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Background Tuberculosis (Tb) is a disease with worldwide presence and a major cause of death in several developing countries. Current diagnostic methodologies often lack specificity and sensitivity, whereas a long time is needed to obtain a conclusive result. Material and methods In an effort to develop better diagnostic methods, this study aimed at the discovery of a biomarker signature for Tb diagnosis using a Nuclear Magnetic Resonance based metabolomics approach. In this study, we acquired 1H NMR spectra of blood serum samples of groups of healthy subjects, individuals with latent Tb and of patients with pulmonary and extra-pulmonary Tb. The resulting data were treated with uni- and multivariate statistical analysis. Results/Conclusion Six metabolites (inosine, hypoxanthine, mannose, asparagine, aspartate and glutamate) were validated by an independent cohort, all of them related with metabolic processes described as associated with TB infection. The findings of the study are according with the WHO Target Product Profile recommendations for a triage test to rule-out active TB.

The 60 kDa heat shock proteins, also known as Cpn60s (GroELs) are components of the essential protein folding machinery of the cell, but are also dominant antigens in many infectious diseases. Although generally essential for cellular survival, in some organisms such as Mycobacterium tuberculosis, one or more paralogous Cpn60s are known to be dispensable. In M. tuberculosis, Cpn60.2 (GroEL2) is essential for cell survival, but the biological role of the non-essential Cpn60.1 (GroEL1) is still elusive. To understand the relevance of Cpn60.1 (GroEL1) in M. tuberculosis physiology, detailed transcriptomic analyses for the wild type H37Rv and cpn60.1 knockout (groEL1-KO) were performed under in vitro stress conditions: stationary phase, cold shock, low aeration, mild cold shock and low pH. Additionally, the survival of the groEL1-KO was assessed in macrophages at multiplicity of infection (MOI) of 1:1 and 1:5. We observed that survival under low aeration was significantly compromised in the groEL1-KO. Further, the gene expression analyses under low aeration showed change in expression of several key virulence factors like two component system PhoP/R and MprA/B, sigma factors SigM and C and adversely affected known hypoxia response regulators Rv0081, Rv0023 and DosR. Our work is therefore suggestive of an important role of Cpn60.1 (GroEL1) for survival under low aeration by affecting the expression of genes known for hypoxia response.

Cyclic AMP (cAMP) receptor protein, which acts as the sensor of cAMP levels in cells, is a well-studied transcription factor that is best known for allosteric changes effected by the binding of cAMP. Although genetic and biochemical data on the protein are available from several sources, structural information about the cAMP-free protein has been lacking. Therefore, the precise atomic events that take place upon binding of cAMP, leading to conformational changes in the protein and its activation to bind DNA, have been elusive. In this work we solved the cAMP-free crystal structure of the Mycobacterium tuberculosis homolog of cAMP receptor protein at 2.9 Å resolution, and carried out normal-mode analysis to map conformational transitions among its various conformational states. In our structure, the cAMP-binding domain holds onto the DNA-binding domain via strong hydrophobic interactions, thereby freezing the latter in a conformation that is not competent to bind DNA. The two domains release each other in the presence of cAMP, making the DNA-binding domain more flexible and allowing it to bind its cognate DNA via an induced-fit mechanism. The structure of the cAMP-free protein and results of the normal-mode analysis therefore highlight an elegant mechanism of the allosteric changes effected by the binding of cAMP.

1L-myo-inositol (inositol) is vital for the biogenesis of mycothiol, phosphatidylinositol and glycosylphosphatidylinositol anchors linked to complex carbohydrates in Mycobacterium tuberculosis. All these cellular components are thought to play important roles in host-pathogen interactions and in the survival of the pathogen within the host. However, the inositol biosynthetic pathway in M. tuberculosis is not known. To delineate the pathways for inositol formation, we employed a unique combination of tertiary structure prediction and yeast-based functional assays. Here, we describe the identification of the gene for mycobacterial INO1 that encodes inositol-1-phosphate synthase distinct in many respects from the eukaryotic analogues.

