Survival of Mycobacterium tuberculosis and Mycobacterium bovis BCG in lysosomes in vivo

Mycobacterium tuberculosis is one of the most successful pathogens known, having infected more than a third of the global population. An important strategy for intracellular survival of pathogenic mycobacteria relies on their capacity to resist delivery to lysosomes, instead surviving within macrophage phagosomes. Several factors of both mycobacterial and host origin have been implicated in this process. However, whether or not this strategy is employed in vivo is not clear. Here we show that in vivo, following intravenous infection, M. tuberculosis and Mycobacterium bovis BCG initially survived by resisting lysosomal transfer. However, after prolonged infection the bacteria were transferred to lysosomes yet continued to proliferate. A M. bovis BCG mutant lacking protein kinase G (PknG), that cannot avoid lysosomal transfer and is readily cleared in vitro, was found to survive and proliferate in vivo. The ability to survive and proliferate in lysosomal organelles in vivo was found to be due to an altered host environment rather than changes in the inherent ability of the bacteria to arrest phagosome maturation. Thus, within an infected host, both M. tuberculosis and M. bovis BCG adapts to infection-specific host responses. These results are important to understand the pathology of tuberculosis and may have implications for the development of effective strategies to combat tuberculosis.     

Is Mycobacterium tuberculosis stressed out? A critical assessment of the genetic evidence

Mycobacterium tuberculosis is an obligate human intracellular pathogen which remains a major killer worldwide. A remarkable feature of M. tuberculosis infection is the ability of the pathogen to persist within the host for decades despite an impressive onslaught of stresses. In this review we seek to outline the host-inflicted stresses experienced by M. tuberculosis, the bacterial strategies used to withstand these stresses, and how this information should guide our efforts to combat this global pathogen.     

Detection and molecular characterization of 9,000-year-old Mycobacterium tuberculosis from a Neolithic settlement in the Eastern Mediterranean

Background

Mycobacterium tuberculosis is the principal etiologic agent of human tuberculosis. It has no environmental reservoir and is believed to have co-evolved with its host over millennia. This is supported by skeletal evidence of the disease in early humans, and inferred from M. tuberculosis genomic analysis. Direct examination of ancient human remains for M. tuberculosis biomarkers should aid our understanding of the nature of prehistoric tuberculosis and the host/pathogen relationship.

Methodology/Principal Findings

We used conventional PCR to examine bone samples with typical tuberculosis lesions from a woman and infant, who were buried together in the now submerged site of Atlit-Yam in the Eastern Mediterranean, dating from 9250-8160 years ago. Rigorous precautions were taken to prevent contamination, and independent centers were used to confirm authenticity of findings. DNA from five M tuberculosis genetic loci was detected and had characteristics consistent with extant genetic lineages. High performance liquid chromatography was used as an independent method of verification and it directly detected mycolic acid lipid biomarkers, specific for the M. tuberculosis complex.

Conclusions/Significance

Human tuberculosis was confirmed by morphological and molecular methods in a population living in one of the first villages with evidence of agriculture and animal domestication. The widespread use of animals was not a source of infection but may have supported a denser human population that facilitated transmission of the tubercle bacillus. The similarity of the M. tuberculosis genetic signature with those of today gives support to the theory of a long-term co-existence of host and pathogen.     

Genes and regulatory networks involved in persistence of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

The causative agent of tuberculosis, Mycobacterium tuberculosis, is one of the most successful of human pathogens. It can evade the host immune response and establish a persistent infection or enter a dormant state within the host which can be reactivated if the host becomes immuno-compromised. Both of these features are major obstacles to tuberculosis eradication. Dormancy and reactivation of M. tuberculosis are tightly coordinated dynamic processes involving numerous genes and their products. Molecular mechanisms underlying M. tuberculosis persistence may provide an opportunity for the discovery of effective drug targets for tuberculosis control. Here, we review the genes required for M. tuberculosis persistence and propose a regulatory network for the action of these genes using text mining. This should provide fresh insights into the persistence mechanisms of M. tuberculosis and suggest candidates for new drug targets and immune intervention.     

