ß-Lactam Resistance in ESKAPE Pathogens Mediated Through Modifications in Penicillin-Binding Proteins: An Overview.

Bacteria acquire ß-lactam resistance through a multitude of mechanisms among which production of ß-lactamases (enzymes that hydrolyze ß-lactams) is the most common, especially in Gram-negatives. Structural changes in the high-molecular-weight, essential penicillin-binding proteins (PBPs) are widespread in Gram-positives and increasingly reported in Gram-negatives. PBP-mediated resistance is largely achieved by accumulation of mutation(s) resulting in reduced binding affinities of ß-lactams. Herein, we discuss PBP-mediated resistance among ESKAPE pathogens that cause diverse hospital- and community-acquired infections globally.

Two new mutations in dnaJ suppress DNA damage hypersensitivity and capsule overproduction phenotypes of Δlon mutant of Escherichia coli by modulating the expression of clpYQ (hslUV) and rcsA genes.

Lon is a major ATP-dependent protease of E. coli involved in degradation of abnormal misfolded proteins and specific regulatory proteins. Absence of Lon in E. coli results in sensitivity to DNA damaging agents and over-production of capsular polysaccharide due to accumulation of Lon substrates, SulA (cell division inhibitor induced upon DNA damage) and RcsA (activator of cps genes), respectively. In a previous study, we identified that a G232D mutation, termed faa (for function affecting alternative-lon-protease), in the E. coli co-chaperone DnaJ, results in suppression of lon mutant phenotypes. Additionally, inactivation of the trans-translation system was found to have an additive effect on faa activity. In the present work, we employed random mutagenesis approach to isolate novel mutations in dnaJ which could phenotypically compensate the absence of Lon. Using a lacZ-based Lon reporter strain, we were able to isolate two new mutations in dnaJ as lon suppressors. These mutations, namely, flm-1 (H33Y) and flm-2 (P34S), affected the highly conserved HPD motif of DnaJ. Both mutations suppressed lon phenotypes to variable extent and the suppression was also differentially modulated by mutations in ssrA that affect trans-translation. We show that ClpYQ protease up-regulated in both mutants should degrade SulA, since inactivation of clpQ abolished the resistance to DNA damaging agents. On the other hand, we found suppression of capsule overproduction phenotype was independent of ClpYQ in both mutants but resulted due to down-regulation of rcsA in flm-1. Thus, our findings highlight the intricate redundancy of cellular proteolysis networks in bacteria which can compensate the absence of Lon via distinct mechanisms.

Unveiling the molecular basis for pleiotropy in selected rif mutants of Escherichia coli: Possible role for Tyrosine in the Rif binding pocket and fast movement of RNA polymerase.

Rifampicin (RIF) is still a first line of antibiotic in the treatment of bacterial diseases, in particular the Mycobacterial infections. The antimicrobial activity of RIF is attributed to its ability to inhibit transcription by binding to the ß subunit of bacterial RNA polymerase (encoded by rpoB). Continued use of this drug resulted in the emergence of RIF resistant rpoB mutations in a high frequency that compels the use of RIF almost exclusively in drug combinations. As of date, a broad array of rif mutations have been isolated and characterized by different research groups. Studies on rpoB mutations strengthen the view that the ß subunit of RNA polymerase (RNAP) is very crucial in modulating transcription thereby leading to differential gene expression. Very recently we have reported the transcriptome profile of rpoB12 mutant that provides molecular evidence that presence of rpoB12 mutation modulates the transcription of about 450 genes. Here we present a maiden report that rpoB mutations that substitute Tyr at the Rif binding pocket (RBP) of ß subunit of RNA polymerase are able to suppress the over-production of colanic acid capsular polysaccharide (Ces phenotype) in Δlon mutant of Escherichia coli. Further analyses of the rif mutants involving their growth pattern on LB at higher temperature (42 °C), LB media without NaCl, survival in LB media with acidic pH (pH - 3) and motility revealed that only rpoB12 (His526Tyr) and rpoB137 (Ser522Tyr) affected all the above mentioned physiological parameters in addition to the elicitation of Ces phenotype. These two rif mutations confer fast movement to RNAP and they bear Tyr as the substituted amino acid in the RBP. This is perhaps the first study that brings out the possible role of Tyr in the RBP and its participation in the global gene expression. This study also envisages the point that amino acid residues that share the properties of Tyr in the RBP can be employed as a tool to bring out differential gene expression which would certainly have basic and applied values for the mankind.

A putative curved DNA region upstream of rcsA in Escherichia coli plays a key role in transcriptional regulation by H-NS.

It is well established that in Escherichia coli, the histone-like nucleoid structuring (H-NS) protein also functions as negative regulator of rcsA transcription. However, the exact mode of regulation of rcsA transcription by H-NS has not been studied extensively. Here, we report the multicopy effect of dominant-negative hns alleles on the transcription of rcsA based on expression of cps-lac transcriptional fusion in ∆lon, ∆lon rpoB12, ∆lon rpoB77 and lon+ strains. Our results indicate that H-NS defective in recognizing curved DNA fails to repress rcsA transcription significantly, while nonoligomeric H-NS molecules still retain the repressor activity to an appreciable extent. Together with bioinformatics analysis, our study envisages a critical role for the putative curved DNA region present upstream of rcsA promoter in the transcriptional regulation of rcsA by H-NS.

Gut microbial degradation of organophosphate insecticides-induces glucose intolerance via gluconeogenesis.

BACKGROUND: Organophosphates are the most frequently and largely applied insecticide in the world due to their biodegradable nature. Gut microbes were shown to degrade organophosphates and cause intestinal dysfunction. The diabetogenic nature of organophosphates was recently reported but the underlying molecular mechanism is unclear. We aimed to understand the role of gut microbiota in organophosphate-induced hyperglycemia and to unravel the molecular mechanism behind this process. RESULTS: Here we demonstrate a high prevalence of diabetes among people directly exposed to organophosphates in rural India (n = 3080). Correlation and linear regression analysis reveal a strong association between plasma organophosphate residues and HbA1c but no association with acetylcholine esterase was noticed. Chronic treatment of mice with organophosphate for 180 days confirms the induction of glucose intolerance with no significant change in acetylcholine esterase. Further fecal transplantation and culture transplantation experiments confirm the involvement of gut microbiota in organophosphate-induced glucose intolerance. Intestinal metatranscriptomic and host metabolomic analyses reveal that gut microbial organophosphate degradation produces short chain fatty acids like acetic acid, which induces gluconeogenesis and thereby accounts for glucose intolerance. Plasma organophosphate residues are positively correlated with fecal esterase activity and acetate level of human diabetes. CONCLUSION: Collectively, our results implicate gluconeogenesis as the key mechanism behind organophosphate-induced hyperglycemia, mediated by the organophosphate-degrading potential of gut microbiota. This study reveals the gut microbiome-mediated diabetogenic nature of organophosphates and hence that the usage of these insecticides should be reconsidered.