Vengadesan Krishnan

Inflammatory bowel disease (IBD), comprising Crohn’s disease and Ulcerative colitis, is a group of multifactorial illnesses with persistent gastrointestinal inflammation and a series of undesirable consequences. IBD patients have a three times higher risk of developing colitis-associated colorectal cancer (CAC). A higher mutational burden due to persistent inflammation acts as a driver of dysplasia and even tumorigenesis. While the pathological sequence is known, the molecular mechanisms underlying the transition from chronic inflammation to CAC remain largely elusive. Post-translational modification- SUMOylation plays an integral role in shaping gut inflammation as well as several forms of cancers, including sporadic colorectal cancer. In this study, the contribution of SUMOylation to CAC pathogenesis was characterized. In the AOM-DSS CAC mice model and human IBD patient specimens, SENP5, but not other deSUMOylases, shows an altered expression. Notably, both SENP5 expression dynamics and alterations in the SUMOylome occur in chronically inflamed and neoplastic colon tissues. SENP5 interactome analysis identified Coronin1A (Coro1A), an actin-binding protein predominantly expressed in immune cells. Coro1A also shows a state-specific, distinct expression pattern in the colon. Interestingly, in contrast to wild-type mice, Coro 1A knockout mice were resistant to polyp formation, with reduced cell proliferation, oncogene expression, and epithelial-to-mesenchymal transition (EMT) gene activation, and reduced extracellular matrix (ECM) development and fibrosis, suggesting its integral role in tumorigenesis. WT mice with chronically inflamed colons and polyps have a higher number of M2-like macrophages, with increased abundance of Coro1A, suggesting a role of Coro1A in modulating the tissue microenvironment toward a pro-tumorigenic state. Mechanistically, Coro1A physically interacts with TGF-β RI and regulates TGF-β-TGF-β RI signalling endosome stability, thereby controlling TGF-β-mediated macrophage polarization. Detailed in vitro experiments revealed stabilization of Coro 1A through its interaction with SUMOylated Raftlin protein. Overall, Coro 1A is necessary and sufficient for TGF-β signalling, macrophage polarization, and tumorigenesis in CAC.

Dental plaque is a complex microbial biofilm that contributes significantly to the onset of oral diseases and related infections. The initial attachment of primary colonizers to the salivary pellicle, which coats the tooth surface, is a critical first step in plaque development. Primary colonizers use pili on their cell surfaces to facilitate attachment to host surfaces and other bacteria. Pili‐mediated adhesion and interbacterial coaggregation promote oral biofilm growth. Recent structural studies of primary colonizers and other oral bacteria have provided new insights into the role of pili in dental plaque formation. Understanding the molecular intricacies of pilus assembly and adhesion paves the way for the development of antiadhesive approaches to control dental plaque formation. Disrupting initial bacterial attachment by targeting pili‐ and pili‐mediated interactions may offer a promising strategy to prevent oral biofilm development and associated infections. This review provides updates on pili and pilus components in oral bacteria, with a focus on the primary colonizers of dental plaque from a structural perspective.

Tuberculosis eradication efforts are severely hampered by antimicrobial resistance and bacterial persistence. We previously reported that the VapBC12 toxin–antitoxin (TA) system in Mycobacterium tuberculosis ( M. tuberculosis ), regulates disease progression and antibiotic persistence. In this study, we investigated the molecular mechanisms underlying VapC12-dependent modulation of bacterial growth and antibiotic susceptibility. We identified a C holesterol R ecognition A mino acid C onsensus (CRAC) motif in the VapC12 toxin that is critical for cholesterol binding and interaction with its cognate antitoxin, VapB12. Heterologous expression of VapC12 in Mycobacterium smegmatis induced toxicity, providing a model to investigate its functional activity. We determined the crystal structure of VapB12 at 1.6 Å resolution and used molecular modelling to predict the structure of the VapBC12 complex. Structure–function analyses identified specific residues critical for complex assembly and toxin activity. Mechanistically, our findings suggest that cholesterol activates VapC12 by competitively displacing VapB12 from the CRAC motif, thereby restricting M. tuberculosis growth. Finally, we demonstrated that pharmacological inhibition of VapC12 alleviates cholesterol-mediated growth restriction and enhances antibiotic activity. Collectively, these findings establish a mechanistic link between cholesterol sensing, toxin–antitoxin regulation, and antibiotic susceptibility in M. tuberculosis , highlighting VapC12 as a potential target for therapeutic strategies to combat tuberculosis persistence. Significance Statement: The rise in MDR and XDR tuberculosis cases poses a critical challenge for disease eradication programs worldwide. This therapeutic failure is driven by the ability of M. tuberculosis to generate metabolically dormant “antibiotic persister” cells that are refractory to anti-TB drugs. We previously established that the utilization of host-cholesterol promotes persister formation during infection, a phenotype dependent on the VapC12 ribonuclease toxin. In this study, we decipher the mechanism by which the VapC12 ribonuclease toxin regulates cholesterol-dependent growth restriction and antibiotic tolerance in the host environment. Importantly, we demonstrate that targeting VapC12 as an adjunct therapy significantly enhances the efficacy of anti-tuberculosis drugs by alleviating cholesterol-mediated growth arrest and reducing phenotypic drug tolerance in M. tuberculosis.

