Development of vaccines against tuberculosis

Development of an effective vaccine against tuberculosis (TB) hinges on an improved understanding of the human immune responses to Mycobacterium tuberculosis. A successful vaccination strategy should be able to stimulate the appropriate arm of the immune system with concomitant generation of the memory cells. In the absence of a perfect strategy, while long term efforts of TB researchers continue to resolve the nature of protective immunity against TB and other related issues, the current approach, dictated by the urgency of a TB vaccine, employs available knowledge and technology to develop new TB vaccines and channel the promising ones to clinical trials. While Indian scientists have contributed in several areas towards the development of a TB vaccine, this review is an attempt to summarize their contributions mainly pertaining to the discovery of new antigens, immune responses elicited by antigens against TB and development of new vaccines and their evaluation in animal models. (C) 2011 Elsevier Ltd. All rights reserved

Schematic representation of the proposed functions of BfrA and BfrB of <i>M</i>. <i>tuberculosis</i>.

<p>The figure depicts that under low iron conditions, BfrA is required for efficient utilization of stored iron while BfrB is better equipped to protect the pathogen from oxidative damage in iron rich conditions.</p

Deletion of ferritins results in diminished survival of <i>M</i>. <i>tuberculosis</i> under hypoxic conditions.

<p><i>M</i>. <i>tuberculosis</i> strains were grown in MB7H9 media in screw capped tight 15 ml falcons as 10 ml aliquots. (a) The aliquots of each strain were kept at 37°C standing and the growth of various strains was monitored by measuring the absorbance at 600 nm at various time points. (b) Few aliquots of each strain were also kept at shaking at 200 rpm. An aliquot of each strain was removed and plated on MB7H11 agar to visualize any change in the growth of the strains. These experiments were performed twice and the mean values ± standard errors of the means are plotted. WT: M.tbH37Rv, BKO: Δ<i>bfrB</i> mutant, AKO: Δ <i>bfrA</i> mutant, DKO: Δ<i>bfrA</i> and Δ<i>bfrB</i> double mutant, Bcomp: Δ<i>bfrB</i> mutant complemented with <i>bfrB</i> gene and Acomp: Δ<i>bfrA</i> mutant complemented with <i>bfrA</i> gene.</p

Differential Roles of Iron Storage Proteins in Maintaining the Iron Homeostasis in <i>Mycobacterium tuberculosis</i>

<div><p>Ferritins and bacterioferritins are iron storage proteins that represent key players in iron homeostasis. Several organisms possess both forms of ferritins, however, their relative physiological roles are less understood. <i>Mycobacterium tuberculosis</i> possesses both ferritin (BfrB) and bacterioferritin (BfrA), playing an essential role in its pathogenesis as reported by us earlier. This study provides insights into the role of these two proteins in iron homeostasis by employing <i>M</i>. <i>tuberculosis bfr</i> mutants. Our data suggests that BfrA is required for efficient utilization of stored iron under low iron conditions while BfrB plays a crucial role as the major defense protein under excessive iron conditions. We show that these two proteins provide protection against oxidative stress and hypoxia. Iron incorporation study showed that BfrB has higher capacity for storing iron than BfrA, which augurs well for efficient iron quenching under iron excess conditions. Moreover, iron release assay demonstrated that BfrA has 3 times superior ability to release stored iron emphasizing its requirement for efficient iron release under low iron conditions, facilitated by the presence of heme. Thus, for the first time, our observations suggest that the importance of BfrA or BfrB separately might vary depending upon the iron situation faced by the cell.</p></div

Iron release capacities of BfrA and BfrB.

<p>Purified proteins (0.1 μM) were incubated separately with 125 μM of iron for 2 hours at room temperature to facilitate the formation of holo-proteins. (a) Iron release was initiated by incubating the holo-proteins with sodium ascorbate and ferrozine and rate of iron release was monitored by measuring the absorbance of the Fe(II)-Ferrozine complex at 570 nm. (b) Figure shows the characteristic brown colour in BfrAWT(1mg/ml) due to the presence of heme whereas BfrAM52L (1 mg/ml) appears similar to BfrBWT (1 mg/ml) due to absence of heme. These experiments were performed twice, however, the figure shown is representative of one experiment.</p

Iron storage capacities of BfrA and BfrB.

