Temporal Profiling of Host Proteome against Different M. tuberculosis Strains Reveals Delayed Epigenetic Orchestration.

Apart from being preventable and treatable, tuberculosis is the deadliest bacterial disease afflicting humankind owing to its ability to evade host defence responses, many of which are controlled by epigenetic mechanisms. Here, we report the temporal dynamics of the proteome of macrophage-like host cells after infecting them for 6, 18, 30, and 42 h with two laboratory strains (H37Ra and H37Rv) and two clinical strains (BND433 and JAL2287) of Mycobacterium tuberculosis (MTB). Using SWATH-MS, the proteins characterized at the onset of infection broadly represented oxidative stress and cell cytoskeleton processes. Intermediary and later stages of infection are accompanied by a reshaping of the combination of proteins implicated in histone stability, gene expression, and protein trafficking. This study provides strain-specific and time-specific variations in the proteome of the host, which might further the development of host-directed therapeutics and diagnostic tools against the pathogen. Also, our findings accentuate the importance of proteomic tools in delineating the complex recalibration of the host defence enabled as an effect of MTB infection. To the best of our knowledge, this is the first comprehensive proteomic account of the host response to avirulent and virulent strains of MTB at different time periods of the life span of macrophage-like cells. The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE repository with the dataset identifier PXD022352.

Effect of herbal extracts and Saroglitazar on high-fat diet-induced obesity, insulin resistance, dyslipidemia, and hepatic lipidome in C57BL/6J mice.

We evaluated the effects of select herbal extracts (Tinospora cordifolia [TC], Tinospora cordifolia with Piper longum [TC + PL], Withania somnifera [WS], Glycyrrhiza glabra [GG], AYUSH-64 [AY-64], and Saroglitazar [S]) on various parameters in a diet-induced obesity mouse model. After 12 weeks of oral administration of the herbal extracts in high-fat diet (HFD)-fed C57BL/6J mice, we analyzed plasma biochemical parameters, insulin resistance (IR), liver histology, and the expression of inflammatory and fibrosis markers, along with hepatic lipidome. We also used a 3D hepatic spheroid model to assess their impact on profibrotic gene expression. Among the extracts, TC + PL showed a significant reduction in IR, liver weight, TNF-α, IL4, IL10 expression, and hepatic lipid levels (saturated triglycerides, ceramides, lysophosphocholines, acylcarnitines, diglycerides, and phosphatidylinositol levels). Saroglitazar reversed changes in body weight, IR, plasma triglycerides, glucose, insulin, and various hepatic lipid species (fatty acids, phospholipids, glycerophospholipids, sphingolipids, and triglycerides). With the exception of GG, Saroglitazar, and other extracts protected against palmitic acid-induced fibrosis marker gene expression in the 3D spheroids. TC + PL and Saroglitazar also effectively prevented HFD-induced insulin resistance, inflammation, and specific harmful lipid species in the liver.

Antimicrobial resistance heterogeneity among multidrug-resistant Gram-negative pathogens: Phenotypic, genotypic, and proteomic analysis.

Microbes evolve rapidly by modifying their genomes through mutations or through the horizontal acquisition of mobile genetic elements (MGEs) linked with fitness traits such as antimicrobial resistance (AMR), virulence, and metabolic functions. We conducted a multicentric study in India and collected different clinical samples for decoding the genome sequences of bacterial pathogens associated with sepsis, urinary tract infections, and respiratory infections to understand the functional potency associated with AMR and its dynamics. Genomic analysis identified several acquired AMR genes (ARGs) that have a pathogen-specific signature. We observed that blaCTX-M-15, blaCMY-42, blaNDM-5, and aadA(2) were prevalent in Escherichia coli, and blaTEM-1B, blaOXA-232, blaNDM-1, rmtB, and rmtC were dominant in Klebsiella pneumoniae. In contrast, Pseudomonas aeruginosa and Acinetobacter baumannii harbored blaVEB, blaVIM-2, aph(3'), strA/B, blaOXA-23, aph(3') variants, and amrA, respectively. Regardless of the type of ARG, the MGEs linked with ARGs were also pathogen-specific. The sequence type of these pathogens was identified as high-risk international clones, with only a few lineages being predominant and region-specific. Whole-cell proteome analysis of extensively drug-resistant K. pneumoniae, A. baumannii, E. coli, and P. aeruginosa strains revealed differential abundances of resistance-associated proteins in the presence and absence of different classes of antibiotics. The pathogen-specific resistance signatures and differential abundance of AMR-associated proteins identified in this study should add value to AMR diagnostics and the choice of appropriate drug combinations for successful antimicrobial therapy.

Pharmacological potential of Withania somnifera (L.) Dunal and Tinospora cordifolia (Willd.) Miers on the experimental models of COVID-19, T cell differentiation, and neutrophil functions.

