Multidrug resistance (MDR) is a major problem in the treatment of infectious diseases and cancer. Accumulating evidence suggests that the cyclooxygenase-2 (COX-2)-specific inhibitor celecoxib would not only inhibit COX-2 but also help in the reversal of drug resistance in cancers by inhibiting the MDR1 efflux pump. Here, we demonstrate that celecoxib increases the sensitivity of bacteria to the antibiotics ampicillin, kanamycin, chloramphenicol, and ciprofloxacin by accumulating the drugs inside the cell, thus reversing MDR in bacteria.

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Although 23% of world population is infected with Mycobacterium tuberculosis (M. tb), only 5-10% manifest the disease. Individuals surely exposed to M. tb that remain asymptomatic are considered potential latent TB (LTB) cases. Such asymptomatic M. tb.-exposed individuals represent a reservoir for active TB cases. Although accurate discrimination and early treatment of patients with active TB and asymptomatic M. tb.-exposed individuals are necessary to control TB, identifying those individuals at risk of developing active TB still remains a tremendous clinical challenge. This study aimed to characterize the differences in the serum metabolic profile specifically associated to active TB infected individuals or to asymptomatic M. tb.-exposed population. Interestingly, significant changes in a specific set of metabolites were shared when comparing either asymptomatic household contacts of active TB patients (HHC-TB) or active TB patients (A-TB) to clinically healthy controls (HC). Furthermore, this analysis revealed statistically significant lower serum levels of aminoacids such as alanine, lysine, glutamate and glutamine, and citrate and choline in patients with A-TB, when compared to HHC-TB. The predictive ability of these metabolic changes was also evaluated. Although further validation in independent cohorts and comparison with other pulmonary infectious diseases will be necessary to assess the clinical potential, this analysis enabled the discrimination between HHC-TB and A-TB patients with an AUC value of 0.904 (confidence interval 0.81-1.00, p-value < 0.0001). Overall, the strategy described in this work could provide a sensitive, specific, and minimally invasive method that could eventually be translated into a clinical tool for TB control.

Metabolic adaptation of Mycobacterium tuberculosis (M. tuberculosis) to microbicidal intracellular environment of host macrophages is fundamental to its pathogenicity. However, an in-depth understanding of metabolic adjustments through key reaction pathways and networks is limited. To understand how such changes occur, we measured the cellular metabolome of M. tuberculosis subjected to four microbicidal stresses using liquid chromatography-mass spectrometric multiple reactions monitoring (LC-MRM/MS). Overall, 87 metabolites were identified. The metabolites best describing the separation between stresses were identified through multivariate analysis. The coupling of the metabolite measurements with existing genome-scale metabolic model, and using constraint-based simulation led to several new concepts and unreported observations in M. tuberculosis; such as (i) the high levels of released ammonia as an adaptive response to acidic stress was due to increased flux through L-asparaginase rather than urease activity; (ii) nutrient starvation-induced anaplerotic pathway for generation of TCA intermediates from phosphoenolpyruvate using phosphoenolpyruvate kinase; (iii) quenching of protons through GABA shunt pathway or sugar alcohols as possible mechanisms of early adaptation to acidic and oxidative stresses; and (iv) usage of alternate cofactors by the same enzyme as a possible mechanism of rewiring metabolic pathways to overcome stresses. Besides providing new leads and important nodes that can be used for designing intervention strategies, the study advocates the strength of applying flux balance analyses coupled with metabolomics to get a global picture of complex metabolic adjustments.

Environmental mycobacteria, highly prevalent in natural and artificial (including chlorinated municipal water) niches, are emerging as new threat to human health, especially to HIV-infected population. These seemingly harmless non-pathogenic mycobacteria, which are otherwise cleared, establish as opportunistic infections adding to HIV-associated complications. Although immune-evading strategies of pathogenic mycobacteria are known, the mechanisms underlying the early events by which opportunistic mycobacteria establish infection in macrophages and influencing HIV infection are unclear. Proteomics of phagosome-enriched fractions from Mycobacterium bovis Bacillus Calmette-Guérin (BCG) mono-infected and HIV-M. bovis BCG co-infected THP-1 cells by LC-MALDI-MS/MS revealed differential distribution of 260 proteins. Validation of the proteomics data showed that HIV co-infection helped the survival of non-pathogenic mycobacteria by obstructing phagosome maturation, promoting lipid biogenesis and increasing intracellular ATP equivalents. In turn, mycobacterial co-infection up-regulated purinergic receptors in macrophages that are known to support HIV entry, explaining increased viral titers during co-infection. The mutualism was reconfirmed using clinically relevant opportunistic mycobacteria, Mycobacterium avium, Mycobacterium kansasii and Mycobacterium phlei that exhibited increased survival during co-infection, together with increase in HIV titers. Additionally, the catalogued proteins in the study provide new leads that will significantly add to the understanding of the biology of opportunistic mycobacteria and HIV coalition.

