Variations in Outer-membrane Characteristics of Two Stem-nodulating Bacteria of <Emphasis Type="Italic">Sesbania rostrata</Emphasis> and its Role in Tolerance Towards Diverse Stress

Outer-membrane characteristics may determine the survivability of rhizobia under diverse abiotic and biotic stresses. Therefore, the role of lipopolysaccharides (LPS) and membrane proteins of two stem-nodulating bacteria of Sesbania rostrata (Azorhizobium caulinodans ORS571 and Rhizobium sp. WE7) in determining tolerance towards abiotic and biotic stresses (hydrophobics and phages) was investigated. Outer-membrane characteristics (LPS and membrane–protein profiles) of ORS571, WE7 and thirteen standard strains were distinct. ORS571 and WE7 also showed susceptibility towards morphologically distinct phages, i.e., ACSR16 (short-tailed) and WESR29 (long-tailed), respectively. ORS571 and WE7 were tolerant to hydrophobic compounds (triton X-100, rifampicin, crystal violet and deoxycholate). To ascertain the role of outer membrane characteristics in stress tolerance, phage-resistant transconjugant mutants of ORS571 (ORS571-M8 and ORS571-M20) and WE7 (WE7-M9) were developed. LPS- and membrane–protein profiles of mutants differed from that of respective wild types (ORS571 and WE7). In in vitro assay, phages got adsorbed onto purified LPS-membrane protein fractions of wild types. Phages did not adsorb onto membrane fraction of mutants and standard strains. Mutant with reduced expression of LPS (ORS571-M20 and WE7-M9) showed reduced tolerance towards hydrophobics. However, the tolerance was unaffected in mutant (ORS571-M8) where expression of LPS was not reduced but pattern was different. The tolerance level of mutants towards hydrophobics varied with the expression of LPS, whereas the specificity towards phages is correlated with the specific LPS pattern.     

N-terminal Gly(224)-Gly(411) domain in Listeria adhesion protein interacts with host receptor Hsp60

Background

Listeria adhesion protein (LAP) is a housekeeping bifunctional enzyme consisting of N-terminal acetaldehyde dehydrogenase (ALDH) and C-terminal alcohol dehydrogenase (ADH). It aids Listeria monocytogenes in crossing the epithelial barrier through a paracellular route by interacting with its host receptor, heat shock protein 60 (Hsp60). To gain insight into the binding interaction between LAP and Hsp60, LAP subdomain(s) participating in the Hsp60 interaction were investigated.

Methods

Using a ModBase structural model, LAP was divided into 4 putative subdomains: the ALDH region contains N1 (Met₁–Pro₂₂₃) and N2 (Gly₂₂₄–Gly₄₁₁), and the ADH region contains C1 (Gly₄₁₂–Val₆₄₈) and C2 (Pro₆₄₉–Val₈₆₆). Each subdomain was cloned and overexpressed in Escherichia coli and purified. Purified subdomains were used in ligand overlay, immunofluorescence, and bead-based epithelial cell adhesion assays to analyze each domain''s affinity toward Hsp60 protein or human ileocecal epithelial HCT-8 cells.

Results

The N2 subdomain exhibited the greatest affinity for Hsp60 with a K _D of 9.50±2.6 nM. The K _D of full-length LAP (7.2±0.5 nM) to Hsp60 was comparable to the N2 value. Microspheres (1 µm diameter) coated with N2 subdomain showed significantly (P<0.05) higher binding to HCT-8 cells than beads coated with other subdomains and this binding was inhibited when HCT-8 cells were pretreated with anti-Hsp60 antibody to specifically block epithelial Hsp60. Furthermore, HCT-8 cells pretreated with purified N2 subdomain also reduced L. monocytogenes adhesion by about 4 log confirming its involvement in interaction with epithelial cells.

Conclusion

These data indicate that the N2 subdomain in the LAP ALDH domain is critical in initiating interaction with mammalian cell receptor Hsp60 providing insight into the molecular mechanism of pathogenesis for the development of potential anti-listerial control strategies.     

Investigation on the role of p53 codon 72 polymorphism and interactions with tobacco, betel quid, and alcohol in susceptibility to cancers in a high-risk population from North East India

