Tale of Twin Bifunctional Second Messenger (p)ppGpp Synthetases and Their Function in Mycobacteria.

M. tuberculosis, an etiological agent of tuberculosis, requires a long treatment regimen due to its ability to respond to stress and persist inside the host. The second messenger (p)ppGpp-mediated stress response plays a critical role in such long-term survival, persistence, and antibiotic tolerance which may also lead to the emergence of multiple drug resistance. In mycobacteria, (pp)pGpp molecules are synthesized predominantly by two bifunctional enzymes-long RSH-Rel and short SAS-RelZ. The long RSH-Rel is a major (p)ppGpp synthetase and hydrolase. How it switches its activity from synthesis to hydrolysis remains unclear. RelMtb mutant has been reported to be defective in biofilm formation, cell wall function, and persister cell formation. The survival of such mutants has also been observed to be compromised in infection models. In M. smegmatis, short SAS-RelZ has RNase HII activity in addition to (pp)Gpp synthesis activity. The RNase HII function of RelZ has been implicated in resolving replication-transcription conflicts by degrading R-loops. However, the mechanism and regulatory aspects of such a regulation remain elusive. In this article, we have discussed (p)ppGpp metabolism and its role in managing the stress response network of mycobacteria, which is responsible for long-term survival inside the host, making it an important therapeutic target.

Multi epitope vaccine candidate design against Streptococcus pneumonia.

Streptococcus pneumonia, the causative agent of sepsis, meningitis and pneumonia, is held responsible for causing invasive diseases predominantly in children along with adults from both developing and developed countries. The available vaccines coverage in the context of different serotypes is limited and emergence of non-vaccine serotypes could further emerge as a threat in future. Advanced immunoinformatics tools have been used for developing a multi epitope subunit vaccine. In the current study we have subjected these four surface antigenic proteins Ply, PsaA, PspA and PspK to construct vaccine designs. We have predicted different B-cell and T-cell epitopes by using NetCTL 1.2, IEDB (Immune Epitope Databases) and ABCpred. An adjuvant (griselimycin) has been added to the vaccine construct sequence in order to improve its immunogenicity. The vaccine construct has been evaluated for its antigenicity, allergenicity, toxicity and different physio-chemical properties. The bioinformatic tools have been used for prediction, refinement and validation of the 3 D structure. Further, the vaccine structure has been docked with a toll-like receptor (TLR-4) by ClusPro 2.0. In conclusion, the proposed multi-epitope vaccine designs could potentially activate both humoral and cellular immune responses and has a potential to be a vaccine candidate against S.pneumoniae, and requires experimental validation for ensuring immunogenicity and safety profile.Communicated by Ramaswamy H. Sarma.

An Unusual Presentation of Superior Mesenteric Venous Occlusion in Mild COVID-19.

SARS-CoV-2, an etiological agent of COVID-19, has been reported to inflict remarkably diverse manifestations in different subjects across the globe. Though patients with COVID-19 predominantly have fever, respiratory and constitutional symptoms, atypical presentations are becoming increasingly evident. COVID-19 may predispose to both venous and arterial thromboembolism due to excessive inflammation, hypoxia, immobilization, and diffuse intravascular coagulation in moderate to severe symptomatic cases. In this case report, we are reporting thromboembolic complications of COVID-19 in a mild symptomatic subject incidentally diagnosed with mesenteric venous occlusion with no abdominal symptoms. Early recognition of the abdominal symptoms, diagnosis, initiation of anticoagulants, and timely surgical intervention may improvise the outcome in a patient with COVID-19 infection-induced mesenteric thrombosis. Superior mesenteric artery and venous thrombosis may lead to subsequent ischemia necessitating emergency laparotomy. Thus, the usage of low-dose anticoagulants in all the patients of COVID-19 irrespective of the categorization into mild, moderate, and severe COVID-19 disease should be considered.

Vitamin C and its therapeutic potential in the management of COVID19.

