Stability of Lysozyme in Aqueous Extremolyte Solutions during Heat Shock and Accelerated Thermal Conditions

The purpose of this study was to investigate the stability of lysozyme in aqueous solutions in the presence of various extremolytes (betaine, hydroxyectoine, trehalose, ectoine, and firoin) under different stress conditions. The stability of lysozyme was determined by Nile red Fluorescence Spectroscopy and a bioactivity assay. During heat shock (10 min at 70°C), betaine, trehalose, ectoin and firoin protected lysozyme against inactivation while hydroxyectoine, did not have a significant effect. During accelerated thermal conditions (4 weeks at 55°C), firoin also acted as a stabilizer. In contrast, betaine, hydroxyectoine, trehalose and ectoine destabilized lysozyme under this condition. These findings surprisingly indicate that some extremolytes can stabilize a protein under certain stress conditions but destabilize the same protein under other stress conditions. Therefore it is suggested that for the screening extremolytes to be used for protein stabilization, an appropriate storage conditions should also be taken into account.     

Compromised base excision repair pathway in Mycobacterium tuberculosis imparts superior adaptability in the host

Tuberculosis caused by Mycobacterium tuberculosis (Mtb) is a significant public health concern, exacerbated by the emergence of drug-resistant TB. To combat the host’s dynamic environment, Mtb encodes multiple DNA repair enzymes that play a critical role in maintaining genomic integrity. Mtb possesses a GC-rich genome, rendering it highly susceptible to cytosine deaminations, resulting in the occurrence of uracils in the DNA. UDGs encoded by ung and udgB initiate the repair; hence we investigated the biological impact of deleting UDGs in the adaptation of pathogen. We generated gene replacement mutants of uracil DNA glycosylases, individually (RvΔung, RvΔudgB) or together (RvΔdKO). The double KO mutant, RvΔdKO exhibited remarkably higher spontaneous mutation rate, in the presence of antibiotics. Interestingly, RvΔdKO showed higher survival rates in guinea pigs and accumulated large number of SNPs as revealed by whole-genome sequence analysis. Competition assays revealed the superior fitness of RvΔdKO over Rv, both in ex vivo and in vivo conditions. We propose that compromised DNA repair results in the accumulation of mutations, and a subset of these drives adaptation in the host. Importantly, this property allowed us to utilize RvΔdKO for the facile identification of drug targets.     

Common variant in FUT2 gene is associated with levels of vitamin B12 in Indian population

Vitamin B₁₂ is an essential micronutrient synthesized by microorganisms. Mammals including humans have evolved ways for transport and absorption of this vitamin. Deficiency of vitamin B₁₂ (either due to low intake or polymorphism in genes involved in absorption and intracellular transport of this vitamin) has been associated with various complex diseases. Genome-wide association studies have recently identified several common single nucleotide polymorphisms (SNPs) in fucosyl transferase 2 gene (FUT2) to be associated with levels of vitamin B₁₂—the strongest association was with a non-synonymous SNP rs602662 in this gene. In the present study, we attempted to replicate the association of this SNP (rs602662) in an Indian population since a significant proportion has been reported to have low levels of vitamin B₁₂ in this population. A total of 1146 individuals were genotyped for this SNP using a single base extension method and association with levels of vitamin B₁₂ was assessed in these individuals. Regression analysis was performed to analyze the association considering various confounding factors like for age, sex, diet, hypertension, diabetes mellitus and coronary artery disease status. We found that the SNP rs602662 was significantly associated with the levels of vitamin B₁₂ (p value < 0.0001). We also found that individuals adhering to a vegetarian diet with GG (homozygous major genotype) have significantly lower levels of vitamin B₁₂ in these individuals. Thus, our study reveals that vegetarian diet along with polymorphism in the FUT2 gene may contribute significantly to the high prevalence of vitamin B₁₂ deficiency in India.     

Interaction between mosquito-larvicidal Lysinibacillus sphaericus binary toxin components: Analysis of complex formation

The two components (BinA and BinB) of Lysinibacillus sphaericus binary toxin together are highly toxic to Culex and Anopheles mosquito larvae, and have been employed world-wide to control mosquito borne diseases. Upon binding to the membrane receptor an oligomeric form (BinA2.BinB2) of the binary toxin is expected to play role in pore formation. It is not clear if these two proteins interact in solution as well, in the absence of receptor. The interactions between active forms of BinA and BinB polypeptides were probed in solution using size-exclusion chromatography, pull-down assay, surface plasmon resonance, circular dichroism, and by chemically crosslinking BinA and BinB components. We demonstrate that the two proteins interact weakly with first association and dissociation rate constants of 4.5 × 10³ M^?1 s^?1 and 0.8 s^?1, resulting in conformational change, most likely, in toxic BinA protein that could kinetically favor membrane translocation of the active oligomer. The weak interactions between the two toxin components could be stabilized by glutaraldehyde crosslinking. The cross-linked complex, interestingly, showed maximal Culex larvicidal activity (LC₅₀ value of 1.59 ng mL^?1) reported so far for combination of BinA/BinB components, and thus is an attractive option for development of new bio-pesticides for control of mosquito borne vector diseases.     

Tryptophan to Glycine mutation in the position 116 leads to protein aggregation and decreases the stability of the LITAF protein

Mutations in the gene-encoding vesicle lipopolysaccharide-induced tumor necrosis factor (LITAF) protein cause Charcot–Marie–Tooth type 1C (CMT1C) disease, a neurological disorder. The LITAF gene is mapped to chromosome number 16 and can be found at cytogenetic location 16p13 of the chromosome. CMT1C-linked small integral membrane protein of lysosome/late endosome mutants are loss-of-function mutants that act in a dominant negative manner to impair endosomal trafficking, leading to prolonged extracellular signal-regulated kinases 1/2 signaling downstream of ErbB activation. Mutation W116G in the LITAF decreases the stability of the protein and also interrupts the functioning of gene. We have analyzed the single nucleotide polymorphism (SNP) results of 28 nsSNPs obtained from dbSNP. We also carried out multiple molecular dynamics simulations of 200 ns and obtained results of root-mean-square deviation, root-mean-square fluctuation, radius of gyration, solvent-accessible surface area, H-bond, and principal component analysis to check and prove the stability of both the wild type and the mutant. The protein was then checked for its aggregation and the results showed loss of helix. The loss of helix leads to the instability of the protein.     

Identification of Penicillin-binding proteins employing support vector machines and random forest

Penicillin-Binding Proteins are peptidases that play an important role in cell-wall biogenesis in bacteria and thus maintaining bacterial infections. A wide class of β-lactam drugs are known to act on these proteins and inhibit bacterial infections by disrupting the cell-wall biogenesis pathway. Penicillin-Binding proteins have recently gained importance with the increase in the number of multi-drug resistant bacteria. In this work, we have collected a dataset of over 700 Penicillin-Binding and non-Penicillin Binding Proteins and extracted various sequence-related features. We then created models to classify the proteins into Penicillin-Binding and non-binding using supervised machine learning algorithms such as Support Vector Machines and Random Forest. We obtain a good classification performance for both the models using both the methods.