<p>Purified proteins were incubated separately with increasing amounts of iron for 2 hours at room temperature to facilitate the storage of iron as ferric mineral and determine the iron storing capacity of the protein. The residual unbound excess iron was detected by the addition of ferrozine reagent which complexes with free ferrous present in the solution forming a purple coloured product that is analyzed at 570 nm. (a) The figure depicts the percentage of iron incorporated by the protein at increasing iron:protein molar ratios. (b) Rate of iron oxidation was monitored by measuring the absorbance at 310 nm by incubating protein and iron in a ratio of 1:500. Rate was calculated as change in absorbance per unit time for both the proteins and was found to be similar. These experiments were performed twice, however, the figure shown is representative of one experiment.</p

Effect of Bfr deletion on the growth of <i>M</i>. <i>tuberculosis</i> under iron starved and iron rich conditions.

<p>Various <i>M</i>. <i>tuberculosis</i> strains were grown in MB7H9 and were passaged twice in MM before inoculating for the growth curves (one passage comprised of the growth of the bacterium from the time of its inoculation at OD_600nm ~0.02 to its growth to reach stationary phase at OD_600nm ~3.0). Subsequently, growth of various <i>M</i>. <i>tuberculosis</i> strains was monitored at indicated time points under various conditions (a) iron starvation and (c) iron excess (250 μM FeCl₃). These experiments were performed thrice and the mean values ± standard errors of the means are plotted. Immunoblotting was performed by using antibodies against BfrA (upper panel) and BfrB (lower panel) in the lysates of <i>M</i>. <i>tuberculosis</i> cells grown under (b) iron starvation and (d) iron excess (250 μM FeCl₃). For internal control, the same lysates were immunoblotted by using antibodies against SigA. The immunoblot shown is representative of one experiment, however, the same pattern was observed across three experiments. WT: M.tbH37Rv, BKO: Δ<i>bfrB</i> mutant, AKO: Δ <i>bfrA</i> mutant, DKO: Δ<i>bfrA</i> and Δ<i>bfrB</i> double mutant, Bcomp: Δ<i>bfrB</i> mutant complemented with <i>bfrB</i> gene and Acomp: Δ<i>bfrA</i> mutant complemented with <i>bfrA</i> gene.</p

Ferritin structure from Mycobacterium tuberculosis: comparative study with homologues identifies extended C-terminus involved in ferroxidase activity.

Ferritins are recognized as key players in the iron storage and detoxification processes. Iron acquisition in the case of pathogenic bacteria has long been established as an important virulence mechanism. Here, we report a 3.0 Å crystal structure of a ferritin, annotated as Bacterioferritin B (BfrB), from Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis that continues to be one of the world's deadliest diseases. Similar to the other members of ferritin family, the Mtb BfrB subunit exhibits the characteristic fold of a four-helical bundle that possesses the ferroxidase catalytic centre. We compare the structure of Mtb BfrB with representatives of the ferritin family belonging to the archaea, eubacteria and eukarya. Unlike most other ferritins, Mtb BfrB has an extended C-terminus. To dissect the role of this extended C-terminus, truncated Mtb BfrB was purified and biochemical studies implicate this region in ferroxidase activity and iron release in addition to providing stability to the protein. Functionally important regions in a protein of known 3D-structure can be determined by estimating the degree of conservation of the amino-acid sites with its close homologues. Based on the comparative studies, we identify the slowly evolving conserved sites as well as the rapidly evolving variable sites and analyze their role in relation to structure and function of Mtb BfrB. Further, electrostatic computations demonstrate that although the electrostatic environment of catalytic residues is preserved within the family, extensive variability is exhibited by residues defining the channels and pores, in all likelihood keeping up with the diverse functions executed by these ferritins in varied environments