COVID19 has emerged as one of the worst pandemics in the history of mankind. Several vaccines have been approved by different government agencies worldwide, but data on their efficacy and safety are limited, and distribution remains a massive challenge. As per WHO, personal immunity is vital for protection against COVID19. Earlier, Vitamin C-mediated pathways have been shown to play critical role in boosting immunity attributed to its antioxidant properties. Recently, the involvement of such pathways in protection against COVID19 has been suggested. The controlled doses of Vitamin C administered through intravenous (IV) injections are being studied for determining its role in the prognosis of COVID19. In this article, we have discussed the potential role of Vitamin C in the management in COVID19 patients and presented recent clinical trials data. Additionally, we have elaborated the possibility of administering Vitamin C through inhalers in order to achieve local high concentration and the challenges of such approach.

The extended (p)ppGpp family: New dimensions in Stress response.

Second messenger (p)ppGpp mediated stress response plays a crucial role in bacterial persistence and multiple drug resistance. In E. coli, (p)ppGpp binds to RNA polymerase and upregulates the transcription of genes essential for stress response while concurrently downregulating the expression of genes critical for growth and metabolism. Recently, the family of alarmone molecules has expanded to pppGpp, ppGpp, pGpp & (pp)pApp as distinct members. These molecules may help in fine-tuning stress responses in different hostile conditions. Do all of these molecules bind to RNA polymerase? Do they compete with each other or complement each other's functions is still not clear. Earlier, others and we have synthesized artificial analogs of (p)ppGpp that inhibited (p)ppGpp synthesis and long-term survival in M. smegmatis and in B. subtilis suggesting that analogs could compete with each other. Understanding the interplay of these molecules will allow deciphering novel pathways that can be potentially subjected to the therapeutic intervention. In this article, we have reviewed newly characterized second messengers and discussed their mode of action. We have also documented the progress made to-date in understanding the molecular basis of regulation of transcription by second messenger ppGpp, pppGpp, and pGpp.

Guidelines on newly identified limitations of diagnostic tools for COVID-19 and consequences.

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.

Noncanonical CTD kinases regulate RNA polymerase II in a gene-class-specific manner.

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 Y1S2P3T4S5P6S7 heptad repeats and sequential phosphorylations of Ser7, Ser5 and Ser2 occur universally at Pol II-transcribed genes. Phosphorylation of Thr4, however, appears to selectively modulate transcription of specific classes of genes. Here, we identify ten 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 3' 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 place phospho-Thr4 marks to regulate expression of targeted genes.

The role of ω-subunit of Escherichia coli RNA polymerase in stress response.

ppGpp, an alarmone for stringent response, plays an important role in the reprogramming of the transcription complex at the time of stress. In Escherichia coli, ppGpp mediates its action by binding to at least two different sites on RNA polymerase (RNAP). One of the sites to which ppGpp binds to RNAP is at the ß'-ω interface; however, the underlying molecular mechanism and the physiological relevance of ppGpp binding to this site remain unclear. In this study, we have performed UV cross-linking experiments using 32 P azido-labeled ppGpp to probe its association with RNAP in the absence and presence of ω, and observed weaker binding of ppGpp to the RNAP without ω. Furthermore, we followed the binding kinetics of ppGpp to RNAP with and without ω by isothermal titration calorimetry and found it to be concurrent with the cross-linking results. Native ω is intrinsically disordered, and we have used a previously characterized structured mutant of ω, which affects the plasticity of the active site of RNAP. Results show that the flexibility conferred by the unstructured ω is a prerequisite for ppGpp binding to RNAP. We have analyzed the stress-associated phenotypes in an E. coli strain devoid of ω (∆rpoZ). ppGpp levels in ∆rpoZ strain were found to be similar to that of the wild-type strain. Interestingly, when the ∆rpoZ strain of E. coli was transferred after nutritional stress to an enriched media, the recovery of growth was compromised. We have identified a new phenotype of ∆rpoZ strain corresponding to defect in biofilm formation in minimal media.

