Ancient origin and gene mosaicism of the progenitor of Mycobacterium tuberculosis

The highly successful human pathogen Mycobacterium tuberculosis has an extremely low level of genetic variation, which suggests that the entire population resulted from clonal expansion following an evolutionary bottleneck around 35,000 y ago. Here, we show that this population constitutes just the visible tip of a much broader progenitor species, whose extant representatives are human isolates of tubercle bacilli from East Africa. In these isolates, we detected incongruence among gene phylogenies as well as mosaic gene sequences, whose individual elements are retrieved in classical M. tuberculosis. Therefore, despite its apparent homogeneity, the M. tuberculosis genome appears to be a composite assembly resulting from horizontal gene transfer events predating clonal expansion. The amount of synonymous nucleotide variation in housekeeping genes suggests that tubercle bacilli were contemporaneous with early hominids in East Africa, and have thus been coevolving with their human host much longer than previously thought. These results open novel perspectives for unraveling the molecular bases of M. tuberculosis evolutionary success.     

A promoter mutation causes differential nitrate reductase activity of Mycobacterium tuberculosis and Mycobacterium bovis

The recent publication of the genome sequence of Mycobacterium bovis showed >99.95% identity to M. tuberculosis. No genes unique to M. bovis were found. Instead numerous single-nucleotide polymorphisms (SNPs) were identified. This has led to the hypothesis that differential gene expression due to SNPs might explain the differences between the human and bovine tubercle bacilli. One phenotypic distinction between M. tuberculosis and M. bovis is nitrate reduction, which not only is an essential diagnostic tool but also contributes to mycobacterial pathogenesis. We previously showed that narGHJI encodes a nitrate reductase in both M. tuberculosis and M. bovis and that NarGHJI-mediated nitrate reductase activity was substantially higher in the human tubercle bacillus. In the present study we used a genetic approach to demonstrate that an SNP within the promoter of the nitrate reductase gene cluster narGHJI is responsible for the different nitrate reductase activity of M. tuberculosis and M. bovis. This is the first example of an SNP that leads to differential gene expression between the human and bovine tubercle bacilli.     

cAMP levels within Mycobacterium tuberculosis and Mycobacterium bovis BCG increase upon infection of macrophages

Adenosine 3',5'-cyclic monophosphate (cAMP)-mediated signal transduction is common in both prokaryotes and eukaryotes, and several bacterial pathogens modulate cAMP signaling pathways of their mammalian hosts during infection. In this study, cAMP levels associated with Mycobacterium tuberculosis and Mycobacterium bovis BCG were measured during macrophage infection. cAMP levels within both bacteria increased c . 50-fold during infection of J774.16 macrophages, relative to the cAMP levels within bacteria incubated in tissue culture media alone. cAMP levels also increased within the macrophage cytoplasm upon uptake of live, but not dead, mycobacteria. The presence of albumin in the absence of oleic acid significantly decreased cAMP secretion and production by both M. tuberculosis and M. bovis BCG. These results suggest that cAMP signaling plays a role in the interaction of tuberculosis-complex mycobacteria with macrophages during infection, and that albumin may be a physiological indicator differentiating host environments during infection.     

Stress responses in mycobacteria

Mycobacterium tuberculosis is a successful pathogen that overcomes numerous challenges presented by the immune system of the host. This bacterium usually establishes a chronic infection in the host where it may silently persist inside a granuloma until, a failure in host defenses, leads to manifestation of the disease. None of the conventional anti-tuberculosis drugs are able to target these persisting bacilli. Development of drugs against such persisting bacilli is a constant challenge since the physiology of these dormant bacteria is still not understood at the molecular level. Some evidence suggests that the in vivo environment encountered by the persisting bacteria is anoxic and nutritionally starved. Based on these assumptions, anaerobic and starved cultures are used as models to study the molecular basis of dormancy. This review outlines the problem of persistence of M. tuberculosis and the various in vitro models used to study mycobacterial latency. The basis of selecting the nutritional starvation model has been outlined here. Also, the choice of M. smegmatis as a model suitable for studying mycobacterial latency is discussed. Lastly, general issues related to oxidative stress and bacterial responses to it have been elaborated. We have also discussed general control of OxyR-mediated regulation and emphasized the processes which manifest in the absence of functional OxyR in the bacteria. Lastly, a new class of protein called Dps has been reviewed for its important role in protecting DNA under stress.     

