Vivek Rao

Disruption of mptpB impairs the ability of Mycobacterium tuberculosis to survive in guinea pigs

Protein tyrosine kinases and tyrosine phosphatases from several bacterial pathogens have been shown to act as virulence factors by modulating the phosphorylation and dephosphorylation of host proteins. The identification and characterization of two tyrosine phosphatases namely MptpA and MptpB from Mycobacterium tuberculosis has been reported earlier. MptpB is secreted by M. tuberculosis into extracellular mileu and exhibits a pH optimum of 5.6, similar to the pH of the lysosomal compartment of the cell. To determine the role of MptpB in the pathogenesis of M. tuberculosis, we constructed a mptpB mutant strain by homologous recombination and compared the ability of parent and the mutant strain to survive intracellularly. We show that disruption of the mptpB gene impairs the ability of the mutant strain to survive in activated macrophages and guinea pigs but not in resting macrophages suggesting the importance of its role in the host–pathogen interaction. Infection of guinea pigs with the mutant strain resulted in a 70‐fold reduction in the bacillary load of spleens in infected animals as compared with the bacillary load in animals infected with the parental strain. Upon reintroduction of the mptpB gene into the mutant strain, the complemented strain was able to establish infection and survive in guinea pigs at rates comparable to the parental strain. These observations demonstrate a   role   of MptpB in the pathogenesis   of M. tuberculosis.

Enhanced and enduring protection against tuberculosis by recombinant BCG-Ag85C and its association with modulation of cytokine profile in lung.

Background The variable efficacy (0–80%) of Mycobacterium bovis Bacille Calmette Guréin (BCG) vaccine against adult tuberculosis (TB) necessitates development of alternative vaccine candidates. Development of recombinant BCG (rBCG) over-expressing promising immunodominant antigens of M. tuberculosis represents one of the potential approaches for the development of vaccines against TB. Methods/Principal Findings A recombinant strain of BCG - rBCG85C, over expressing the antigen 85C, a secretory immuno-dominant protein of M. tuberculosis, was evaluated for its protective efficacy in guinea pigs against M. tuberculosis challenge by aerosol route. Immunization with rBCG85C resulted in a substantial reduction in the lung (1.87 log10, p<0.01) and spleen (2.36 log10, p<0.001) bacillary load with a commensurate reduction in pathological damage, when compared to the animals immunized with the parent BCG strain at 10 weeks post-infection. rBCG85C continued to provide superior protection over BCG even when post-challenge period was prolonged to 16 weeks. The cytokine profile of pulmonary granulomas revealed that the superior protection imparted by rBCG85C was associated with the reduced levels of pro-inflammatory cytokines - interleukin (IL)-12, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, moderate levels of anti-inflammatory cytokine - transforming growth factor (TGF)-β along with up-regulation of inducible nitric oxide synthase (iNOS). In addition, the rBCG85C vaccine induced modulation of the cytokine levels was found to be associated with reduced fibrosis and antigen load accompanied by the restoration of normal lung architecture. Conclusions/Significance These results clearly indicate the superiority of rBCG85C over BCG as a promising prophylactic vaccine against TB. The enduring protection observed in this study gives enough reason to postulate that if an open-ended study is carried out with low dose of infection, rBCG85C vaccine in all likelihood would show enhanced survival of guinea pigs.

Modulation of Host Immune Responses by Overexpression of Immunodominant Antigens of Mycobacterium tuberculosis in Bacille Calmette–Guérin

Based on their immunodominant nature and ability to induce appropriate immune responses in the host, several antigens of Mycobacterium tuberculosis have shown promise of protection. However, most of the candidate vaccines developed by employing various strategies have afforded protection that is at best comparable with bacillus Calmette–Guérin (BCG) in animal models. Due to the inherent ability of BCG to prime cellular responses in the host, it has become an attractive vehicle for development of a vaccine against intracellular infections. In this study, we have cloned the genes of three immunodominant antigens of M. tuberculosis viz. the ESAT6 (Rv3875), the 19 kDa lipoprotein (Rv3763) and the 38 kDa antigen (Pst homolog) (Rv0934) in pSD5 under the transcriptional control of Trrn, a strong mycobacterial promoter, and expressed them in BCG. The19 kDa antigen and the 38 kDa antigen were expressed at levels that were approximately five and eightfolds higher in the cytosols of recombinant BCG strains rBCG19T and rBCG38T, respectively, as compared with their corresponding levels in M. bovis BCG. Both these antigens were also secreted into the extracellular medium at enhanced levels (19 kDa antigen fourfold and 38 kDa antigen twofold) by rBCG strains in comparison with the wild type BCG. ESAT6 antigen, which is absent in M. bovis BCG, was also expressed at a very high level in the cytosol of the rBCG strain (rBCGE6T). Evaluation of immune responses induced by these three rBCG strains in mice shows a markedly different pattern. The rBCG strain overexpressing the 38 kDa antigen exhibited a predominant T helper 1 (Th1) response with high levels of interferon‐γ (IFN‐γ) production, whereas overexpression of the 19 kDa antigen resulted in completely polarized Th2 responses against the BCG sonicate. The rBCG‐expressing ESAT6 antigen induced a mixed Th1/Th2 response. Our observations suggest that the 38 kDa antigen may hold excellent promise in the rBCG approach for the development of a vaccine against tuberculosis.