Vitamin C: A Natural Inhibitor of Cell Wall Functions and Stress Response in Mycobacteria.

Tuberculosis, caused by Mycobacterium tuberculosis, has re-emerged as a threat to human race. Conventional antibiotic treatments are failing due to different stress response strategies adopted by bacterial pathogens. Since time immemorial, Vitamin C is known to protect against pathogens by boosting immunity in humans. Recently, Vitamin C has been shown to directly kill M. tuberculosis including multiple drug-resistant strains by generation of oxidative radicals through Fenton's reaction. Concurrently, it inhibits (p)ppGpp-mediated stringent response thus effectively shutting down long-term survival and persistence in mycobacteria. Here, we have discussed historical perspective and recent evidences on Vitamin C-mediated inhibition of several key pathways of M. tuberculosis such as (p)ppGpp synthesis and mycobacterial cell wall function. Several cell wall components including mycolic acids are critical for mycobacterial virulence. We observed downregulation of various mycolic acids in M. smegmatis upon treatment with Vitamin C, and data have been presented here. Vitamin C has been shown to inhibit the biofilm growth as well as disrupt the formed biofilm in mycobacteria. Additionally, Vitamin C role in cell-mediated and humoral immunity has been elucidated. Vitamin C is toxic at high concentration; therefore we have proposed the idea of derivatizing Vitamin C in order to lower the minimal inhibition concentration (MIC) necessary to target M. tuberculosis.

Synthetic Arabinomannan Heptasaccharide Glycolipids Inhibit Biofilm Growth and Augment Isoniazid Effects in Mycobacterium smegmatis.

Biofilm formation, involving attachment to an adherent surface, is a critical survival strategy of mycobacterial colonies in hostile environmental conditions. Here we report the synthesis of heptasaccharide glycolipids based on mannopyranoside units anchored on to a branched arabinofuranoside core. Two types of glycolipids-2,3-branched and 2,5-branched-were synthesized and evaluated for their efficacies in inhibiting biofilm growth by the non-pathogenic mycobacterium variant Mycobacterium smegmatis. Biofilm formation was inhibited at a minimum biofilm growth inhibition concentration (MBIC) of 100 µg mL-1 in the case of the 2,5-branched heptasaccharide glycolipid. Further, we were able to ascertain that a combination of the drug isoniazid with the branched heptasaccharide glycolipid (50 µg mL-1 ) potentiates the drug, making it three times more effective, with an improved MBIC of 30 µg mL-1 . These studies establish that synthetic glycolipids not only act as inhibitors of biofilm growth, but also provide a synergistic effect when combined with significantly lowered concentrations of isoniazid to disrupt the biofilm structures of the mycobacteria.

Novel Method for Quantitative Estimation of Biofilms.

Biofilm protects bacteria from stress and hostile environment. Crystal violet (CV) assay is the most popular method for biofilm determination adopted by different laboratories so far. However, biofilm layer formed at the liquid-air interphase known as pellicle is extremely sensitive to its washing and staining steps. Early phase biofilms are also prone to damage by the latter steps. In bacteria like mycobacteria, biofilm formation occurs largely at the liquid-air interphase which is susceptible to loss. In the proposed protocol, loss of such biofilm layer was prevented. In place of inverting and discarding the media which can lead to the loss of the aerobic biofilm layer in CV assay, media was removed from the formed biofilm with the help of a syringe and biofilm layer was allowed to dry. The staining and washing steps were avoided, and an organic solvent-tetrahydrofuran (THF) was deployed to dissolve the biofilm, and the absorbance was recorded at 595 nm. The protocol was tested for biofilm estimation of E. coli, B. subtilis and M. smegmatis, and compared with the traditional CV assays. Isoniazid drug molecule, a known inhibitor of M. smegmatis biofilm, was tested and its inhibitory effects were quantified by the proposed protocol. For ease in referring, this method has been described as the Syal method for biofilm quantification. This new method was found to be useful for the estimation of early phase biofilm and aerobic biofilm layer formed at the liquid-air interphase. The biofilms formed by all three tested bacteria-B. subtilis, E. coli and M. smegmatis, were precisely quantified.

