The World Health Organization has identified India as a major hot-spot region for Mycobacterium tuberculosis infection. We have characterized the sequences of the loci associated with multidrug resistance in 126 clinical isolates of M. tuberculosis from India to identify the respective mutations. The loci selected were rpoB (rifampin), katG and the ribosomal binding site of inhA (isoniazid), gyrA and gyrB (ofloxacin), and rpsL and rrs (streptomycin). We found known as well as novel mutations at these loci. Few of the mutations at the rpoB locus could be correlated with the drug resistance levels exhibited by the M. tuberculosis isolates and occurred with frequencies different from those reported earlier. Missense mutations at codons 526 to 531 seemed to be crucial in conferring a high degree of resistance to rifampin. We identified a common Arg463Leu substitution in the katG locus and certain novel insertions and deletions. Mutations were also mapped in the ribosomal binding site of the inhA gene. A Ser95Thr substitution in the gyrA locus was the most common mutation observed in ofloxacin-resistant isolates. A few isolates showed other mutations in this locus. Seven streptomycin-resistant isolates had a silent mutation at the lysine residue at position 121. While certain mutations are widely present, pointing to the magnitude of the polymorphisms at these loci, others are not common, suggesting diversity in the multidrug-resistant M. tuberculosis strains prevalent in this region. Our results additionally have implications for the development of methods for multidrug resistance detection and are also relevant in the shaping of future clinical treatment regimens and drug design strategies.

The variation in sequence and length in the C-terminal region among members of the unique PE (Pro-Glu) and PPE (Pro-Pro-Glu) protein families of Mycobacterium tuberculosis is a likely source of antigenic variation, giving rise to the speculation that these protein families could be immunologically important. Based on in silico analysis, we selected a hypothetical open reading frame (ORF) encoding a protein belonging to the PPE family and having epitopes with predictably higher antigenic indexes. Reverse transcriptase PCR using total RNA extracted from in vitro-cultured M. tuberculosis H37Rv generated an mRNA product corresponding to this gene, indicating the expression of this ORF (Rv2430c) at the mRNA level. Recombinant protein expressed in Escherichia coli was used to screen the sera of M. tuberculosis-infected patients, as well as those of clinically healthy controls (n ‫؍‬ 10), by enzyme-linked immunosorbent assay. The panel of patient sera comprised sera from fresh infection cases (category 1; n ‫؍‬ 32), patients with relapsed tuberculosis (category 2; n ‫؍‬ 30), and extrapulmonary cases (category 3; n ‫؍‬ 30). Category 2 and 3 sera had strong antibody responses to the PPE antigen, equal to or higher than those to other well-known antigens, such as Hsp10 or purified protein derivative (PPD). However, a higher percentage of patients belonging to category 1, as opposed to clinically healthy controls, showed stronger antibody response against the PPE protein when probed with anti-immunoglobulin M (IgM) (71 versus 37.5%) or anti-IgG (62.5 versus 28.12%). Our results reveal that this PPE ORF induces a strong B-cell response compared to that generated by M. tuberculosis Hsp10 or PPD, pointing to the immunodominant nature of the protein.About 10% of the genome of Mycobacterium tuberculosis codes for the PE and PPE families of proteins (7), which are glycine rich and are exclusive to M. tuberculosis. The 69 members of the PPE protein family have a conserved N-terminal domain that comprises ϳ180 amino acids followed by C-terminal segments that vary markedly in sequence and length. These proteins fall into three groups, one of which constitutes the MPTR class characterized by the presence of multiple tandem copies of the motif Asn-X-Gly-X-Gly-Asn-X-Gly. The second subgroup contains a characteristic well-conserved motif, Gly-X-X-Ser-Val-Pro-X-X-Trp, around position 350. The proteins in the third group are unrelated except for the presence of the common PPE domain. The subcellular locations of a few PPE proteins are known (6, 25), and in only one case (7), that of a lipase (Rv3097), has a function been suggested. There are few studies supporting the notion that PE and PPE proteins could be of functional importance (7,23). It is widely speculated that they could be responsible for generating antigenic variation (1,4,6,8,12,27). However, the effects the PPE family proteins, unique in their protein sequences and possible structure, may have on the immune system have not been well documented. Furthermore, a qualitative and...

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Caseinolytic protein, ClpC is a general stress protein which belongs to the heat shock protein HSP100 family of molecular chaperones. Some of the Clp group proteins have been identified as having a role in the pathogenesis of many bacteria. The Mycobacterium tuberculosis genome demonstrates the presence of a ClpC homolog, ClpC1. M. tuberculosis ClpC1 is an 848‐amino acid protein, has two repeat sequences at its N‐terminus and contains all the determinants to be classified as a member of the HSP100 family. In this study, we overexpressed, purified and functionally characterized M. tuberculosis ClpC1. Recombinant M. tuberculosis ClpC1 showed an inherent ATPase activity, and prevented protein aggregation. Furthermore, to investigate the contribution made by the N‐terminal repeats of ClpC1 to its functional activity, two deletion variants, ClpC1Δ1 and ClpC1Δ2, lacking N‐terminal repeat I and N‐terminal repeat I along with the linker between N‐terminal repeats I and II, respectively were generated. Neither deletion affected the ATPase activity. However, ClpC1Δ1 was structurally altered, less stable and was unable to prevent protein aggregation. Compared with wild‐type protein, ClpC1Δ2 was more active in preventing protein aggregation and displayed higher ATPase activity at high pH values and temperatures. The study demonstrates that M. tuberculosis ClpC1 manifests chaperone activity in the absence of any adaptor protein and only one of the two N‐terminal repeats is sufficient for the chaperone activity. Also, an exposed repeat II makes the protein more stable and functionally more active.

The production of correctly folded protein in Escherichia coli is often challenging because of aggregation of the overexpressed protein into inclusion bodies. Although a number of general and protein-specific techniques are available, their effectiveness varies widely. We report a novel method for enhancing the solubility of overexpressed proteins. Presence of a dipeptide, glycylglycine, in the range of 100 mM to 1 M in the medium was found to significantly enhance the solubility (up to 170-fold) of the expressed proteins. The method has been validated using mycobacterial proteins, resulting in improved solubilization, which were otherwise difficult to express as soluble proteins in E. coli. This method can also be used to enhance the solubility of other heterologous recombinant proteins expressed in a bacterial system.