Activation of the complement system on host cells is regulated primarily by a family of proteins termed as regulators of complement activation (RCA). A characteristic feature of the RCA family of proteins is the presence of repeating complement control protein (CCP) domains. Recent advances in genome sequencing resulted in availability of a large pool of sequences encoding CCP domain-containing proteins. However, their functional annotation could not be performed as CCP domains are not exclusive to RCA family (e.g., also present in proteins involved in complement activation, cell adhesion, coagulation, neurotransmission, cytokine signalling and blood clotting) and not all RCA family members are capable of regulating complement (e.g., Complement Receptor 2). In the present study, we therefore, sought to identify whether RCA proteins with complement regulatory function contain any sequence motif indicative of their function. In silico analysis of three N-terminal CCP domains of mammalian and viral RCA proteins resulted in identification of five motifs. These motifs are present in a specific order and are conserved spatially. Phylum-wide analysis of genomes for the presence of proteins with these motifs showed that they are present in CCP domain-containing proteins of a wide variety of organisms ranging from mammalia to nematoda, which included annotated and unannotated proteins for complement regulatory function. Thus, this approach seems to have broad-range sensitivity. To experimentally validate whether these motifs are indeed critical for complement regulatory activities, we performed functional characterization of human SVEP1 (Sushi, von Willebrand factor type A, EGF and pentraxin domain-containing protein 1)/Polydom expressed in placenta, which contains these motifs, but has not been characterised to have complement regulatory function. The protein displayed the complement regulatory activities. We therefore propose that the sequence motifs we have identified are capable of distinguishing between regulatory and non-regulatory CCP domains and thus are apt for identifying new complement regulatory proteins in a continually expanding dataset of CCP containing proteins.

We focused on the temperature adaptation of triosephosphate isomerase (TIM; E.C. 5.3.1.1.) by comparing the structure of TIMs isolated from bacterial organisms living in either cold or hot environments. The TIM gene from psychrophilic bacteria Moraxella sp. TA137 was cloned and its nucleotide sequence determined. Its deduced amino acid sequence revealed 34% identity with the thermophilic bacteria Bacillus stearothermophilus TIM. Expression vectors were constructed and recombinant Moraxella TA137 and Bacillus stearothermophilus TIMs were overproduced and purified to homogeneity. Recombinant TIM inactivation constants (Ki), measured at various temperatures, compared to those of the mesophilic Escherichia coli recombinant TIM clearly show that Moraxella TA137 and B. stearothermophilus TIMs have respectively psychrophilic and thermophilic characteristics. To try to elucidate the structure-thermolability and structure-thermostability relationship, factors affecting the overall stability of these two TIMs were examined, based on the alignment with the mesophilic chicken TIM, the three-dimensional structure of which is already known. From this comparison, it appears that the adaptability of TIM to high temperature is favored by better stabilizing residues for the helix dipole as well as better helix-forming residues whereas the adaptability of TIM to low temperature seems to reside in the nature of helix-capping residues.

Introduction: Though PE and PPE families have been thought to play important roles in mycobacterial pathogenesis from an antigenic as well as immunological view point, there has not been much information available regarding their physiological relevance, more so in the light of the fact that mycobacteria have been highly successful in devising strategies of immune evasion. One of the PPE proteins, Rv1196 (PPE18 or mtb39a) was found to be expressed during infection and present exclusively in M. tuberculosis and M. bovis BCG but not in other mycobacterial species. Further, the microarray studies indicate that Rv1196 is one of the proteins involved in maintaining long term survival of M. tuberculosis within the host. Since Th2 plays an important role in activating M. tuberculosis pathogenesis, we have evaluated the role of Rv1196 protein to interact with macrophage and affect the Th2 balance. Method: Cytokine induction was measured by enzyme immunoassay (EIA). Interacting receptor on macrophages were identified by flow cytometry and coimmunoprecipitation assay using specific antibody. The p38 MAPK and ERK 1/2 and JNK signaling was examined by Western blotting and flow cytometry Results: We demonstrate that the PPE protein, Rv1196 stimulates macrophages to secrete interleukin (IL)-10 cytokine, which favors a Th2 T-cell response congenial for mycobacterial survival. However, it does not significantly affect the production of inflammatory cytokines like IL-12, TNFas well as nitric oxide production. IL-10 induction was found to be dependent on specific interaction of Rv1196 protein to the toll-like receptor (TLR) 2 on membrane. We found that an early and sustained activation of p38 MAPK (but not ERK 1/2 and JNK) downstream of TLR2 was critical for triggering of IL-10 by Rv1196. Conclusion: Our data suggest a unique role of the M. tuberculosis PPE protein in eliciting an anti-Th1 response critical for survival of the bacteria.

X-ray intensity data from the native orthorhombic crystals of peanut lectin have been collected using oscillation photography. Rotation function studies using data up to a resolution of 4.5 A indicate that the four subunits in the molecule, which constitute the asymmetric unit in the crystals, are related to one another by three mutually perpendicular noncrystallographic 2-fold axes. Chemical cross-linking experiments in solution followed by sodium dodecyl sulfate gel electrophoresis, carried out in parallel, suggest that there is more than one type of intersubunit approach in the molecule. Rotation function and cross-linking studies thus show that the tetrameric molecule of peanut lectin is a dimer of a dimer. The two monomers in a dimer are related by a 2-fold axis. The two dimers are in turn related by another 2-fold axis perpendicular to the one that relates the two monomers in the dimer, endowing the molecule with 222 (D2) symmetry.