Cytokine release syndrome (CRS) due to severe acute respiratory coronavirus-2 (SARS-CoV-2) infection leads to life-threatening pneumonia which has been associated with coronavirus disease (COVID-19) pathologies. Centuries-old Asian traditional medicines such as Withania somnifera (L.) Dunal (WS) and Tinospora cordifolia (Willd.) Miers (TC) possess potent immunomodulatory effects and were used by the AYUSH ministry, in India during the COVID-19 pandemic. In the present study, we investigated WS and TC's anti-viral and immunomodulatory efficacy at the human equivalent doses using suitable in vitro and in vivo models. While both WS and TC showed immuno-modulatory potential, WS showed robust protection against loss in body weight, viral load, and pulmonary pathology in the hamster model of SARS-CoV2. In vitro pretreatment of mice and human neutrophils with WS and TC had no adverse effect on PMA, calcium ionophore, and TRLM-induced ROS generation, phagocytosis, bactericidal activity, and NETs formation. Interestingly, WS significantly suppressed the pro-inflammatory cytokines-induced Th1, Th2, and Th17 differentiation. We also used hACE2 transgenic mice to further investigate the efficacy of WS against acute SARS-CoV2 infection. Prophylactic treatment of WS in the hACE2 mice model showed significant protection against body weight loss, inflammation, and the lung viral load. The results obtained indicate that WS promoted the immunosuppressive environment in the hamster and hACE2 transgenic mice models and limited the worsening of the disease by reducing inflammation, suggesting that WS might be useful against other acute viral infections. The present study thus provides pre-clinical efficacy data to demonstrate a robust protective effect of WS against COVID-19 through its broader immunomodulatory activity.

Prophylactic treatment of Glycyrrhiza glabra mitigates COVID-19 pathology through inhibition of pro-inflammatory cytokines in the hamster model and NETosis.

Severe coronavirus disease (COVID-19) is accompanied by acute respiratory distress syndrome and pulmonary pathology, and is presented mostly with an inflammatory cytokine release, a dysregulated immune response, a skewed neutrophil/lymphocyte ratio, and a hypercoagulable state. Though vaccinations have proved effective in reducing the COVID-19-related mortality, the limitation of the use of vaccine against immunocompromised individuals, those with comorbidity, and emerging variants remains a concern. In the current study, we investigate for the first time the efficacy of the Glycyrrhiza glabra (GG) extract, a potent immunomodulator, against SARS-CoV-2 infection in hamsters. Prophylactic treatment with GG showed protection against loss in body weight and a 35%-40% decrease in lung viral load along with reduced lung pathology in the hamster model. Remarkably, GG reduced the mRNA expression of pro-inflammatory cytokines and plasminogen activator inhibitor-1 (PAI-1). In vitro, GG acted as a potent immunomodulator by reducing Th2 and Th17 differentiation and IL-4 and IL-17A cytokine production. In addition, GG also showed robust potential to suppress ROS, mtROS, and NET generation in a concentration-dependent manner in both human polymorphonuclear neutrophils (PMNs) and murine bone marrow-derived neutrophils (BMDNs). Taken together, we provide evidence for the protective efficacy of GG against COVID-19 and its putative mechanistic insight through its immunomodulatory properties. Our study provides the proof of concept for GG efficacy against SARS-CoV-2 using a hamster model and opens the path for further studies aimed at identifying the active ingredients of GG and its efficacy in COVID-19 clinical cases.

Strategy for the Enrichment of Protein Biomarkers from Diverse Bacterial Select Agents.

BACKGROUND: Some pathogenic bacteria can be potentially used for nefarious applications in the event of bioterrorism or biowarfare. Accurate identification of biological agent from clinical and diverse environmental matrices is of paramount importance for implementation of medical countermeasures and biothreat mitigation. OBJECTIVE: A novel methodology is reported here for the development of a novel enrichment strategy for the generally conserved abundant bacterial proteins for an accurate downstream species identification using tandem MS analysis in biothreat scenario. METHODS: Conserved regions in the common bacterial protein markers were analyzed using bioinformatic tools and stitched for a possible generic immuno-capture for an intended downstream MS/MS analysis. Phylogenetic analysis of selected proteins was carried out and synthetic constructs were generated for the expression of conserved stitched regions of 60 kDa chaperonin GroEL. Hyper-immune serum was raised against recombinant synthetic GroEL protein. RESULTS: The conserved regions of common bacterial proteins were stitched for a possible generic immuno-capture and subsequent specific identification by tandem MS using variable regions of the molecule. Phylogenetic analysis of selected proteins was carried out and synthetic constructs were generated for the expression of conserved stitched regions of GroEL. In a proof-of-concept study, hyper-immune serum raised against recombinant synthetic GroEL protein exhibited reactivity with ~60 KDa proteins from the cell lysates of three bacterial species tested. CONCLUSION: The envisaged methodology can lead to the development of a novel enrichment strategy for the abundant bacterial proteins from complex environmental matrices for the downstream species identification with increased sensitivity and substantially reduce the time-to-result.

Protein biomarker elucidation for the verification of biological agents in the taxonomic group of Gammaproteobacteria using tandem mass spectrometry.

