B cell responses to a peptide epitope. X. Epitope selection in a primary response is thermodynamically regulated

We examine the etiological basis of hierarchical immunodominance of B cell epitopes on a multideterminant Ag. A model T-dependent immunogen, containing a single immunodominant B cell epitope, was used. The primary IgM response to this peptide included Abs directed against diverse determinants presented by the peptide. Interestingly, affinity of individual monomeric IgM Abs segregated around epitope recognized and was independent of their clonal origins. Furthermore, affinity of Abs directed against the immunodominant epitope were markedly higher than that of the alternate specificities. These studies suggested that the affinity of an epitope-specific primary response, and variations therein, may be determined by the chemical composition of epitope. This inference was supported by thermodynamic analyses of monomer IgM binding to Ag, which revealed that this interaction occurs at the expense of unfavorable entropy changes. Permissible binding required compensation by net enthalpic changes. Finally, the correlation between chemical composition of an epitope, the resultant affinity of the early primary humoral response, and its eventual influence on relative immunogenicity could be experimentally verified. This was achieved by examining the effect of various amino-terminal substitutions on immunogenicity of a, hitherto cryptic, amino-terminal determinant. Such experiments permitted delineation of a hierarchy of individual amino acid residues based on their influence; which correlated well with calculated Gibbs-free energy changes that individual residue side chains were expected to contribute in a binding interaction. Thus, maturation of a T-dependent humoral response is initiated by a step that is under thermodynamic control.     

Phosphorylation regulates mycobacterial proteasome

Mycobacterium tuberculosis possesses a proteasome system that is required for the microbe to resist elimination by the host immune system. Despite the importance of the proteasome in the pathogenesis of tuberculosis, the molecular mechanisms by which proteasome activity is controlled remain largely unknown. Here, we demonstrate that the α-subunit (PrcA) of the M. tuberculosis proteasome is phosphorylated by the PknB kinase at three threonine residues (T84, T202, and T178) in a sequential manner. Furthermore, the proteasome with phosphorylated PrcA enhances the degradation of Ino1, a known proteasomal substrate, suggesting that PknB regulates the proteolytic activity of the proteasome. Previous studies showed that depletion of the proteasome and the proteasome-associated proteins decreases resistance to reactive nitrogen intermediates (RNIs) but increases resistance to hydrogen peroxide (H₂O₂). Here we show that PknA phosphorylation of unprocessed proteasome β-subunit (pre-PrcB) and α-subunit reduces the assembly of the proteasome complex and thereby enhances the mycobacterial resistance to H₂O₂ and that H₂O₂ stress diminishes the formation of the proteasome complex in a PknA-dependent manner. These findings indicate that phosphorylation of the M. tuberculosis proteasome not only modulates proteolytic activity of the proteasome, but also affects the proteasome complex formation contributing to the survival of M. tuberculosis under oxidative stress conditions.     

Characterization of a chromosomal toxin-antitoxin, Rv1102c-Rv1103c system in Mycobacterium tuberculosis

Toxin-antitoxin systems, ubiquitous in prokaryotic genomes, have been proposed to play an important role in several stress responses. While Mycobacterium tuberculosis contains more than 80 putative TA loci, the roles they play in this pathogen are yet to be studied. Here, we characterize a chromosomal Rv1102c-Rv1103c TA system in M. tuberculosis. We found that the Rv1102c toxin interacts with the Rv1103c antitoxin in a pull-down assay and the yeast two-hybrid system. Rv1102c cleaved the era mRNA in Escherichia coli, and cleavage was inhibited by co-expression of Rv1103c. Heterologous expression of Rv1102c led to growth arrest in E. coli, which was fully recovered only when Rv1103c was co-expressed in cis with Rv1102c, suggesting that the production and assembly of Rv1102c and Rv1103c are tightly linked. Our additional results indicate that translational coupling of the Rv1102c and Rv1103c genes is important for Rv1102c-Rv1103c binding. Finally, we discovered that the expression of Rv1102c induced growth arrest and increased the level of persister cells in Mycobacterium smegmatis. These results suggest that the Rv1102c-Rv1103c TA system could play a role in M. tuberculosis pathogenesis via generating bacilli that survive in the face of multidrug therapy.     

Peptidoglycan in Mycobacteria: chemistry,biology and intervention

Peptidoglycan, a major glycoconjugate in the mycobacterial cell envelope provides strength to resist osmotic stress and plays a pivotal role in maintaining the cellular morphology. Several unique growth stage specific structural alterations occur in its constituent monosaccharides and peptides that allow Mycobacterium to survive nutrient starvation and environmental stress. Here, we discuss the enzymes involved in its intricate biosynthesis that are novel targets for therapeutic intervention and provide an opportunity for potential antibiotic adjuvants. We also revisit the enzymatic steps which are critical for maintaining the equilibrium between peptidoglycan synthesis and hydrolysis during cellular growth and division specifically focused on the importance of cell wall remodelling during “exit from dormancy” in Mycobacterium, a phenomenon with tremendous physiological and therapeutic importance for intervention in mycobacterial infections.     

Biosynthesis of eukaryotic cell surface glycosphingolipids using solubilized glycosyltransferases

Two fucsyltransferases (FucT-2 and FucT-3) have been solubilized from Golgi-rich membrane fraction of bovine spleen, using a cationic detergent. FucT-3 was distinguished from FucT-2 by comparing their kinetic parameters and heat stability. FucT-2 and FucT-3 lost activity (85 %) and (5 %), respectively, when heated at 55°C for 10 sec. Two galactosyltransferases (GalT-3 and GalT-4) and two sialyltransferases (SAT-2 and SAT-3) have also been solubilized from embryonic chicken brain membranes using nonionic detergents. Affinity chromatography and microisoelectric focusing were used to separate these enzymes into functionally pure fractions. Anomeric and positional linkages in some of the products (LM1 and LD1c) have also been established. The terminal NeuAc(α2-8) linkage in GD3 and LD1c was established by identification of the partially methylated penultimate [Ac-¹⁴C]sialic acid.     

Identification of the regeneration potential of embryo derived calluses from various Indica rice varieties

The regeneration response of mature and immature rice embryos from 15 rice varieties (Oryza sativa L. var. indica) was separately measured in terms of regeneration per cent and total number of regenerated plantlets obtained for a fixed sample size per variety, when cultured on standard MS media. The response of the rice varieties was classed as high, medium or poor, based on the above criteria. The regeneration per cent of genotypes showing high, medium and poor response ranged from 67 to 97, 38 to 63 and 0 to 36 respectively. The variation in the regeneration response amongst the defined groups was found to be statistically significant in terms of regeneration per cent and total plantlets for a constant sample size when subjected to one way analysis of variance and Duncan's multiple range test. Only small influences on regeneration frequency were found when the most and least responsive genotypes, Binnatoa and BR-23 were cultured on N6 and MS media with additional or different supplements.