O-acetylation is a crucial substitution found in hemicelluloses and pectin, which are necessary for maintaining the flexibility and structural integrity of the cell. Cell wall acetyl transferases and esterases maintain balanced polysaccharide O-acetylation levels; however, the role of esterases in cell wall polysaccharide O-acetylation metabolism is not well explored. Therefore, we investigated the role of the Arabidopsis (Arabidopsis thaliana) GDSL Esterase/Lipase Protein (GELP) family member AtGELP53. Here, we show that AtGELP53 is localized in the plasma membrane. Analysis of AtGELP53-overexpressing independent transgenic lines revealed a decrease in xyloglucan acetylation, changes in acetylation of other polysaccharides, and alterations in cell wall composition. Molecular docking and in vitro protein activity assays showed that AtGELP53 might deacetylate xyloglucan and xylan. Elicitor-based, transcriptomic, and proteomic analyses in AtGELP53-overexpressing lines suggested that oligosaccharide-mediated signaling activates the cell wall and defense-related genes primarily through xyloglucan deacetylation. Furthermore, AtGELP53-overexpressing plants showed resistance against Pseudomonas syringae and Ralstonia solanacearum through activation of elicitor-mediated defense responses. Overall, our findings outline the role of AtGELP53 in polysaccharide acetylation, cell wall remodeling, and defense through oligosaccharide signaling.

Enterococcus faecalis is an opportunistic pathogen that causes various clinically significant infections, including infective endocarditis and catheter‐associated urinary tract infections. E. faecalis assembles hair‐like appendages termed endocarditis and biofilm‐associated pili (Ebp), crucial for adherence, colonization, biofilm formation, and virulence. The Ebp pilus comprises three pilin subunits (EbpA, EbpB, and EbpC) encoded by the ebpABC pilus operon. EbpC forms the Ebp pilus backbone decorated with EbpA and EbpB at the tip and base for adhesion and anchoring, respectively. Experimental structures are not yet available for any of the Ebp pilins. Herein, we report the crystal structures of EbpC, EbpB, and their homologs from E. faecium . The structures of EbpC and EbpB reveal four and three linearly arranged immunoglobulin‐like domains, respectively. The basal pilin EbpB structure fully mimics the features of backbone pilins with a typical pilin motif and CnaB–CnaA–CnaB fold, each with an intact isopeptide bond. The rigid C‐terminal fragment of the backbone pilin EbpC, containing three domains, exhibits a CnaB–CnaA–CnaB fold, each with an intradomain isopeptide bond. The flexible N‐terminal domain with the CnaB fold in EbpC lacks an isopeptide bond but appears to form in a full‐length structure that shows a ‘beads on a string’ architecture, previously unobserved for four‐domain backbone pilins. Structural analysis helped us to identify residues of internal isopeptide bonds and pilin motifs harboring lysine residues responsible for intermolecular covalent links during sortase‐mediated pilus assembly and propose a structural model for the Ebp pilus. This knowledge may be useful for developing structure‐based approaches targeting pilins and pili in enterococcal infections.

O -acetylation is a crucial substitution found in hemicelluloses and pectin which are necessary for maintaining the flexibility and structural integrity of the cell. A balanced polysaccharide O -acetylation level is maintained by cell wall acetyl transferases and esterases present in different cell organelles. Specifically, the role of esterases in cell wall acetylation metabolism is less explored. Therefore, we investigated the role of At GELP53 from the GELP family of esterases or lipases. Here, we show that At GELP53 is localized in the plasma membrane. Analysis of At GELP53 overexpressing independent transgenic lines revealed a decrease in xyloglucan acetylation, changes in other polysaccharide acetylation and alteration in cell wall composition. The series of elicitor-based, transcriptomic and proteomic analyses in At GELP53 overexpressing lines suggested that oligosaccharide-mediated signalling activates the cell wall and defence-related genes primarily because of xyloglucan deacetylation. Furthermore, At GELP53 overexpression plants showed resistance against Pseudomonas syringae and Ralstonia solanacearum through activation of elicitor-mediated defense response. Overall, our findings outline the role of At GELP53 in polysaccharide acetylation, cell wall remodelling and defense through the activation of plant cell wall integrity.

