitro. Here we show that it inhibits M. tuberculosis proliferation more potently in infected human THP-1 cells and peripheral monocytes. We identify the human quinone oxidoreductase NQO1 as a nitazoxanide target and propose, based on experiments with cells expressing NQO1 or not, that NQO1 inhibition is partly responsible for mTORC1 inhibition and enhanced autophagy. The dual action of nitazoxanide on both the bacterium and the host cell response to infection may lead to improved tuberculosis treatment

Maturation of Rhodococcus equi-containing vacuoles is arrested after completion of the early endosome stage.

Rhodococcus equi is a facultative intracellular bacterium that can cause bronchopneumonia in foals and AIDS patients. Here, we have analyzed R. equi-containing vacuoles (RCVs) in murine macrophages by confocal laser scanning microscopy, by transmission electron microscopy and by immunochemistry upon purification. We show that RCVs progress normally through the early stages of phagosome maturation acquiring PI3P, early endosome antigen-1, and Rab5, and loosing all or much of them within minutes. Although mature RCVs possess the normally late endocytic markers, lysosome-associated membrane proteins, lysobisphosphatidic acid and Rab7, they lack other hallmark features of late endocytic organelles such as possession of cathepsin D, acid beta-glucuronidase, proton-pumping ATPase and the ability to fuse with prelabeled lysosomes. Bacterial strains possessing a virulence-associated plasmid maintain a nonacidified compartment for 48 h, whereas isogenic strains lacking such plasmids acidify progressively. In summary, RCVs represent a novel phagosome maturation stage positioned after completion of the early endosome stage and before reaching a fully mature late endosome compartment. In addition, vacuole biogenesis can be influenced by bacterial plasmids

Activation of macrophages and interference with CD4(+) T-cell stimulation by Mycobacterium avium subspecies paratuberculosis and Mycobacterium avium subspecies avium

Mycobacterium avium subspecies paratuberculosis (M. ptb) and M. avium subspecies avium (M. avium) are closely related but exhibit significant differences in their interaction with the host immune system. The macrophage line, J774, was infected with M. ptb and M. avium and analysed for cytokine production and stimulatory capacity towards antigen-specific CD4(+) T cells. Under all conditions J774 cells were activated to produce proinflammatory cytokines. No influence on the expression of major histocompatibility complex (MHC) class II, intracellular adhesion molecule-1 (ICAM-1), B7.1, B7.2 or CD40 was found. However, the antigen-specific stimulatory capacity of J774 cells for a CD4(+) T-cell line was significantly inhibited after infection with M. ptb, but not with M. avium. When a T-cell hybridoma expressing a T-cell receptor identical to that of the T-cell line was used, this inhibition was not observed, suggesting that costimulation which is essential for the CD4(+) T-cell line is influenced by the pathogenic bacterium M. ptb