Institute of Microbial Technology

Serratiopeptidase, a proteolytic enzyme with therapeutic applications, is traditionally produced from the bacterium Serratia marcescens. Recombinant production of serratiopeptidase in Escherichia coli offers a safer alternative to the biosafety concerns of the producer. Present study involves cloning and heterologous expression of thermoactive serratiopeptidase gene from S. marcescens AD-W2 in E. coli K12 in pET28a plasmid. Optimized expression conditions i.e. 37°C, OD600 5, 5% L-rhamnose, 5mM IPTG, and 50% dissolved oxygen led to the final yield of 190mg/g serratiopeptidase (4747mg protein/L within 9h in a bioreactor. Purification and refolding of recombinant serratiopeptidase was performed in a single step using Tangential Flow Filtration (TFF) and diafiltration process. The purified recombinant serratiopeptidase exhibited specific activity of 1800 Units/mg protein, with an optimal activity at pH 9.0 and temperature 50°C. The value of kinetic constant Km was calculated as 1.38mg/mL for casein. The recombinant serratiopeptidase demonstrated comparable anti-inflammatory activity to the commercially available serratiopeptidase, inhibiting nitric oxide release and pro-inflammatory cytokine production in LPS-stimulated murine macrophage cell line RAW 264.7. The study reveals that the recombinant serratiopeptidase produced in E. coli K12 holds promising source of safe and effective anti-inflammatory agent.

Abstract In siRNA-based applications, cellular delivery remains one of the main hurdles. Many formulations are tested for the same and peptides came up as one of the optimal options. The latter have various advantages like natural biological presence, high specificity, and natural metabolism etc. siRNA in conjugation with peptides have exhibited enhanced mRNA silencing. Peptides aid siRNAs in condensation to smaller volumes, enhance nuclease protection, increase half-life, promote cell specific binding as well as endosomal escape and release in cytosol. Despite its prime importance, no resource is available for the peptide-based delivery of siRNAs, therefore to fill the gap we developed PEPsRNA web server. It includes 2266 entries of 270 different kinds of peptides, 106 different types of siRNAs and shRNAs along with more than 80 conjugate molecules targeting 55 different genes, experimentally tested for the delivery of the siRNAs. To provide the detailed insights of the procedure, we have incorporated analysis of the peptides (e.g. secondary structure, amino acid composition, polarity, hydrophobicity etc.), siRNAs (e.g. secondary structures with minimum free energies etc.) and associated conjugate molecules (e.g. structure, SMILES, Inchl). We have derived these values using various other tools and resources to make the web server comprehensive. We further compared various physicochemical properties with the efficacy of the peptide based on the target gene silencing, but these properties do not shown any distinct conclusive relationship. The data is available for browsing, searching and downloading freely on the web server with URL: http://bioinfo.imtech.res.in/manojk/pepsirna . Highlights ➣ PEPsRNA is the first database of experimentally tested peptides for siRNA delivery ➣ It comprised of 2266 entries with 270 peptides and about 80 conjugate molecules ➣ Analysis of peptides, siRNAs and details of conjugate molecules are provided ➣ Browse, search and various tools are incorporated for data retrieval and usage

Mycobacterium tuberculosis (Mtb) effectively suppresses host immunity to ensure its survival. We have earlier shown that the Acr1 protein of Mtb can inhibit the differentiation of dendritic cells (DCs). Consequently, in the current study, we examined the role of Acr1 in mitigating autoimmunity. Initially, we observed that Acr1 skews the differentiation of DCs into functionally competent myeloid-derived suppressor cells (MDSCAcr1) that chiefly secrete immunosuppressive molecules, expand regulatory T cells (TregAcr1) and attenuate inflammatory responses. Further, MDSCAcr1 suppress Th17 cells. Acr1 expanded MDSCs with a concurrent increase in myelin oligodendrocyte glycoprotein (MOG)-specific Tregs and a decline in Th17 cells in a murine experimental autoimmune encephalomyelitis (EAE) model and prevented the onset of the disease. These results were further validated in the prophylactic model of EAE. Mechanistically, Acr1 activates Tregs and MDSCs via the TLR-4 pathway, implicating innate immune recognition in Mtb-induced suppression. The results indicate a potential role of Acr1 against autoimmune diseases.