Mycobacterium tuberculosis (Mtb) is the most common co-infection in HIV patients and a serious co-epidemic. Apart from increasing the risk of reactivation of latent tuberculosis (TB), HIV infection also permits opportunistic infection of environmental non-pathogenic mycobacteria. To gain insights into mycobacterial survival inside host macrophages and identify mycobacterial proteins or processes that influence HIV propagation during co-infection, we employed proteomics approach to identify differentially expressed intracellular mycobacterial proteins during mono- and HIV co-infection of human THP-1 derived macrophage cell lines. Of the 92 proteins identified, 30 proteins were upregulated during mycobacterial mono-infection and 40 proteins during HIV-mycobacteria co-infection. We observed down-regulation of toxin-antitoxin (TA) modules, up-regulation of cation transporters, Type VII (Esx) secretion systems, proteins involved in cell wall lipid or protein metabolism, glyoxalate pathway and branched chain amino-acid synthesis during co-infection. The bearings of these mycobacterial factors or processes on HIV propagation during co-infection, as inferred from the proteomics data, were validated using deletion mutants of mycobacteria. The analyses revealed mycobacterial factors that possibly via modulating the host environment, increased viral titers during co-infection. The study provides new leads for investigations towards hitherto unknown molecular mechanisms explaining HIV-mycobacteria synergism, helping address diagnostics and treatment challenges for effective co-epidemic management.

Mycobacterium smegmatis, the saprophytic soil mycobacterium, is routinely used as a surrogate system to study the human pathogen Mycobacterium tuberculosis. It has also been reported as an opportunistic pathogen in immunocompromised hosts. In addition, it can exist in several ecological setups, thereby suggesting its capacity to adapt to a variety of environmental cues. In this study, we employed untargeted proton nuclear magnetic resonance ( 1 H-NMR)-based metabolomics to identify metabolites and metabolic pathways critical for early adaptive responses to acidic stress, oxidative stress, and nutrient starvation in Mycobacterium smegmatis. We identified 31, 20, and 46 metabolites that showed significant changes in levels in response to acidic, oxidative, and nutrient starvation stresses, respectively. Pathway analyses showed significant perturbations in purine-pyrimidine, amino-acid, nicotinate-nicotinamide, and energy metabolism pathways. Besides these, differential levels of intermediary metabolites involved in ␣-glucan biosynthesis pathway were observed. We also detected high levels of organic osmolytes, methylamine, and betaine during nutrient starvation and oxidative stress. Further, tracing the differential levels of these osmolytes through computational search tools, gene expression studies (using reverse transcription-PCR [RT-PCR]), and enzyme assays, we detected the presence of a putative pathway of biosynthesis of betaine, methylamine, and dimethylamine previously unreported in Mycobacterium smegmatis. IMPORTANCE Alterations in metabolite levels provide fast and direct means to regulate enzymatic reactions and, therefore, metabolic pathways. This study documents, for the first time, the metabolic changes that occur in Mycobacterium smegmatis as a response to three stresses, namely, acidic stress, oxidative stress, and nutrient starvation. These stresses are also faced by intracellular mycobacteria during infection and therefore may be extended to frame therapeutic interventions for pathogenic mycobacteria. In addition to the purine-pyrimidine, amino acid, nicotinate-nicotinamide, and energy metabolism pathways that were found to be affected in response to different stresses, a novel putative methylamine biosynthesis pathway was identified to be present in Mycobacterium smegmatis.

BackgroundAdvanced stages of leprosy show T cell unresponsiveness and lipids of mycobacterial origin are speculated to modulate immune responses in these patients. Present study elucidates the role of phenolicglycolipid (PGL-1) and Mannose-capped lipoarabinomannan (Man-LAM) on TCR- and TCR/CD28- mediated signalling.ResultsWe observed that lipid antigens significantly inhibit proximal early signalling events like Zap-70 phosphorylation and calcium mobilization. Interestingly, these antigens preferentially curtailed TCR-triggered early downstream signalling events like p38 phosphorylation whereas potentiated that of Erk1/2. Further, at later stages inhibition of NFAT binding, IL-2 message, CD25 expression and T-cell blastogenesis by PGL-1 and Man-LAM was noted.ConclusionAltogether, we report that Man-LAM and PGL-1 preferentially interfere with TCR/CD28-triggered upstream cell signalling events, leading to reduced IL-2 secretion and T-cell blastogenesis which potentially could lead to immunosupression and thus, disease exacerbation, as noted in disease spectrum.