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It is instrumental for the Mycobacterium tuberculosis (Mtb) to persist within its host in dormancy. Mtb represses most of its metabolic machinery during latency, but upregulates the expression of latency-associated protein alpha-crystallin protein (Acr1). Therefore, it is imperative to understand how throughout dormancy, Mtb employs Acr1 to regulate the host immunity. This study reveals that Acr1 exhibits divergent effect on the pre- and post-maturation stages of dendritic cells (DCs). In the current study, we demonstrate that early encounter of bone marrow cells with Acr1 while differentiating into DCs (AcrDC pre ), leads to impairment in their maturation. In contrast, when exposed to Acr1 after maturation (AcrDC post ), DCs show augmentation in their activity, secretion of TNF-α, IL-12, IL-6, and activation of T cells. Additionally, AcrDC post promoted the polarization of naïve CD4 T cells to Th1 cells and Th17 cells and restricted the intracellular growth of Mtb . Furthermore, these DCs upregulated the expression of CCR7 and exhibited enhanced migratory capabilities. The discrete impact of Acr1 on DCs is mediated through a mechanism involving STAT-1, SOCS-3, ERK, TLR-4, and NF-κB signaling pathways. This study reveals the unprecedented role of Acr1 in distinctly modulating the function of DCs at different stages of maturation.

Tuberculosis continues to be one of the most devastating global health problem. Its diagnosis will benefit in timely initiation of the treatment, cure and therefore reduction in the transmission of the disease. Tests are available, but none can be comprehensively relied on for its diagnosis; especially in TB-endemic zones. PhoP is a key player in Mycobacterium tuberculosis virulence but nothing has been known about its role in the diagnosis of TB. We monitored the presence of anti-PhoP antibodies in the healthy, patients and their contacts. In addition, we also measured antibodies against early secretory antigens ESAT-6 and CFP-10, and latency associated antigen Acr-1 to include proteins that are associated with the different stages of disease progression. Healthy subjects showed high antibody titer against PhoP than patients and their contacts. In addition, a distinct pattern in the ratio of Acr-1/PhoP was observed among all cohorts. This study for the first time demonstrates a novel role of anti-PhoP antibodies, as a possible marker for the diagnosis of TB and therefore will contribute in the appropriate action and management of the disease.

Bacillus Calmette-Guérin (BCG) remains the only available and most widely administered vaccine against Mycobacterium tuberculosis (Mtb), yet it fails to protect vaccinated individuals either from primary infection or reactivation of latent tuberculosis (TB). Despite BCG's variable efficacy against TB, the fact remains that BCG imparts protection in children against the disease, indicating that BCG possesses a wide protective antigenic repertoire. However, its failure to impart protection in adulthood can be linked to its failure to generate long-lived memory response and elicitation of an inadequate immune response against latency-associated antigens. Therefore, to improve the protective efficacy of BCG, a novel vaccination strategy is required. Consequently, in the present study, we have exploited the vaccination potential of liposomized α-crystalline 1 (Acr1L), a latency-associated antigen to induce enduring protective immunity against Mtb in BCG-primed animals. It is noteworthy that an increase in the multi-functional [interferon (IFN)-γ(hi) /tumour necrosis factor (TNF)-α(hi) ] CD4 and CD8 T cells were observed in BCG-primed and Acr1L-boosted (BCG-Acr1L) animals, compared to BCG alone. Further, substantial expansion of both central memory (CD44(hi) /CD62L(hi) ) and effector memory (CD44(hi) /CD62L(lo) ) populations of CD4 and CD8 T cells was noted. Importantly, BCG-Acr1L exhibited significantly better protection than BCG, as evidenced by a reduction in the bacterial burden and histopathological data of the lungs. In essence, BCG-Acr1L could be a potent future vaccination strategy to reinvigorate BCG potency., (© 2015 Institute of Microbial Technology.)

An efficient and reproducible method for transformation depends on the competency of the organism. We have developed a simple method for the preparation of competent Escherichia coli, Kluyveromyces lactis, and Bifidobacterium sp. by using a buffer containing cetyl trimethyl ammonium bromide (CTAB) and permits efficient uptake of plasmid DNA and ligation-reaction products. Cells are harvested, washed, mixed with 1-10 μg/ml CTAB, incubated, and followed by a buffer wash. For long-term storage of competent cells, bacteria may be frozen in 10% glycerol without the addition of other components. The transformation process is very simple; plasmid DNA and the cells are mixed and incubated for 5-60 min at 4 °C; no heat pulse is required, and the duration of incubation at 4 °C is not crucial.

Mycobacterium tuberculosis (M. tuberculosis) in latently infected individuals survives and thwarts the attempts of eradication by the immune system. During latency, Acr1 is predominantly expressed by the bacterium. However, whether M. tuberculosis exploits its Acr1 in impairing the host immunity remains widely unexplored. Hence, currently we have investigated the role of Acr1 in influencing the differentiation and function of dendritic cells (DCs), which play a cardinal role in innate and adaptive immunity. Therefore, for the first time, we have revealed a novel mechanism of mycobacterial Acr1 in inhibiting the maturation and differentiation of DCs by inducing tolerogenic phenotype by modulating the expression of PD-L1; Tim-3; indoleamine 2, 3-dioxygenase (IDO); and interleukin 10. Furthermore, Acr1 interferes in the differentiation of DCs by targeting STAT-6 and STAT-3 pathways. Continuous activation of STAT-3 inhibited the translocation of NF-κB in Acr1-treated DCs. Furthermore, Acr1 also augmented the induction of regulatory T cells. These DCs displayed decline in their antigen uptake capacity and reduced ability to help T cells. Interestingly, M. tuberculosis exhibited better survival in Acr1-treated DCs. Thus, this study provides a crucial insight into a strategy adopted by M. tuberculosis to survive in the host by impairing the function of DCs.

One of the main reasons considered for BCG failure in tuberculosis-endemic areas is impediment by environmental mycobacteria in its processing and generation of memory T-cell response. To overcome this problem, we developed a unique lipopeptide (L91) by linking the promiscuous peptide (sequence 91-110) of 16 kDa antigen of Mycobacterium tuberculosis to Pam2Cys. L91 does not require extensive antigen processing and generates enduring Th1 memory response. This is evidenced by the fact that L91 significantly improved the activation, proliferation, and generation of protective T cells. Furthermore, L91 surmounts the barrier of major histocompatibility complex polymorphism and induces better protection than BCG. This peptide has self-adjuvanting properties and activates dendritic cells. Importantly, L91 activates T cells isolated from purified protein derivative-positive healthy volunteers that responded weakly to free peptide (F91). In essence, L91 can be a potent future vaccine candidate against tuberculosis.

Heat shock proteins (HSPs) are conserved and ubiquitous house keeping entities that act as molecular chaperones, which protect the cell from damage during stress. One such HSP, the 16 kDa antigen, from Mycobacterium tuberculosis (Mtb) has received considerable attention due to its importance in tuberculosis latency and immunodominant property. In this article, we discuss about the potential role of 16 kDa antigen of Mtb in latency, its expression, regulation, and implication in host immune response. We also highlight the scope of employing 16 kDa in early diagnosis, development of vaccine and as a potential drug target.

The formation constants of the complexes of adenyl-5'-yl imidodiphosphate with H+ Mg2+, Ca2+ and a number of bivalent transition-metal ions were measured potentionmetrically. The complexes are generally a little more stable than the analogous complexes of ATP. By measuring the formation constants at two temperatures, this increase in stability was shown to result from an increased enthalpy change on complex-formation.