ABSTRACT Carbapenem-resistant Acinetobacter baumannii (CRAB) is a major public health threat and a key contributor to the global antimicrobial resistance (AMR) crisis. This study applied a bacterial genome-wide association study (bGWAS) framework to identify genetic factors beyond the canonical carbapenemase OXA-23, that may contribute to carbapenem resistance. A. baumannii isolates with publicly available whole-genome sequencing (WGS) data and carbapenem-specific antimicrobial susceptibility testing (AST) results were compiled (n = 1,601). Unitigs were extracted from de novo assemblies, and single-nucleotide polymorphisms (SNPs) were called from raw sequence reads mapped to seven selected reference genomes. The population was highly clonal and comprised ten lineages, including three predominantly resistant ST2 lineages. The known CRAB-associated loci , including OXA-23, the RND efflux pump regulator AdeL, and an OXA-66 substitution (Leu167Val), were readily identified. Novel resistance-associated mutations were detected in outer-membrane proteins, including porins (OprD, DcaP) and proteins (PqiB, LptD) involved in lipopolysaccharide (LPS) biogenesis. Notably, the detection of OprD and DcaP was reference-dependent, indicating that a multi-reference bGWAS framework improves identification of resistance-associated variants that may be missed in single-reference analyses. Structural mapping of the identified mutations suggested plausible effects on protein function.

Antimicrobial resistance (AMR) is a global health concern that requires innovative therapeutic strategies to improve clinical outcomes. Bacterial priority pathogens have developed multiple mechanisms for AMR. To develop therapeutics to overcome AMR, it is essential to explore, understand, and identify effective molecular targets. Iron is crucial for survival and pathogenesis in bacterial physiology. Reports indicate that iron supports vital cellular functions and modulates bacterial virulence and pathogenicity. Due to the bacterial requirement for iron during infection, antimicrobials are being developed by targeting iron homeostasis pathways in pathogens. In this review, we explore the virulence, AMR, and iron homeostatic mechanisms in bacterial pathogens, as well as the functional role of iron in regulating virulence, antimicrobial resistance, and other aspects of bacterial physiology, to elucidate potential therapeutic strategies against drug-resistant pathogens. This review briefly discusses therapeutic interventions based on iron homeostasis mechanisms and the associated translational challenges.

Endogenous intracellular allosteric modulators of GPCRs remain largely unexplored, with limited binding and phenotype data available. This gap arises from the lack of robust computational methods for unbiased cavity identification, cavity-specific ligand design, synthesis, and validation across GPCR topology. Here, we developed Gcoupler, an AI-driven generalized computational toolkit that leverages an integrative approach combining de novo ligand design, statistical methods, Graph Neural Networks, and bioactivity-based ligand prioritization for rationally predicting high-affinity ligands. Using Gcoupler, we interrogated intracellular metabolites that target and regulate the GPCR–Gα interface (Ste2p–Gpa1p), affecting pheromone-induced programmed cell death in yeast. Our computational analysis, complemented by experimental validations, including genetic screening, multi-omics, site-directed mutagenesis, biochemical assays, and physiological readouts, identified endogenous hydrophobic metabolites, notably sterols, as direct intracellular allosteric modulators of Ste2p. Molecular simulations coupled with biochemical signaling assessment in site-directed Ste2p mutants further confirmed that metabolites binding to GPCR–Gα obstruct downstream signaling, possibly via a cohesive effect. Finally, by utilizing isoproterenol-induced, GPCR-mediated human and neonatal rat cardiac hypertrophy models, we observed that elevated metabolite levels attenuate hypertrophic response, reinforcing the evolutionary relevance of this mechanism.

The online version contains supplementary material available at 10.1007/s13205-025-04621-x.