Studies on neutral exopolysaccharides produced by the ectomycorrhiza Thelephora terrestris

We developed an in vitro tissue-culture model to analyze the process involved in mycobacterial spread through lung epithelial cell monolayers. A549 cells were infected with low numbers of viable Mycobacterium tuberculosis bacilli expressing the gfp gene. Subsequent addition of a soft agarose overlay prevented the dispersal of the bacilli from the initial points of attachment. By fluorescence microscopy the bacteria were observed to infect and grow within the primary target cells; this was followed by lysis of the infected cells and subsequent infection of adjacent cells. This process repeated itself until an area of clearing (plaque formation) was observed. The addition of amikacin after initial infection did not prevent intracellular growth; however, subsequent plaque formation was not observed. Plaque formation was also observed after infection with Mycobacterium bovis BCG bacilli, but the plaques were smaller than those formed after infection with M. tuberculosis. These observations reinforce the possibility that cell-to-cell spreading of M. tuberculosis bacilli, particularly early in the course of infection within lung macrophages, pneumocytes, and other cells, may be an important component in the infectious process.     

Enhanced mortality despite control of lung infection in mice aerogenically infected with a Mycobacterium tuberculosis mce1 operon mutant

Mycobacterium tuberculosis causes a variety of host clinical outcomes. We previously showed that M. tuberculosis disrupted in an operon called mce1 proliferates unchecked in BALB/c mouse lungs. The observed outcome could be attributed either to the mutant bacterial burden or to the host immunopathologic response. To differentiate these possibilities, we studied the outcomes of infection in a mouse strain (C57BL/6) less susceptible to M. tuberculosis than BALB/c. We found that the mutant infection reached a plateau in the lungs at a rate similar to that of the wild type. All mice infected with the mutant, but only half of the groups of mice infected with the wild type or complemented strain, died by 40 weeks (p<0.05). At 12-21 weeks of infection, histological examination of the lungs of mice infected with the mutant showed a diffuse pattern of lymphocyte infiltration, while that of mice infected with the other strains exhibited a nodular cellular infiltration pattern. Surprisingly, the number of bacilli recovered from the lungs was similar in all three groups. These observations suggest that rather than the bacterial burden, products of the mce1 operon may directly or indirectly modulate the host immune response that is protective to both the tubercle bacilli and the host.     

Inhibition of bfl-1/A1 by siRNA inhibits mycobacterial growth in THP-1 cells by enhancing phagosomal acidification

Virulent tubercle bacilli inhibit apoptosis to establish a safe environment within the host cells. Here, we report that NF-kappaB dependent antiapoptotic protein bfl-1/A1 plays an important role in this process. Both virulent and avirulent mycobacteria bearing THP-1 cells expressed considerable amount of bfl-1/A1 after 4 h of infection. However, after 48 h of infection, bfl-1/A1 expression was evident only in Mycobacterium tuberculosis H37Rv but not in M. tuberculosis H37Ra infected cells. When parallel experiments were performed with Human monocyte-derived macrophages (MDMs), differential expression of bfl-1/A1 mRNA was observed in case of M. tuberculosis H37Rv and M. tuberculosis H37Ra infection. siRNA mediated inhibition of bfl-1/A1 induced apoptosis in M. tuberculosis H37Rv infected THP-1 and MDMs. Reduction in intracellular mycobacterial growth was observed in bfl-1/A1 siRNA transfected, M. tuberculosis H37Rv infected THP-1 cells. Enhancement of phagosome-lysosome fusion was observed in bfl-1/A1 siRNA treated and M. tuberculosis H37Rv infected THP-1 cells. These results clearly indicated that differential expression of bfl-1/A1 in M. tuberculosis H37Rv and M. tuberculosis H37Ra infected THP-1 cells probably account for the difference in infection outcome.     

Location of intra- and extracellular M. tuberculosis populations in lungs of mice and guinea pigs during disease progression and after drug treatment