Skewing of the Th1/Th2 responses in mice due to variation in the level of expression of an antigen in a recombinant BCG system

In spite of rapid developments in the study of mycobacteria during the last two decades, tuberculosis (TB) has maintained its status as the leading killer among all infectious diseases. Extensive evidence exists to support a central role for a T-helper type 1 (Th1) immune response for protection against TB in mice and humans. Bacille Calmette-Guerin (BCG), the only vaccine against TB, although not perfect in its ability to protect against the adult form of TB, is a strong inducer of Th1 responses and is being increasingly used as a delivery vehicle for the presentation of foreign antigens to the immune system. It has been proposed that expression of immunodominant antigens or cytokine genes in BCG can enhance the ability of BCG to induce a Th1 immune response. Since dose of the antigen is considered as one of the parameters that influence the Th cell responses, the level of expression of the candidate antigen should influence the final Th response against the recombinant BCG (rBCG). In the present study, the effect of over-expression of a candidate antigen Antigen 85B (Ag 85B) in a rBCG system, on the Th-priming ability of BCG has been investigated in the murine model. BALB/c mice were immunized with three different rBCG constructs expressing Ag 85B to various levels. Induction of Th1/Th2 responses was analyzed by measuring levels of interferon-gamma (Th1) and interleukin-10 (Th2) in antigen-stimulated splenocyte cultures and by quantifying the antigen-specific IgG2a (Th1) and IgG1 (Th2) antibody responses. By varying the level of expression of Ag 85B, specific immune responses against Ag 85B were observed to range from mixed Th1/Th2 to Th1. However, the BCG-specific immune responses in case of all rBCG-immunized animals remained predominantly Th1.

Increased expression of Mycobacterium tuberculosis 19 kDa lipoprotein obliterates the protective efficacy of BCG by polarizing host immune responses to the Th2 subtype

Mycobacterium tuberculosis can not only neutralize immune effector functions, but also has the ability to modulate host‐signalling cascades involved in the development of these responses. The 19 kDa antigen (Rv3763), a lipoprotein of M. tuberculosis, elicits high levels of interleukin (IL)‐12 from macrophages in addition to its powerful immunomodulatory properties, leading to suppression of antigen‐presentation signalling cascades. The present study was aimed at analysing the effect of overexpression of this antigen on the immunostimulatory properties of M. bovis Bacille Calmette–Guérin (BCG). We have constructed a recombinant BCG strain (rBCG19N) producing higher levels of the 19 kDa antigen in both the cytoplasmic (approximately eightfold) and extracellular (approximately fivefold) fractions as compared to the wildtype BCG. Immunization of mice with rBCG19N elicited high levels of interferon‐gamma (IFN‐γ) and relatively low levels of IL‐10 against the purified 19 kDa antigen. However, in response to total BCG sonicate, mice immunized with rBCG19N produced significantly high levels of IL‐10 with relatively very low levels of IFN‐γ. This polarization of the host immune responses towards T‐helper 2 subtype resulted in complete abrogation of the protective efficacy of BCG, when rBCG19N was used as a live vaccine against M. tuberculosis challenge in guinea pigs.

Immunogenicity of recombinant BCG vaccine strains overexpressing components of the antigen 85 complex of Mycobacterium tuberculosis

The components of antigen 85 complex of Mycobacterium tuberculosis (Ag 85A, Ag 85B and Ag 85C), due to their immunodominant and secretory nature, represent promising protective antigen candidates and have been used in numerous vaccine preparations. We have used recombinant Bacille Calmette Guerin (BCG) strains overexpressing Ag 85A and Ag 85C to immunize BALB/c mice to investigate the immunogenicity of these strains. Mice immunized with recombinant BCG strains exhibited an increased humoral immune response when compared to mice immunized with wild-type BCG. The recombinant BCG strain overexpressing Ag 85A also induced an increased Th1-like response, characterized by elevated levels of IFN-γ in antigen stimulated splenocyte cultures and a strong IgG2a antibody response, when compared to wild-type BCG. Immunization with recombinant BCG strain overexpressing Ag 85C, on the other hand did not elicit increased IFN-γ secretion on restimulation of splenocytes in vitro.