Some pathogenic microbes can be used for nefarious applications and instigate population-based fear. In a bio-threat scenario, rapid and accurate methods to detect biological agents in a wide range of complex environmental and clinical matrices, is of paramount importance for the implementation of mitigation protocols and medical countermeasures. This study describes targeted and shot-gun tandem MS based approaches for the verification of biological agents from the environmental samples. The marker proteins and peptides were elucidated by an exhaustive literature mining, in silico analysis of prioritized proteins, and MS/MS analysis of abundant proteins from selected bacterial species. For the shot-gun methodology, tandem MS analysis of abundant peptides was carried from spiked samples. The validation experiments employing a combination of shot-gun tandem MS analysis and a targeted search reported here is a proof of concept to show the applicability of the methodology for the unambiguous verification of biological agents at sub-species level, even with limited fractionation of crude protein extracts from environmental samples.

Putative serum protein biomarkers for epsilon toxin exposure in mouse model using LC-MS/MS analysis.

Epsilon toxin (ETX), produced by Clostridium perfringens Type B or type D strains, is a potential biological and toxin warfare (BTW) agent, largely for its very high toxicity. The toxin is implicated in several animal diseases. Using LC-MS/MS analysis, we report here elucidation of putative serum maker proteins for ETX exposure with an objective of the early diagnosis of intoxication. Of 166 consensus proteins (488 peptides), showing ETX-induced alterations, 119 proteins exhibited increase and 47 proteins showed decreased abundance in serum, as revealed by SWATH (DIA) acquisition on LC-MS/MS and label free quantitative analysis of control and test samples. Complement and coagulation cascade, nitrogen metabolism, negative regulation of peptidase activity, and response to ROS were among the biological processes and pathways perturbed by the ETX exposure. Interaction network indicated enzyme inhibitor activity, detoxification of ROS, and steroid binding functions were the major interaction networks for the proteins with increased abundance, while, hemostasis and structural molecule activity were the prominent networks for the down-regulated proteins. Validation studies were carried out by immunoprecipitation, ELISA, and Western blot analysis of selected proteins to demonstrate diagnostic potential of the putative marker proteins of ETX exposure.

Elucidation of protein biomarkers for verification of selected biological warfare agents using tandem mass spectrometry.

Some pathogens and toxins have the potential to be used as weapons of mass destruction and instigate population-based fear. Efforts to mitigate biothreat require development of efficient countermeasures which in turn relies on fast and accurate methods to detect the biological agents in a range of complex matrices including environmental and clinical samples. We report here an mass spectrometry (MS) based methodology, employing both targeted and shot-gun approaches for the verification of biological agents from the environmental samples. Our shot-gun methodology relied on tandem MS analysis of abundant peptides from the spiked samples, whereas, the targeted method was based on an extensive elucidation of marker proteins and unique peptides resulting in the generation of an inclusion list of masses reflecting relevant peptides for the unambiguous identification of nine bacterial species [listed as priority agents of bioterrorism by Centre for Disease Control and Prevention (CDC)] belonging to phylogenetically diverse genera. The marker peptides were elucidated by extensive literature mining, in silico analysis, and tandem MS (MS/MS) analysis of abundant proteins of the cultivated bacterial species in our laboratory. A combination of shot-gun MS/MS analysis and the targeted search using a panel of unique peptides is likely to provide unambiguous verification of biological agents at sub-species level, even with limited fractionation of crude protein extracts from environmental samples. The comprehensive list of peptides reflected in the inclusion list, makes a valuable resource for the multiplex analysis of select biothreat agents and further development of targeted MS/MS assays.

Elucidation of protein biomarkers in plasma and urine for epsilon toxin exposure in mouse model.

Epsilon toxin (ETX) is the major virulence determinant of C. perfringens type B or type D strains, causing diseases in animals, besides being a listed biological and toxin warfare (BTW) agent. Keeping in mind the high lethality and the rapid onset of clinical manifestations, early diagnosis of epsilon toxin exposure is of paramount importance for implementation of appropriate medical countermeasures. Using a 2DE-MS approach, the present study is the first comprehensive proteomic elucidation of ETX-induced protein markers in the mouse model, providing putative targets for early diagnosis of ETX exposure. A total of 52 unique proteins showing ETX-induced modulations were identified in plasma and urine samples. Fibrinogen, apolipoprotein, serum amyloid protein, plasminogen, serum albumin, glutathione peroxidase, transferrin, major urinary protein 2, haptoglobin, transthyretin, and vitamin D-binding protein were among the proteins observed in more than one dataset with altered abundance after the ETX-intoxication. The predicted localization, function, and interaction of the ETX-modulated proteins in the plasma and urine indicated involvement of multiple pathways; extracellular proteins, followed by macromolecular complexes associated with blood coagulation and plasminogen activating cascade, being the most prominent among others. The putative markers elucidated here warrants further validation and can be of immense value for the early diagnosis of ETX exposure.