The lengthy treatment regimen for tuberculosis is necessary to eradicate a small sub-population of M. tuberculosis that persists in certain host locations under drug pressure. Limited information is available on persisting bacilli and their location within the lung during disease progression and after drug treatment. Here we provide a comprehensive histopathological and microscopic evaluation to elucidate the location of bacterial populations in animal models for TB drug development.To detect bacilli in tissues, a new combination staining method was optimized using auramine O and rhodamine B for staining acid-fast bacilli, hematoxylin QS for staining tissue and DAPI for staining nuclei. Bacillary location was studied in three animal models used in-house for TB drug evaluations: C57BL/6 mice, immunocompromised GKO mice and guinea pigs. In both mouse models, the bacilli were found primarily intracellularly in inflammatory lesions at most stages of disease, except for late stage GKO mice, which showed significant necrosis and extracellular bacilli after 25 days of infection. This is also the time when hypoxia was initially visualized in GKO mice by 2-piminidazole. In guinea pigs, the majority of bacteria in lungs are extracellular organisms in necrotic lesions and only few, if any, were ever visualized in inflammatory lesions. Following drug treatment in mice a homogenous bacillary reduction across lung granulomas was observed, whereas in guinea pigs the remaining extracellular bacilli persisted in lesions with residual necrosis. In summary, differences in pathogenesis between animal models infected with M. tuberculosis result in various granulomatous lesion types, which affect the location, environment and state of bacilli. The majority of M. tuberculosis bacilli in an advanced disease state were found to be extracellular in necrotic lesions with an acellular rim of residual necrosis. Drug development should be designed to target this bacillary population and should evaluate drug regimens in the appropriate animal models.     

Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis.

The live attenuated bacillus Calmette-Guérin (BCG) vaccine for the prevention of disease associated with Mycobacterium tuberculosis was derived from the closely related virulent tubercle bacillus, Mycobacterium bovis. Although the BCG vaccine has been one of the most widely used vaccines in the world for over 40 years, the genetic basis of BCG's attenuation has never been elucidated. We employed subtractive genomic hybridization to identify genetic differences between virulent M. bovis and M. tuberculosis and avirulent BCG. Three distinct genomic regions of difference (designated RD1 to RD3) were found to be deleted from BCG, and the precise junctions and DNA sequence of each deletion were determined. RD3, a 9.3-kb genomic segment present in virulent laboratory strains of M. bovis and M. tuberculosis, was absent from BCG and 84% of virulent clinical isolates. RD2, a 10.7-kb DNA segment containing a novel repetitive element and the previously identified mpt-64 gene, was conserved in all virulent laboratory and clinical tubercle bacilli tested and was deleted only from substrains derived from the original BCG Pasteur strain after 1925. Thus, the RD2 deletion occurred after the original derivation of BCG. RD1, a 9.5-kb DNA segment found to be deleted from all BCG substrains, was conserved in all virulent laboratory and clinical isolates of M. bovis and M. tuberculosis tested. The reintroduction of RD1 into BCG repressed the expression of at least 10 proteins and resulted in a protein expression profile almost identical to that of virulent M. bovis and M. tuberculosis, as determined by two-dimensional gel electrophoresis. These data indicate a role for RD1 in the regulation of multiple genetic loci, suggesting that the loss of virulence by BCG is due to a regulatory mutation. These findings may be applicable to the rational design of a new attenuated tuberculosis vaccine and the development of new diagnostic tests to distinguish BCG vaccination from tuberculosis infection.     

Identification of variable regions in the genomes of tubercle bacilli using bacterial artificial chromosome arrays

Whole-genome comparisons of the tubercle bacilli were undertaken using ordered bacterial artificial chromosome (BAC) libraries of Mycobacterium tuberculosis and the vaccine strain, Mycobacterium bovis BCG-Pasteur, together with the complete genome sequence of M. tuberculosis H37Rv. Restriction-digested BAC arrays of M. tuberculosis H37Rv were used in hybridization experiments with radiolabelled M. bovis BCG genomic DNA to reveal the presence of 10 deletions (RD1-RD10) relative to M. tuberculosis. Seven of these regions, RD4-RD10, were also found to be deleted from M. bovis, with the three M. bovis BCG-specific deletions being identical to the RD1-RD3 loci described previously. The distribution of RD4-RD10 in Mycobacterium africanum resembles that of M. tuberculosis more closely than that of M. bovis, whereas an intermediate arrangement was found in Mycobacterium microti, suggesting that the corresponding genes may affect host range and virulence of the various tubercle bacilli. Among the known products encoded by these loci are a copy of the proposed mycobacterial invasin Mce, three phospholipases, several PE, PPE and ESAT-6 proteins, epoxide hydrolase and an insertion sequence. In a complementary approach, direct comparison of BACs uncovered a third class of deletions consisting of two M. tuberculosis H37Rv loci, RvD1 and RvD2, deleted from the genome relative to M. bovis BCG and M. bovis. These deletions affect a further seven genes, including a fourth phospholipase, plcD. In summary, the insertions and deletions described here have important implications for our understanding of the evolution of the tubercle complex.     