Boosting with a DNA vaccine expressing ESAT-6 (DNAE6) obliterates the protection imparted by recombinant BCG (rBCGE6) against aerosol Mycobacterium tuberculosis infection in guinea pigs

Owing to its highly immunodominant nature and ability to induce long-lived memory immunity, ESAT-6, a prominent antigen of Mycobacterium tuberculosis, has been employed in several approaches to develop tuberculosis vaccines. Here, for the first time, we combined ESAT-6 based recombinant BCG (rBCG) and DNA vaccine (DNAE6) in a prime boost approach. Interestingly, in spite of inducing an enhanced antigen specific IFN-gamma response in mice, a DNAE6 booster completely obliterated the protection imparted by rBCG against tuberculosis in guinea pigs. Analysis of immunopathology and cytokine responses suggests involvement of an exaggerated immunity behind the lack of protection imparted by this regimen.

Expression profiling of lymph nodes in tuberculosis patients reveal inflammatory milieu at site of infection.

Extrapulmonary manifestations constitute 15 to 20% of tuberculosis cases, with lymph node tuberculosis (LNTB) as the most common form of infection. However, diagnosis and treatment advances are hindered by lack of understanding of LNTB biology. To identify host response, Mycobacterium tuberculosis infected lymph nodes from LNTB patients were studied by means of transcriptomics and quantitative proteomics analyses. The selected targets obtained by comparative analyses were validated by quantitative PCR and immunohistochemistry. This approach provided expression data for 8,728 transcripts and 102 proteins, differentially regulated in the infected human lymph node. Enhanced inflammation with upregulation of T-helper1-related genes, combined with marked dysregulation of matrix metalloproteinases, indicates tissue damage due to high immunoactivity at infected niche. This expression signature was accompanied by significant upregulation of an immunoregulatory gene, leukotriene A4 hydrolase, at both transcript and protein levels. Comparative transcriptional analyses revealed LNTB-specific perturbations. In contrast to pulmonary TB-associated increase in lipid metabolism, genes involved in fatty-acid metabolism were found to be downregulated in LNTB suggesting differential lipid metabolic signature. This study investigates the tissue molecular signature of LNTB patients for the first time and presents findings that indicate the possible mechanism of disease pathology through dysregulation of inflammatory and tissue-repair processes.

Adipocyte Model of Mycobacterium tuberculosis Infection Reveals Differential Availability of Iron to Bacilli in the Lipid-Rich Caseous Environment

ABSTRACT Mycobacterium tuberculosis, a successful human pathogen, utilizes multiple carbon sources from the host but adapts to a fatty-acid-rich environment in vivo. We sought to delineate the physiologic response of M. tuberculosis to a lipid-rich environment by using differentiated adipocytes as a model system. Global transcriptome profiling based on RNA sequencing was performed for bacilli from infected adipocytes and preadipocytes. Genes involved in de novo fatty acid synthesis were downregulated, while those predicted to be involved in triglyceride biosynthesis were upregulated, in bacilli isolated from adipocytes, indicating reliance on host-derived fatty acids. Transcription factor network analysis indicated suppression of IdeR-regulated genes, suggesting decreased iron uptake by M. tuberculosis in the adipocyte model. This suppression of iron uptake coincided with higher ferritin and iron levels in adipocytes than in preadipocytes. In accord with the role of iron in mediating oxidative stress, we observed upregulation of genes involved in mitigating oxidative stress in M. tuberculosis isolated from adipocytes. We provide evidence that oleic acid, a major host-derived fatty acid, helps reduce the bacterial cytoplasm, thereby providing a safe haven for an M. tuberculosis mutant that is sensitive to iron-mediated oxidative stress. Via an independent mechanism, host ferritin is also able to rescue the growth of this mutant. Our work highlights the inherent synergy between macronutrients and micronutrients of the host environment that converge to provide resilience to the pathogen. This complex synergy afforded by the adipocyte model of infection will aid in the identification of genes required by M. tuberculosis in a caseous host environment.