Mycobacterium tuberculosis persistence in various adipose depots of infected mice and the effect of anti-tubercular therapy

The adipocytes are one of the non-professional phagocytes postulated to be a haven for Mycobacterium tuberculosis during persistence in the human host. The adipocyte – M. tuberculosis interaction data available to date are ex vivo. The present study was primarily aimed to investigate M. tuberculosis infection of adipocytes in course of infection of mouse model. Using primary murine adipocytes, the study first confirmed the infection and immunomodulation of natural adipocytes by M. tuberculosis. The bacilli could be isolated form visceral, subcutaneous, peri renal and mesenteric adipose depots of immunocompetent mice infected with M. tuberculosis intravenously. The bacilli could be isolated from adipocytes and the stromal vascular fraction, even though the numbers were significantly higher in the latter. The bacterial burden in the adipose depots was comparable to those in lungs in the early phase of infection. But with time, the burden in the adipose depots was either decreased or kept under control, despite the increasing burden in the lungs. Infected mice treated with standard anti tubercular drugs, despite effective elimination of bacterial loads in the lungs, continued to harbour M. tuberculosis in adipose depots at loads similar to untreated mice in the late infection phase.     

Design,synthesis, and biological evaluation of dihydroartemisinin–fluoroquinolone conjugates as a novel type of potential antitubercular agents

Tuberculosis remains a global public health problem in recent years. To develop novel type of potential antitubercular agents, twelve novel dihydroartemisinin–fluoroquinolone (DHA–FQ) conjugates (three types of molecules) were gradually designed and conveniently synthesized. All the newly synthesized conjugates were well characterized and evaluated against different Mycobacterium tuberculosis strains in vitro. The screening results showed that five DHA–FQ conjugates were active toward M. tuberculosis H37Rv, and compound 3a exhibited the strongest inhibitory activity (MIC = 0.0625 μg/mL), which was comparable to the positive control Moxifloxacin and even stronger than Ofloxacin. Conjugates 2a and 3a also displayed comparable activities against various clinically isolated sensitive and resistant M. tuberculosis strains (MIC = 0.125–16 μg/mL) to Moxifloxacin. All target compounds possessed selective anti-M. tuberculosis ability. Preliminary structure–activity relationship demonstrated that short linker between DHA and FQ was favorable for strong antitubercular activity. This study provides a new clue for the development of novel antitubercular lead molecules.     

Triacylglycerol: nourishing molecule in endurance of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

The ability of Mycobacterium tuberculosis (M. tuberculosis) to accumulate lipid-rich molecules as an energy source obtained from host cell debris remains interesting. Additionally, the potential of M. tuberculosis to survive under different stress conditions leading to its dormant state in pathogenesis remains elusive. The exact mechanism by which these lipid bodies generated in M. tuberculosis infection and utilized by bacilli inside infected macrophage for its survival is still not understood. In this, during bacillary infection, many metabolic pathways are involved that influence the survival of M. tuberculosis for their own support. However, the exact energy source derived from infecting host cells remain elusive. Therefore, this study highlights several alternative energy sources in the form of triacylglycerol (TAG) and fatty acids, i.e. oleic acids accumulation, which are essential in dormancy-like state under M. tuberculosis infection. The prominent stage in tuberculosis (TB) infection is re-establishment of M. tuberculosis under stress conditions and deployment of a confined strategy to utilize these biomolecules for its persistence survival. So, growing in our understanding of these pathways will help us in accelerating therapies, which could reduce TB prevalence world widely.     

Discovery of dipiperidines as new antitubercular agents

As part of our ongoing research effort to develop new therapeutics for treatment of tuberculosis (TB), we synthesized a combinatorial library of 10,358 compounds on solid support using a pool-and-split technique and tested the resulting compounds for activity against Mycobacterium tuberculosis. Structure–activity relationship (SAR) evaluation identified new compounds with antitubercular activity, including a novel hit series that is structurally unrelated to any existing antitubercular drugs, dipiperidines. Dipiperidine representatives exhibited MIC values as low as 7.8 μM, the ability to induce promoter Rv0341 activated in response to cell wall biosynthesis inhibition, relatively low nonspecific cellular toxicity in the range of 30–162 μM, and log P values less than 4.     

Cyclic Amp-Dependent Resuscitation of Dormant Mycobacteria by Exogenous Free Fatty Acids

