Expression profile of mce4 operon of Mycobacterium tuberculosis following environmental stress

Background: The mce4 operon is one of the four mce operons with eight genes [yrbE4A, yrbE4B, mce4A, mce4B, mce4C, mce4D, mce4E and mce4F] of Mycobacterium tuberculosis. It expresses in the later phase of infection and imports cholesterol for long term survival of the bacilli. To cause latent infection, M. tuberculosis undergoes metabolic reprogramming of its genes to survive in the hostile environment like low availability of oxygen and nutrition depletion inside the host

Objective: To analyze real time expression profile of mce4 operon under various stress conditions

Methods: M. tuberculosis H37Rv was exposed to surface stress [0.1% SDS for 30 min and 90min in late log and stationary phase of culture], hypoxia [5, 10, 15 and 20 days] and grown in the presence of either glycerol or cholesterol as sole source of carbon. The expression profile of genes of mce4 operon was analyzed by real time PCR

Results: Surface stress induced expression of mce4C and yrbE4B in late log phase on 30 min and 90 min exposure respectively. The SDS exposure for 30 min induced mce4C, mce4D and mce4F in stationary phase. All eight genes were induced significantly on 10th and 15th days of hypoxia and in the presence of cholesterol

Conclusion: Hypoxia and cholesterol are potent factors for the expression of mce4 operon of M. tuberculosis

Differential signaling of inducible nitric oxide synthase induction in Mycobacterium tuberculosis infected alveolar epithelial cell line A549 in response to cytokines IFN-gamma, TNF-alpha and IL-1beta

In earlier studies, it was shown that ex vivo Mycobacterium tuberculosis-infected type II alveolar epithelial cells generate de novo nitric oxide [NO], but the mycobactericidal quantity of NO was released only by stimulation of these cells with proinflammatory cytokines, i.e. IFN-gamma, TNF-alpha and IL-1beta. In the present communication, it was demonstrated that M. tuberculosis-infected/mycobacterial antigens stimulated cells utilize both, JAK-STAT and NF-kappaB pathways for the induction of inducible Nitric Oxide Synthase [iNOS] mRNA and NO production. Alveolar epithelial cell line A549 were either infected with M. tuberculosis or stimulated with M. tuberculosis components. Confocal microscopy, NO estimation and EMSA were performed on the infected/stimulated A549 cells. Nuclear extracts prepared from M. tuberculosis infected A549 cells alone or stimulated with IFN-gamma or a combination of three cytokines [IFN-gamma, TNF-alpha and IL-1beta] formed DNA protein complexes with probes from both -5.2 kb region [specific for binding of STAT-1 protein] and -5.8 kb region [specific for binding of both STAT-1 and NF-kappaB] of the iNOS promoter. However, TNF-alpha or IL-1beta stimulated M. tuberculosis-infected A549 cells showed no protein DNA complexes with construct from -5.2 kb region. This differential response indicated that TNF-alpha/IL-1beta does not allow STAT-1 production or its translocation to nucleus in M. tuberculosis-infected A549 cells in the absence of IFN-gamma. This differential signaling of iNOS induction in M. tuberculosis-infected alveolar epithelial cells by cytokines may be responsible for controlled production of NO intracellularly.

Are we overlooking infections owing to non-tuberculous mycobacteria during routine conventional laboratory investigations?

A large number of potentially pathogenic non-tuberculous mycobacteria [NTM] encountered in the clinical laboratory makes it necessary to identify their species to ensure appropriate treatment. However, labor-intensive conventional methods of speciation are not used in every laboratory, and hence NTM infections are often ignored. Polymerase chain reaction [PCR] restriction analysis [PRA] was applied in this study for early identification and speciation of mycobacterial species on 306 cultures of acid-fast bacilli isolated from patients suspected of suffering from tuberculosis. Mycobacterium tuberculosis was identified in 85.6% of the isolates. The NTM isolated most commonly was Mycobacterium kansasii/gastri group [3.5%], followed by Mycobacterium fortuitum [3.2%]. Four of the M. fortuitum were grown from cultures obtained on the same day, but from samples from different patients and were probably laboratory contaminants. Mycobacterium intracellulare and Mycobacterium avium were identified in 2.94% and 2.28% of the isolates, respectively. Three isolates of M. avium and two isolates of M. intracellulare were obtained in repeated cultures from sputum samples of the same patients and were thus pathogenic. A single isolate of Mycobacterium abscessus was obtained from a breast abscess. A rare pathogen Mycobacterium phocaicum was isolated from one patient with epididymitis. However, whether it was the causative agent of epididymitis in this patient remains doubtful. The results of this study highlight the importance of speciation of mycobacteria for appropriate diagnosis and the importance of including molecular assays to augment conventional methods of diagnosis of mycobacterial diseases for rapid identification of NTM so that these potential pathogens are not overlooked in routine diagnostic procedures.