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...

The function of the PE/PPE families of proteins, which represent approximately 10% of the coding capacity of the Mycobacterium tuberculosis genome, has remained relatively unknown. We earlier described a PPE family member, Rv2430c, as an immunodominant antigen. We now report another PPE family gene, Rv2608, a member of the major polymorphic tandem repeat subfamily, for its ability to elicit a high humoral and a low T cell response. Rv2608 was also found to be polymorphic in different clinical isolates of M. tuberculosis, as determined by polymerase chain reaction-restriction fragment-length polymorphism analysis. A total of 51 clinically confirmed patients with tuberculosis (TB), belonging to 3 different categories--fresh infection (n=22), relapsed infection (n=21), and extrapulmonary infection (n=8)--and 10 healthy control subjects were included in the study. Recombinant Rv2608 protein showed positive reactivity to patients' serum samples. Enzyme-linked immunosorbent assays and T cell-proliferation assays with synthetic peptides corresponding to predicted regions of high antigenicity showed a predominantly humoral response in patients with relapsed TB. We additionally identified the Gly-X-Gly-Asn-X-Gly repeat motifs as being primarily responsible for eliciting a humoral immune response.

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.

Background: The diphtheria toxin repressor, DtxR, of Corynebacterium diphtheriae has been shown to be an iron-activated transcription regulator that controls not only the expression of diphtheria toxin but also of iron uptake genes. This study aims to identify putative binding sites and operons controlled by DtxR to understand the role of DtxR in patho-physiology of Corynebacterium diphtheriae.

The identification of Mycobacterium tuberculosis by Robert Koch in 1882 as the causative agent of tuberculosis, the release of the drug rifampicin in 1970 and the sequencing of the M. tuberculosis genome in 1998 are three major events that have revolutionized tuberculosis research. In spite of these breakthroughs, the continued status of tuberculosis as the largest killer amongst infectious diseases is an issue of major concern. Although directly observed short course chemotherapy exists to treat the disease, the emergence of drug-resistant strains has severely threatened the efficacy of the treatment. The recent sequencing of the M. tuberculosis genome holds promise for the development of new vaccines and the design of new drugs. This is all the more possible when the information from the genome sequence is combined with proteomics and structural and functional genomics. Such an integrated approach has led to the birth of a new field of research christened ‘postgenomics’ that holds substantial promise for the identification of novel drug targets and the potential to aid the development of new chemotherapeutic compounds to treat tuberculosis. The challenge before the scientific community therefore lies in elucidation of the wealth of information provided by the genome sequence and its translation into the design of novel therapies for the disease. All the major developments in the field of tuberculosis research after the sequencing of the M. tuberculosis genome will be discussed in this review.