One third of the world population carries a latent tuberculosis (TB) infection, which may reactivate leading to active disease. Although TB latency has been known for many years it remains poorly understood. In particular, substances of host origin, which may induce the resuscitation of dormant mycobacteria, have not yet been described. In vitro models of dormant (“non-culturable”) cells of Mycobacterium smegmatis (mc²155) and Mycobacterium tuberculosis H37Rv were used. We found that the resuscitation of dormant M. smegmatis and M. tuberculosis cells in liquid medium was stimulated by adding free unsaturated fatty acids (FA), including arachidonic acid, at concentrations of 1.6–10 µM. FA addition enhanced cAMP levels in reactivating M. smegmatis cells and exogenously added cAMP (3–10 mM) or dibutyryl-cAMP (0.5–1 mM) substituted for FA, causing resuscitation of M. smegmatis and M. tuberculosis dormant cells. A M. smegmatis null-mutant lacking MSMEG_4279, which encodes a FA-activated adenylyl cyclase (AC), could not be resuscitated by FA but it was resuscitated by cAMP. M. smegmatis and M. tuberculosis cells hyper-expressing AC were unable to form non-culturable cells and a specific inhibitor of AC (8-bromo-cAMP) prevented FA-dependent resuscitation. RT-PCR analysis revealed that rpfA (coding for resuscitation promoting factor A) is up-regulated in M. smegmatis in the beginning of exponential growth following the cAMP increase in lag phase caused by FA-induced cell activation. A specific Rpf inhibitor (4-benzoyl-2-nitrophenylthiocyanate) suppressed FA-induced resuscitation. We propose a novel pathway for the resuscitation of dormant mycobacteria involving the activation of adenylyl cyclase MSMEG_4279 by FAs resulted in activation of cellular metabolism followed later by increase of RpfA activity which stimulates cell multiplication in exponential phase. The study reveals a probable role for lipids of host origin in the resuscitation of dormant mycobacteria, which may function during the reactivation of latent TB.     

Resuscitation Promoting Factors: a Family of Microbial Proteins in Survival and Resuscitation of Dormant Mycobacteria

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is an extraordinarily successful pathogen of humankind. It has been estimated that up to one-third of the world’s population is infected with M. tuberculosis, and this population is an important reservoir for disease reactivation. Resuscitation promoting factor (Rpf) is a secretory protein, which was first reported in Micrococcus luteus. There are five functionally redundant Rpf-like proteins found in M. tuberculosis. Rpf promotes the resuscitation of dormant bacilli to yield normal, viable colony forming bacteria. All Rpfs share a conserved domain of about 70 amino acids and possess a lysozyme-like activity. The structural studies of the conserved domain suggest that Rpfs could be considered as a c-type lysozyme and lytic transglycosylases. Recently a novel class of nitrophenylthiocyanates (NPT) inhibitors of the muralytic activity of Rpf were reported which opens a new approach in the study of cell-wall hydrolyzing enzymes. This review describes molecular and structural studies conducted on Rpf proteins, their role in the resuscitation of dormant bacteria, in the reactivation of latent infection and identification of low molecular weight inhibitors of resuscitation promoting factors.     

Molecular mechanism of interaction of Mycobacterium tuberculosis with host macrophages under high glucose conditions

Mycobacterium tuberculosis has the potential to escape various cellular defense mechanisms for its survival which include various oxidative stress responses, inhibition of phagosome-lysosomes fusion and alterations in cell death mechanisms of host macrophages that are crucial for its infectivity and dissemination. Diabetic patients are more susceptible to developing tuberculosis because of impairement of innate immunity and prevailing higher glucose levels. Our earlier observations have demonstrated alterations in the protein profile of M. tuberculosis exposed to concurrent high glucose and tuberculosis conditions suggesting a crosstalk between host and pathogen under high glucose conditions. Since high glucose environment plays crucial role in the interaction of mycobacterium with host macrophages which provide a niche for the survival of M. tuberculosis, it is important to understand various interactive mechanisms under such conditions. Initial phagocytosis and containment of M. tuberculosis by macrophages, mode of macrophage cell death, respiratory burst responses, Mycobacterium and lysosomal co-localization were studied in M. tuberculosis H37Rv infected cells in the presence of varied concentrations of glucose in order to mimic diabetes like conditions. It was observed that initial attachment, phagocytosis and later containment were less effective under high glucose conditions in comparison to normal glucose. Mycobacterium infected cells showed more necrosis than apoptosis as cell death mechanism during the course of infection under high glucose concentrations. Co-localization and respiratory burst assay also indicated evasion strategies adopted by M. tuberculosis under such conditions. This study by using THP1 macrophage model of tuberculosis and high glucose conditions showed immune evasion strategies adapted during co-pathogenesis of tuberculosis and diabetes.     

Mycobacterium tuberculosis TlyA Protein Negatively Regulates T Helper (Th) 1 and Th17 Differentiation and Promotes Tuberculosis Pathogenesis

Mycobacterium tuberculosis, the causative agent of tuberculosis, is an ancient pathogen and a major cause of death worldwide. Although various virulence factors of M. tuberculosis have been identified, its pathogenesis remains incompletely understood. TlyA is a virulence factor in several bacterial infections and is evolutionarily conserved in many Gram-positive bacteria, but its function in M. tuberculosis pathogenesis has not been elucidated. Here, we report that TlyA significantly contributes to the pathogenesis of M. tuberculosis. We show that a TlyA mutant M. tuberculosis strain induces increased IL-12 and reduced IL-1β and IL-10 cytokine responses, which sharply contrasts with the immune responses induced by wild type M. tuberculosis. Furthermore, compared with wild type M. tuberculosis, TlyA-deficient M. tuberculosis bacteria are more susceptible to autophagy in macrophages. Consequently, animals infected with the TlyA mutant M. tuberculosis organisms exhibited increased host-protective immune responses, reduced bacillary load, and increased survival compared with animals infected with wild type M. tuberculosis. Thus, M. tuberculosis employs TlyA as a host evasion factor, thereby contributing to its virulence.     