The association of TP53 codon 72 polymorphism with cancer susceptibility remains uncertain and varies with ethnicity. Northeast India represents a geographically, culturally, and ethnically isolated population. The area reports high rate of tobacco usage in a variety of ways of consumption, compared with the rest of Indian population. A total of 411 cancer patients (161 lung, 134 gastric, and 116 oral) and 282 normal controls from the ethnic population were analyzed for p53 codon 72 polymorphism by polymerase chain reaction-restriction fragment length polymorphism. No significant difference in genotypic distribution of p53 between cases and controls was observed. Results suggested betel quid chewing as a major risk factor for all the three cancers (odds ratio [OR]=3.54, confidence interval [CI]=2.01-6.25, p<0.001; OR=1.74, CI=1.04-2.92, p=0.03; and OR=1.85, CI=1.02-3.33, p=0.04 for lung, gastric, and oral cancers, respectively). Tobacco smoking was associated with risk of lung and oral cancers (OR=1.88, CI=1.11-3.19, p=0.01 and OR=1.68, CI=1.00-2.81, p=0.04). Interactions between p53 genotypes and risk factors were analyzed to look for gene-environment interactions. Interaction of smoking and p53 genotype was significant only for oral cancer. Interactions of betel quid with p53 genotypes in lung cancer showed significant increase for all the three genotypes, indicating a major role of betel quid (OR=5.90, CI=1.67-20.81, p=0.006; OR=5.44, CI=1.67-17.75, p=0.005; and OR=5.84, CI=1.70-19.97, p=0.005 for Arg/Arg, Arg/Pro, and Pro/Pro, respectively). In conclusion, high incidence of these cancers in northeast India might be an outcome of risk habits; further, tissue- and carcinogen-specific risk modification by p53 gene is probable.     

Glucose deprivation induced upregulation of phosphoenolpyruvate carboxykinase modulates virulence in Leishmania donovani

Various physiological stimuli trigger the conversion of noninfective Leishmania donovani promastigotes to the infective form. Here, we present the first evidence of the effect of glucose starvation, on virulence and survival of these parasites. Glucose starvation resulted in a decrease in metabolically active parasites and their proliferation. However, this was reversed by supplementation of gluconeogenic amino acids. Glucose starvation induced metacyclogenesis and enhanced virulence through protein kinase A regulatory subunit (LdPKAR1) mediated autophagy. Glucose starvation driven oxidative stress upregulated the antioxidant machinery, culminating in increased infectivity and greater parasitic load in primary macrophages. Interestingly, phosphoenolpyruvate carboxykinase (LdPEPCK), a gluconeogenic enzyme, exhibited the highest activity under glucose starvation to regulate growth of L. donovani by alternatively utilising amino acids. Deletion of LdPEPCK (Δpepck) decreased virulent traits and parasitic load in primary macrophages but increased autophagosome formation in the mutant parasites. Furthermore, Δpepck parasites failed to activate the Pentose Phosphate Pathway shunt, abrogating NADPH/NADP⁺ homoeostasis, conferring increased susceptibility towards oxidants following glucose starvation. In conclusion, this study showed that L. donovani undertakes metabolic rearrangements via gluconeogenesis under glucose starvation for acquiring virulence and its survival in the hostile environment.     

Sequence specificity of Z-DNA formation in oligonucleotides

The sequence specific requirement for B----Z transition in solution was examined in d(CGTGCGCACG), d(CGTACGTACG), d(ACGTACGT) in presence of various Z-inducing factors. Conformational studies show that inspite of the alternating nature of purines and pyrimidines, the aforementioned sequences do not undergo B----Z transition under the influence of NaCl, hexamine cobalt chloride and ethanol. A comparison with the crystal structures of an assorted array of purine and pyrimidine sequences show that the sequence requirement for B----Z transition is much more stringent in solution as compared to the solid state. The disruptive influence of AT base pairs in B to Z transition is discussed.     

Interaction of l-Prolyl-l-Leucyl Glycinamide with Dopamine D2 Receptor: Evidence for Modulation of Agonist Affinity States in Bovine Striatal Membranes

The role of the hypothalamic tripeptide L-prolyl-L-leucyl-glycinamide (PLG) in modulating the agonist binding to bovine striatal dopamine D2 receptor was investigated using a selective high-affinity agonist, n-propylnorapomorphine (NPA). PLG caused an enhancement in [3H]NPA binding in striatal membranes in a dose-dependent manner, the maximum effect being observed at 10(-7)-10(-6) M concentration of the tripeptide. The Scatchard analysis of [3H]NPA binding to membranes preincubated with 10(-6) M PLG revealed a significant increase in the affinity of the agonist binding sites. In contrast, there was no effect of PLG on the binding pattern of the antagonist [3H]spiroperidol. The antagonist versus agonist competition curves analyzed for agonist high- and low-affinity states of the receptor displayed an increase in the population and affinity of the high-affinity form of the receptor with PLG treatment. The low-affinity sites concomitantly decreased with relatively small change in the affinity for the agonists. Almost similar results were obtained when either NPA or apomorphine was used in the competition experiments. A partial antagonistic effect of PLG on 5'-guanylylimidodiphosphate [Gpp(NH)p]-induced inhibition of high-affinity agonist binding was also observed, as the ratio of high- to low-affinity forms of the receptor was significantly higher in the PLG-treated membranes compared to the controls. Direct [3H]NPA binding experiments demonstrated that PLG attenuated the Gpp(NH)p-induced inhibition of agonist binding by increasing the EC50 of the nucleotide (concentration that inhibits 50% of the specific binding). No effect of PLG on high-affinity [3H]NPA binding, however, could be observed when the striatal membranes were preincubated with Gpp(NH)p.(ABSTRACT TRUNCATED AT 250 WORDS)     