Functional characterization of the phospholipase C activity of Rv3487c and its localization on the cell wall of Mycobacterium tuberculosis

Mycobacterium tuberculosis survives and persists for prolonged periods within its host in an asymptomatic,latent state and can reactivate years later if the host's immune system weakens. The dormant bacilli synthesize and accumulate triacylglycerol, reputed to be an energy source during latency. Among the phospholipases, phospholipase C plays an important role in the pathogenesis. Mutations in a known phospholipase C, plcC, of M.tuberculosis attenuate its growth during the late phase of infection in mice. Hydrolysis of phospholipids by phospholipase C generates diacylglycerol, a well-known signalling molecule that participates in the activation of extracellular signal-regulated kinases (ERK) through protein kinase C leading to macrophage activation. In the present study, we show that M.tuberculosis possesses an additional cell wall-associated protein, Rv3487c, with phospholipase C activity. The recombinant Rv3487c hydrolyses the substrate phosphatidylcholine and generates diacylglycerol by removing the phosphocholine. Furthermore, Rv3487c is expressed during infection as it exhibits significant humoral immunoreactivity with sera from children with tuberculosis, but not with that from adult patients.     

Nitric oxide scavenging and detoxification by the Mycobacterium tuberculosis haemoglobin,HbN in Escherichia coli

Nitric oxide (NO), generated in large amounts within the macrophages, controls and restricts the growth of internalized human pathogen, Mycobacterium tuberculosis H37Rv. The molecular mechanism by which tubercle bacilli survive within macrophages is currently of intense interest. In this work, we have demonstrated that dimeric haemoglobin, HbN, from M. tuberculosis exhibits distinct nitric oxide dioxygenase (NOD) activity and protects growth and cellular respiration of heterologous hosts, Escherichia coli and Mycobacterium smegmatis, from the toxic effect of exogenous NO and the NO-releasing compounds. A flavohaemoglobin (HMP)-deficient mutant of E. coli, unable to metabolize NO, acquired an oxygen-dependent NO consumption activity in the presence of HbN. On the basis of cellular haem content, the specific NOD activity of HbN was nearly 35-fold higher than the single-domain Vitreoscilla haemoglobin (VHb) but was sevenfold lower than the two-domain flavohaemoglobin. HbN-dependent NO consumption was sustained with repeated addition of NO, demonstrating that HbN is catalytically reduced within E. coli. Aerobic growth and respiration of a flavohaemoglobin (HMP) mutant of E. coli was inhibited in the presence of exogenous NO but remained insensitive to NO inhibition when these cells produced HbN, VHb or flavohaemoglobin. M. smegmatis, carrying a native HbN very similar to M. tuberculosis HbN, exhibited a 7.5-fold increase in NO uptake when exposed to gaseous NO, suggesting NO-induced NOD activity in these cells. In addition, expression of plasmid-encoded HbN of M. tuberculosis in M. smegmatis resulted in 100-fold higher NO consumption activity than the isogenic control cells. These results provide strong experimental evidence in support of NO scavenging and detoxification function for the M. tuberculosis HbN. The catalytic NO scavenging by HbN may be highly advantageous for the survival of tubercle bacilli during infection and pathogenesis.     

Multiscale computational modeling reveals a critical role for TNF-α receptor 1 dynamics in tuberculosis granuloma formation

Multiple immune factors control host responses to Mycobacterium tuberculosis infection, including the formation of granulomas, which are aggregates of immune cells whose function may reflect success or failure of the host to contain infection. One such factor is TNF-α. TNF-α has been experimentally characterized to have the following activities in M. tuberculosis infection: macrophage activation, apoptosis, and chemokine and cytokine production. Availability of TNF-α within a granuloma has been proposed to play a critical role in immunity to M. tuberculosis. However, in vivo measurement of a TNF-α concentration gradient and activities within a granuloma are not experimentally feasible. Further, processes that control TNF-α concentration and activities in a granuloma remain unknown. We developed a multiscale computational model that includes molecular, cellular, and tissue scale events that occur during granuloma formation and maintenance in lung. We use our model to identify processes that regulate TNF-α concentration and cellular behaviors and thus influence the outcome of infection within a granuloma. Our model predicts that TNF-αR1 internalization kinetics play a critical role in infection control within a granuloma, controlling whether there is clearance of bacteria, excessive inflammation, containment of bacteria within a stable granuloma, or uncontrolled growth of bacteria. Our results suggest that there is an interplay between TNF-α and bacterial levels in a granuloma that is controlled by the combined effects of both molecular and cellular scale processes. Finally, our model elucidates processes involved in immunity to M. tuberculosis that may be new targets for therapy.     