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.     

Human Granuloma In Vitro Model,for TB Dormancy and Resuscitation

Tuberculosis (TB) is responsible for death of nearly two million people in the world annually. Upon infection, Mycobacterium tuberculosis (Mtb) causes formation of granuloma where the pathogen goes into dormant state and can live for decades before resuscitation to develop active disease when the immune system of the host is weakened and/or suppressed. In an attempt to better understand host-pathogen interactions, several groups have been developing in vitro models of human tuberculosis granuloma. However, to date, an in vitro granuloma model in which Mtb goes into dormancy and can subsequently resuscitate under conditions that mimic weakening of the immune system has not been reported. We describe the development of a biomimetic in vitro model of human tuberculosis granuloma using human primary leukocytes, in which the Mtb exhibited characteristics of dormant mycobacteria as demonstrated by (1) loss of acid-fastness, (2) accumulation of lipid bodies (3) development of rifampicin-tolerance and (4) gene expression changes. Further, when these micro granulomas were treated with immunosuppressant anti-tumor necrosis factor-alpha monoclonal antibodies (anti-TNFα mAbs), resuscitation of Mtb was observed as has been found in humans. In this human in vitro granuloma model triacylglycerol synthase 1deletion mutant (Δtgs1) with impaired ability to accumulate triacylglycerides (TG), but not the complemented mutant, could not go into dormancy. Deletion mutant of lipY, with compromised ability to mobilize the stored TG, but not the complemented mutant, was unable to come out of dormancy upon treatment with anti-TNFα mAbs. In conclusion, we have developed an in vitro human tuberculosis granuloma model that largely exhibits functional features of dormancy and resuscitation observed in human tuberculosis.     

Intracellular replication of attenuated Mycobacterium tuberculosis phoP mutant in the absence of host cell cytotoxicity

Intracellular pathogen Mycobacterium tuberculosis survives and replicates in macrophages but limited information is available on its replication into non-phagocytic cells. Here we study the role of the M. tuberculosis virulence gene phoP in the intracellular growth with rat and human lung fibroblasts. In contrast to macrophages, attenuated M. tuberculosis phoP mutant was able to multiply intracellularly in fibroblasts at the same level as the virulent M. tuberculosis. However, when M. tuberculosis virulence was studied using human foetal lung fibroblasts, MRC-5 cell line, the virulent strain caused a significant damage in cells compared with attenuated strains BCG and M. tuberculosis phoP mutant. We analysed the effect of cytoskeleton inhibitors in NRK-49F fibroblasts. M. tuberculosis invasion was not inhibited, suggesting that mycobacterial uptake was microtubule and microfilament independent. Our results suggest that PhoP in M. tuberculosis does not regulate intracellular replication in fibroblasts, contrary to what happens in macrophages. The ability of M. tuberculosis phoP mutant to replicate within non-phagocytic cells, such as fibroblasts, without causing damage, could be a potential advantage for a live attenuated vaccine against tuberculosis.     

Mycobacterium tuberculosis and Copper: A Newly Appreciated Defense against an Old Foe?

Several independent studies have recently converged upon the conclusion that the human bacterial pathogen Mycobacterium tuberculosis encounters copper during infections. At least three independently regulated pathways respond to excess copper and are required for the full virulence of M. tuberculosis in animals. In this review, I will discuss the functions of the best-characterized copper-responsive proteins in M. tuberculosis, the potential sources of copper during an infection, and remaining questions about the interface between copper and tuberculosis.     

Expression,purification and characterization of UvrD2 helicase from Mycobacterium tuberculosis

Helicases appear to be important for genome stability of dormant Mycobacterium tuberculosis responsible for latent tuberculosis infection and big proportion of active disease cases caused by reactivation. It was demonstrated that in both M. tuberculosis and Mycobacterium smegmatis, a helicase termed UvrD2 is essential for bacterial growth making it a promising target to fight tuberculosis. In many cases expression of soluble and active mycobacterial proteins in Escherichia coli is a complicated issue. In this work we for the first time report a non-trivial expression procedure in E. coli, leading to soluble UvrD2 from M. tuberculosis which possesses DNA-dependent ATP-ase activity.