Designing B‐ and T‐cell multi‐epitope based subunit vaccine using immunoinformatics approach to control Zika virus infection

The Zika virus is a rapidly spreading Aedes mosquito‐borne sickness, which creates an unanticipated linkage birth deformity and neurological turmoil. This study represents the use of the combinatorial immunoinformatics approach to develop a multiepitope subunit vaccine using the structural and nonstructural proteins of the Zika virus. The designed subunit vaccine consists of cytotoxic T‐lymphocyte and helper T‐lymphocyte epitopes accompanied by suitable adjuvant and linkers. The presence of humoral immune response specific B‐cell epitopes was also confirmed by B‐cell epitope mapping among vaccine protein. Further, the vaccine protein was characterized for its allergenicity, antigenicity, and physiochemical parameters and found to be safe and immunogenic. Molecular docking and molecular dynamics studies of the vaccine protein with the toll‐like receptor‐3 were performed to ensure the binding affinity and stability of their complex. Finally, in silico cloning was performed for the effective expression of vaccine construct in the microbial system (Escherichia coli K12 strain). Aforementioned approaches result in the multiepitope subunit vaccine which may have the ability to induce cellular as well as humoral immune response. Moreover, this study needs the experimental validation to prove the immunogenic and protective behavior of the developed subunit vaccine.     

Angiotensin-Converting Enzyme Gene Insertion/Deletion Polymorphism and Cardiometabolic Risk Factors: A Study Among Bhil Tribal Population from Two Environmental Settings

Studies have investigated the association between angiotensin-converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism and cardiometabolic risk factors (CMRFs), however with varying results, which could be due to ethnicity differences. Therefore, the present study was conducted among Bhil tribal population (a mendelian population with the common gene pool and same sociocultural attributes), residing in two different environmental settings. The study attempts to understand the distribution and extent of association of ACE I/D gene polymorphism with cardiometabolic risk factors among Bhils from rural and urban settings. All the obesity and blood pressure variables were collected form 432 recruited subjects from both sexes aged 25–65 years and ACE I/D polymorphism was analysed on 299 subjects. Almost all the studied CMRFs were found to be significantly higher among urban Bhils. ACE gene was found to be polymorphic in the studied groups. DD genotype was found to pose more than threefold significant risk for low HDLC only in rural area. Estimate change analysis revealed an increasing D allele dose leads to more than one unit increase in Blood Pressure, and more than three units decrease in HDLC. The study highlights the differential effect of ACE I/D gene polymorphism in different environmental settings.     

Biological, Serological and Coat Protein Properties of a Strain of Turnip Mosaic Virus Causing a Mosaic Disease of Brassica campestris and B. juncea in India

Biological, serological and coat protein properties of a potyvirus (Poty-Rape) causing a mosaic disease of Brassica campestris and B. juncea in India were investigated. The virus readily infected 4 of the 5 plant species in the family Brassicaceae in which it induced severe systemic mosaic symptoms; it also induced chlorotic and necrotic local lesions in Chenopodium amaranticolor , but failed to infect 4 other species of Chenopodiaceae or 20 species of Amaranthaceae, Apiaceae, Canabinaceae, Compositae, Cucurbitaceae, Euphorbiaceae, Leguminosae and Solanaceae. The virus was transmitted in a non-persistant manner by Myzus persicae, Brevicoryne brassicae and Aphis gossypii. The Average size, of the virus particles in a purified preparation was 740 nm × 12 nm. SDS-PAGE analysis of the viral coat protein showed two major bands of approximately 37 kDa and 31 kDa, a pattern very similar to that of a reference isolate of turnip mosaic virus (TuMV) from the U.S. In Western-blot immunoassay, an antiserum to TuMV reacted with both the coat protein bands of the Poty-Rape islate and the reference TuMV, but not with the coat proteins of four other potyviruses. The high performance liquid chromatographic profile of tryptic peptides from the coat protein of Poty-Rape was found to be very similar to that of the reference TuMV, but differed substantially from those of four other potyviruses. The Poty-Rape isolate is considered to be a distinct strain of, TuMV.     

Microbial βγ-crystallins

βγ-Crystallins have emerged as a superfamily of structurally homologous proteins with representatives across the domains of life. A major portion of this superfamily is constituted by members from microorganisms. This superfamily has also been recognized as a novel group of Ca²⁺-binding proteins with huge diversity. The βγ domain shows variable properties in Ca²⁺ binding, stability and association with other domains. The various members present a series of evolutionary adaptations culminating in great diversity in properties and functions. Most of the predicted βγ-crystallins are yet to be characterized experimentally. In this review, we outline the distinctive features of microbial βγ-crystallins and their position in the βγ-crystallin superfamily.