Synthetic (p)ppGpp Analogue Is an Inhibitor of Stringent Response in Mycobacteria.

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 RelMsm in the presence of designed inhibitors in a dose-dependent 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.

Vitamin C targets (p)ppGpp synthesis leading to stalling of long-term survival and biofilm formation in Mycobacterium smegmatis.

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 RelMSM 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 RelMSM by isothermal titration calorimetry. Enzyme kinetics was followed where K0.5 was found to be increased with the concomitant reduction of Vmax 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.

Synthetic arabinomannan glycolipids impede mycobacterial growth, sliding motility and biofilm structure.

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.

Differential binding of ppGpp and pppGpp to E. coli RNA polymerase: photo-labeling and mass spectral studies.

(p)ppGpp, a secondary messenger, is induced under stress and shows pleiotropic response. It binds to RNA polymerase and regulates transcription in Escherichia coli. More than 25 years have passed since the first discovery was made on the direct interaction of ppGpp with E. coli RNA polymerase. Several lines of evidence suggest different modes of ppGpp binding to the enzyme. Earlier cross-linking experiments suggested that the ß-subunit of RNA polymerase is the preferred site for ppGpp, whereas recent crystallographic studies pinpoint the interface of ß'/ω-subunits as the site of action. With an aim to validate the binding domain and to follow whether tetra- and pentaphosphate guanosines have different location on RNA polymerase, this work was initiated. RNA polymerase was photo-labeled with 8-azido-ppGpp/8-azido-pppGpp, and the product was digested with trypsin and subjected to mass spectrometry analysis. We observed three new peptides in the trypsin digest of the RNA polymerase labeled with 8-azido-ppGpp, of which two peptides correspond to the same pocket on ß'-subunit as predicted by X-ray structural analysis, whereas the third peptide was mapped on the ß-subunit. In the case of 8-azido-pppGpp-labeled RNA polymerase, we have found only one cross-linked peptide from the ß'-subunit. However, we were unable to identify any binding site of pppGpp on the ß-subunit. Interestingly, we observed that pppGpp at high concentration competes out ppGpp bound to RNA polymerase more efficiently, whereas ppGpp cannot titrate out pppGpp. The competition between tetraphosphate guanosine and pentaphosphate guanosine for E. coli RNA polymerase was followed by gel-based assay as well as by a new method known as DRaCALA assay.

Novel pppGpp binding site at the C-terminal region of the Rel enzyme from Mycobacterium smegmatis.

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.

VDR, RXR, Coronin-1 and Interferonγ Levels in PBMCs of Type-2 Diabetes Patients: Molecular Link between Diabetes and Tuberculosis.

Diabetes and tuberculosis are world's most deadly epidemics. People suffering from diabetes are susceptible to tuberculosis. Molecular link between the two is largely unknown. It is known that Vitamin A receptor (RXR) heterodimerizes with Vitamin D receptor (VDR) and Peroxisome proliferator-activator receptor-γ (PPARγ) to regulate Tryptophan-aspartate containing coat protein (TACO) expression and fatty acid metabolism respectively, so it would be interesting to check the expression of these genes in diabetes mellitus (DM) patients which might explain the susceptibility of diabetics to tuberculosis. In this study, we checked the expression of RXR, VDR, TACO and Interferon-γ (IFNγ) genes in type-2 DM patients for understanding the link between the two diseases. We observed down regulation of RXR gene and corresponding up regulation of TACO gene expression. We have not observed significant change in expression of VDR and IFNγ genes in type-2 DM patients. Repression of RXR gene could hamper VDR-RXR heterodimer formation and thus would up regulate TACO gene expression which may predispose the type-2 DM patients to tuberculosis. Also, decrease in RXR-PPARγ heterodimer could be involved in DM.