No abstract

Bacteria elicit an adaptive response against hostile conditions such as starvation and other kinds of stresses. Their ability to survive such conditions depends, in part, on stringent response pathways. (p)ppGpp, considered to be the master regulator of the stringent response, is a novel target for inhibiting the survival of bacteria. In mycobacteria, the (p)ppGpp synthetase activity of bifunctional Rel is critical for stress response and persistence inside a host. Our aim was to design an inhibitor of (p)ppGpp synthesis, monitor its efficiency using enzyme kinetics, and assess its phenotypic effects in mycobacteria. As such, new sets of inhibitors targeting (p)ppGpp synthesis were synthesized and characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. We observed significant inhibition of (p)ppGpp synthesis by Rel Msm in the presence of designed inhibitors in a dosedependent manner, which we further confirmed by monitoring the enzyme kinetics. The Rel enzyme inhibitor binding kinetics were investigated by isothermal titration calorimetry. Subsequently, the effects of the compounds on long-term persistence, biofilm formation, and biofilm disruption were assayed in Mycobacterium smegmatis, where inhibition in each case was observed. In vivo, (p)ppGpp levels were found to be downregulated in M. smegmatis treated with the synthetic inhibitors. The compounds reported here also inhibited biofilm formation by the pathogen Mycobacterium tuberculosis. The compounds were tested for toxicity by using an MTT assay with H460 cells and a hemolysis assay with human red blood cells, for which they were found to be nontoxic. The permeability of compounds across the cell membrane of human lung epithelial cells was also confirmed by mass spectrometry.

Phosphorylation of the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) governs stage-specific interactions with different cellular machines. The CTD consists of Y 1 S 2 P 3 T 4 S 5 P 6 S 7 heptad repeats, and sequential phosphorylations of Ser7, Ser5 and Ser2 occur universally across Pol II-transcribed genes. Phosphorylation of Thr4, however, appears to selectively modulate transcription of specific classes of genes. Here, we identify 10 new Thr4 kinases from different kinase structural groups. Irreversible chemical inhibition of the most active Thr4 kinase, Hrr25, reveals a novel role for this kinase in transcription termination of specific class of noncoding snoRNA genes. Genome-wide profiles of Hrr25 reveal a selective enrichment at 3ʹ regions of noncoding genes that display termination defects. Importantly, phospho-Thr4 marks placed by Hrr25 are recognized by Rtt103, a key component of the termination machinery. Our results suggest that these uncommon CTD kinases selectively place phospho-Thr4 marks to regulate expression of targeted genes.

Earlier, vitamin C was demonstrated to sterilize Mycobacterium tuberculosis culture via Fenton's reaction at high concentration. It alters the regulatory pathways associated with stress response and dormancy. Since (p)ppGpp is considered to be the master regulator of stress response and is responsible for bacterial survival under stress, we tested the effect of vitamin C on the formation of (p)ppGpp. In vivo estimation of (p)ppGpp showed a decrease in (p)ppGpp levels in vitamin C-treated M. smegmatis cells in comparison to the untreated cells. Furthermore, in vitro (p)ppGpp synthesis using Rel enzyme was conducted in order to confirm the specificity of the inhibition in the presence of variable concentrations of vitamin C. We observed that vitamin C at high concentration can inhibit the synthesis of (p)ppGpp. We illustrated binding of vitamin C to Rel by isothermal titration calorimetry. Enzyme kinetics was followed where K was found to be increased with the concomitant reduction of V value suggesting mixed inhibition. Both long-term survival and biofilm formation were inhibited by vitamin C. The experiments suggest that vitamin C has the potential to be developed as the inhibitor of (p)ppGpp synthesis and stress response, at least in the concentration range used here.

Mycobacterium tuberculosis elicits the stringent response under unfavorable growth conditions, such as those encountered by the pathogen inside the host. The hallmark of this response is production of guanosine tetra-and pentaphosphates, collectively termed (p)ppGpp, which have pleiotropic effects on the bacterial physiology. As the stringent response is connected to survival under stress, it is now being targeted for developing inhibitors against bacterial persistence. The Rel enzyme in mycobacteria has two catalytic domains at its N-terminus that are involved in the synthesis and hydrolysis of (p)ppGpp, respectively. However, the function of the C-terminal region of the protein remained unknown. Here, we have identified a binding site for pppGpp in the C-terminal region of Rel. The binding affinity of pppGpp was quantified by isothermal titration calorimetry. The binding site was determined by crosslinking using the nucleotide analog azido-pppGpp, and examining the crosslink product by mass spectrometry. Additionally, mutations in the Rel protein were created to confirm the site of pppGpp binding by isothermal titration calorimetry. These mutants showed increased pppGpp synthesis and reduced hydrolytic activity. We believe that binding of pppGpp to Rel provides a feedback mechanism that allows the protein to detect and adjust the (p)ppGpp level in the cell. Our work suggests that such sites should also be considered while designing inhibitors to target the stringent response.

Coronavirus disease 2019 (COVID‐19) caused by coronavirus has spread worldwide and has become the deadliest pandemic of the 21st century. Such rapid spread is predominantly attributed to the poor diagnosis and its asymptomatic transmission. In the absence of treatment regime, timely diagnosis is the best available remedy that can restrict its spread. An early diagnosis of COVID‐19 is critical for determining the line of treatment and preventing long term complications in the infected subject. Unfortunately, available rapid antigen and antibody kits are known to be erroneous whereas reverse transcription polymerase chain reaction based tests are expensive, viral load dependent and at times inconclusive. In current scenario, the false‐negative results imposed a major risk to the individual patient care and also to the efforts for containing the spread at the population level, where as false positives are traumatic for families and can lead to improper treatment resulting in severe complications. In this article, the limitations of available diagnostic procedures have been elaborated and plausible combination approach has been advised.

No abstract

Mycobacterium has evolved distinct cell wall and strategies such as biofilm formation, which helps it to survive in hostile conditions. We have reported previously that arabinofuranoside containing glycolipids exhibit inhibition activities against the above functions of the mycobacterial species M. smegmatis. In search for activities mediated by oligosaccharide glycolipids, we report herein the inhibitory activities of a linear and a branched pentasaccharides having arabinan and mannan moieties. In the presence of the pentasaccharide glycolipids, a significant reduction in mycobacterial growth is observed, concomitant with reductions in sliding motility and colonization through biofilm formation, at the optimal glycolipid concentrations of 50-100 μg mL(-1). Especially the biofilm coat is ruptured by ~80-85 % in the presence of glycolipids. Pentasaccharides alone without the lipidic chain show only a weak effect. The glycolipids are non-toxic, as evaluated through their effect on RBCs. Analysis of the mycolic acid profile of glycolipid treated biofilm shows that α- and epoxy mycolic acids are downregulated significantly, in comparison to glycolipid untreated biofilms. Lipidomics profile analysis through mass spectrometry further reveals profound downregulation of phosphatidylinositol mannosides, acylatedphosphoglycerols and mycolic acid family, namely, keto-, alpha- and methoxymycolic acids.