Sortase-dependent pili are long surface appendages that mediate attachment, colonization and biofilm formation in certain genera and species of Gram-positive bacteria. Ligilactobacillus ruminis is an autochthonous gut commensal that relies on sortase-dependent LrpCBA pili for host adherence and persistence. X-ray crystal structure snapshots of the backbone pilin LrpA were captured in two atypical bent conformations leading to a zigzag morphology in the LrpCBA pilus structure. Small-angle X-ray scattering and structural analysis revealed that LrpA also adopts the typical linear conformation, resulting in an elongated pilus morphology. Various conformational analyses and biophysical experiments helped to demonstrate that a hinge region located at the end of the flexible N-terminal domain of LrpA facilitates a new closure-and-twist motion for assembling dynamic pili during the assembly process and host attachment. Further, the incongruent combination of flexible domain-driven conformational dynamics and rigid isopeptide bond-driven stability observed in the LrpCBA pilus might also extend to the sortase-dependent pili of other bacteria colonizing a host.

Prostate cancer, the second most prevalent malignancy among men, poses a significant threat to affected patients’ well-being due to its poor prognosis. Novel biomarkers are required to enhance clinical outcomes and tailor personalized treatments. Herein, we describe our research to explore the prognostic value of long non-coding RNAs (lncRNAs) deregulated by copy number variations (CNVs) in prostate cancer. The study employed an integrative multi-omics data analysis of the prostate cancer transcriptomic, CNV and methylation datasets to identify prognosis-related subtypes. Subtype-specific expression profiles of protein-coding genes (PCGs) and lncRNAs were determined. We analysed CNV patterns of lncRNAs across the genome to identify subtype-specific lncRNAs with CNV changes. LncRNAs exhibiting significant amplification or deletion and a positive correlation were designated CNV-deregulated lncRNAs. A prognostic risk score model was subsequently developed using these CNV-driven lncRNAs. Six molecular subtypes of prostate cancer were identified, demonstrating significant differences in prognosis (P = 0.034). The CNV profiles of subtype-specific lncRNAs were examined, revealing their correlation with CNV amplification or deletion. Six lncRNAs (CCAT2, LINC01593, LINC00276, GACAT2, LINC00457, LINC01343) were selected based on significant CNV amplifications or deletions using a rigorous univariate Cox proportional risk regression model. A robust risk score model was developed, stratifying patients into high-risk and low-risk categories. Notably, our prognostic model based on these six lncRNAs exhibited exceptional predictive capabilities for recurrence-free survival (RFS) in prostate cancer patients (P = 0.024). Our study successfully identified a prognostic risk score model comprising six CNV-driven lncRNAs that could potentially be prognostic biomarkers for prostate cancer. These lncRNA signatures are closely associated with RFS, providing promising prospects for improved patient prognostication and personalized therapeutic strategies for novel prostate cancer treatment.

Editorial: Functional insights into the probiotic mechanisms of surface protein action

Surface proteins and pili (or pilus) anchored on the Gram-positive bacterial cell wall play a vital role in adhesion, colonization, biofilm formation, and immunomodulation. The pilus consists of building blocks called pilins or pilus subunits. The surface proteins and pilins share some common sequences and structural features. They contain an N-terminal signal sequence and the C-terminal cell wall sorting region, enabling their transportation across the membrane and covalent attachment to the bacterial cell wall, respectively. The transpeptidase enzymes called sortases facilitate the covalent links between the pilins during the pilus assembly and between surface proteins or basal subunits of pili and peptidoglycan-bridge during the cell wall anchoring. Thus, elucidating three-dimensional structures for the surface proteins and pilins at the atomic level is essential for understanding the mechanism of adhesion, pilus assembly, and host interaction. This chapter aims to provide a general protocol for crystal structure determination of surface proteins and pilins anchored on the Gram-positive bacterial cell wall and substrates for sortases. The protocol involves the production of recombinant protein, crystallization, and structure determination by X-ray crystallography technique. Key words Pili Pilins Sortase Surface proteins Purification Crystallization X-ray crystallography