Remarkable conservation of distinct nonclassical MHC class I lineages in divergent amphibian species

Pulmonary surfactant contains homeostatic and antimicrobial hydrolases. When Mycobacterium tuberculosis is initially deposited in the terminal bronchioles and alveoli, as well as following release from lysed macrophages, bacilli are in intimate contact with these lung surfactant hydrolases. We identified and measured several hydrolases in human alveolar lining fluid and lung tissue that, at their physiological concentrations, dramatically modified the M. tuberculosis cell envelope. Independent of their action time (15 min to 12 h), the effects of the hydrolases on the M. tuberculosis cell envelope resulted in a significant decrease (60-80%) in M. tuberculosis association with, and intracellular growth of the bacteria within, human macrophages. The cell envelope-modifying effects of the hydrolases also led to altered M. tuberculosis intracellular trafficking and induced a protective proinflammatory response to infection. These findings add a new concept to our understanding of M. tuberculosis-macrophage interactions (i.e., the impact of lung surfactant hydrolases on M. tuberculosis infection).     

Bfl-1/A1 acts as a negative regulator of autophagy in mycobacteria infected macrophages

Expression of Bcl-2 family protein, Bfl-1/A1 has been found to differ considerably amongst macrophages infected with virulent Mycobacterium tuberculosis H37Rv or with avirulent M. tuberculosis H37Ra. Present work was undertaken to deduce the significance of differential expression of Bfl-1/A1 in the outcome of mycobacterial infection. We have studied the role of Bfl-1/A1 particularly in autophagy formation in tubercle bacilli infected cells since autophagy has been recognized as a component of innate immunity against pathogenic mycobacteria. First, we have confirmed that upon infection virulent strain H37Rv retain Bfl-1/A1 for longer period and impose autophagosome maturation block within infected cells as evident from confocal microscopy. Moreover, down regulation of Bfl-1/A1 by siRNA induced autophagy formation and reduced bacterial growth. Furthermore, even the avirulent strain H37Ra resist autophagosome maturation and survive if the cellular level of Bfl-1 is maintained in THP-1 cells by stable transfection (Bfl-1 overexpressing cells). No noteworthy difference in mTOR expression was observed between normal THP-1 and Bfl-1 overexpressing THP-1 cells infected with either strain of mycobacteria. Interestingly, we found that not only mTOR but also Bfl-1/A1 is involved in rapamycin induced autophagy in mycobacteria infected macrophages. We have found that Bfl-1 physically interacts with Beclin 1 in Bfl-1 overexpressing THP-1 as well as in H37Rv infected THP-1 cells as they co-precipitated. Taken together, our results clearly demonstrated that Bfl-1/A1 negatively regulates autophagy and expression of Bfl-1/A1 in H37Rv infected macrophages provides the bacteria a survival strategy to overcome host defense.     

Tuberculosis: a problem with persistence

Mycobacterium tuberculosis is one of most successful pathogens of mankind, infecting one-third of the global population and claiming two million lives every year. The ability of the bacteria to persist in the form of a long-term asymptomatic infection, referred to as latent tuberculosis, is central to the biology of the disease. The persistence of bacteria in superficially normal tissue was recognized soon after the discovery of the tubercle bacillus, and much of our knowledge about persistent populations of M. tuberculosis dates back to the first half of the last century. Recent advances in microbial genetics and host immunity provide an opportunity for renewed investigation of this persistent threat to human health.     

Local ischemia and increased expression of vascular endothelial growth factor following ocular dissemination of Mycobacterium tuberculosis

The pathogenesis of intraocular tuberculosis remains poorly understood partly due to the lack of adequate animal models that accurately simulate human disease. Using a recently developed model of ocular tuberculosis following aerosol infection of guinea pigs with Mycobacterium tuberculosis, we studied the microbiological, histological, and clinical features of intraocular tuberculosis infection. Viable tubercle bacilli were cultivated from all eyes by Day 56 after aerosol delivery of ～200 bacilli to guinea pig lungs. Choroidal tuberculous granulomas showed reduced oxygen tension, as evidenced by staining with the hypoxia-specific probe pimonidazole, and expression of vascular endothelial growth factor (VEGF) was detected in the retinal pigment epithelium (RPE) and photoreceptors. Fundoscopic examination of M. tuberculosis-infected guinea pig eyes revealed altered vascular architecture and chorioretinal hemorrhage by Day 56 after infection. This model may be useful in further elucidating the pathogenesis of ocular tuberculosis, as well as in developing tools for diagnosis and assessment of antituberculosis treatment responses in the eye.