Biology subjects

Gunasekaran, P.

Publications and source records attributed to Gunasekaran, P..

4 recordsLinked to original sources

bioRxiv2025

Surfaceome CRISPR activation screening uncovers ligands regulating tumor sensitivity to NK cell killing

Natural killer (NK) cell-based immunotherapies represent a promising avenue for cancer treatment due to their ability to eliminate cancer cells independently of antigen presentation and potential for "off-the-shelf" use. However, the molecular determinants governing tumor cell susceptibility to NK cell-mediated cytotoxicity remain incompletely understood. Here we employed CRISPR activation (CRISPRa) screening to systematically identify cancer cell surface regulators of NK cell killing across multiple cancer types. Using a comprehensive surfaceome-focused library, we screened human and murine cancer cell lines co-cultured with NK cells, identifying both known and novel ligands that modulate NK cell cytotoxicity. Our screens revealed established factors including CD43 (encoded by SPN), while uncovering previously uncharacterized regulators such as CD44, PDPN, and Siglec-1/CD169. Validation through complementary cDNA overexpression and genetic knockout approaches confirmed that disruption of CD43, CD44, PDPN, and Siglec-1 significantly altered cancer cell susceptibility to NK killing both in vitro and in humanized mouse models. Analysis of clinical datasets show that expression of identified factors correlates with patient survival outcomes in an NK-context dependent manner supporting their therapeutic relevance. Most notably, our mechanistic studies demonstrate that CD43-mediated NK cell resistance operates independently of its previously proposed interaction with Siglec-7 on NK cells. Furthermore, we find that targeting CD43 on either NK cells or engineered T cells substantially enhances their cytotoxic activity against leukemia cell lines. These results establish gain-of-function screening as a powerful approach for discovering immunoregulatory surface proteins and identify multiple promising targets for enhancing NK cell-based cancer immunotherapies.

immunology↗

bioRxiv2021

Genome-wide identification of genetic requirements of Pseudomonas aeruginosa PAO1 for rat cardiomyocyte (H9C2) infection by insertion sequencing

Pseudomonas aeruginosa is the major infectious agent among Gram-negative bacteria which causes both acute and chronic infections without any tissue specificity. Infections due to P. aeruginosa are hard to treat, as it entails various strategies like virulence factors synthesis, drug efflux systems & resistance and protein secretion systems during pathogenesis. Despite extensive research in Pseudomonas pathogenesis, novel drug targets and potential therapeutic strategies are inevitable. In this study, we investigated the genetic requirements of P.aeruginosa PAO1 for rat cardiomyocyte (H9C2) infection by insertion sequencing (INSeq). A mutant library comprising ~70,000 mutants of PAO1 was generated and the differentiated form of H9C2 cells (d-H9C2) was infected with the library. The infected d-H9C2 cells were maintained with antibiotic-protection and without any antibiotics in the growth media for 24 h. Subsequently, DNA library for INSeq was prepared, sequenced and fitness analysis was performed. A-One hundred and thirteen mutants were negatively selected in the infection condition with antibiotic-protection, whereas 143 mutants were negatively selected in antibiotic-free condition. Surprisingly, a higher number of mutants showed enriched fitness than the mutants of reduced fitness during the infection. We demonstrated that the genes associated with flagella and T3SS are important for adhesion and invasion of cardiomyocytes, while pili and proteases are conditionally essential during host cell lysis. Take away{checkmark} Fitness of P.aeruginosa mutants were analyzed during cardiomyocyte infection {checkmark}Genes involve amino acid transport & metabolism and signal transduction are important during intracellular lifestyle {checkmark}OMVs play a crucial role during infection and pathogenesis {checkmark}Flagella and T3SS are conditionally essential for adhesion and invasion, whereas pili and proteases are conditionally essential during host cell lysis

microbiology↗

bioRxiv2019

Transcriptome responses of intestinal epithelial cells induced by membrane vesicles of Listeria monocytogenes unveil novel insights into the host-pathogen cross talk

Membrane vesicles (MVs) serve as a vital source of virulence factors in many pathogenic organisms. The release of MVs by Listeria monocytogenes is only recently recognized, but its role in the pathogenesis is poorly understood. Here, we investigated the role of MVs of L. monocytogenes in virulence and host interactions. Proteomic analyses of whole cells and MVs of L. monocytogenes were performed using LC/MS/MS. A total of 1376 and 456 proteins were identified in the L. monocytogenes cells and MVs, respectively. Also, we have found that MVs contains active pore-forming listeriolysin (LLO), internalin B (inlB), phosphatidylinositol-specific phospholipase C (PI-PLC-A). We have previously reported that MVs of L. monocytogenes can infect and induce cytotoxicity in Caco-2 cells. In this study, we report the transcriptome response of Caco-2 cells upon infection with MVs as well as L. monocytogenes. In particular, we observed the up-regulation of autophagy-related genes in the early phase of infection with MVs. Transcription of inflammatory cytokines (CCL2, CXCL6, CXCL8, CXCL15, CXCL5, CXCL10) peaked at four h of infection. A large number of differentially expressed genes was associated with actin cytoskeleton rearrangement, autophagy, cell cycle arrest, and induction of oxidative stress. At a later time point, transcriptional programs generated upon infection with MVs point toward to evade innate immune signals, by modulating the expression of anti-inflammatory genes. KEGG pathway enrichment analysis revealed that MVs induce several signaling pathways such as PI3k-Akt signaling pathway, mitogen-activated protein kinase (MAPK) pathway, NOD-like receptor signaling pathway, cAMP signaling pathway, TNF, and NF-kB signaling pathway. Moreover, MVs induced the expression of cell cycle regulatory genes, which may result in the ability to prolong host cell survival, thus protecting the replicative niche for L. monocytogenes. Notably, we identified several non-coding RNAs (ncRNAs) are regulated during infection, suggesting that an early manipulation of the host gene expression may be essential for L. monocytogenes persistence and replication in host cells.

microbiology↗

bioRxiv2018

Evaluation of InSeq to Identify Genes Essential for Pseudomonas aeruginosa PGPR2 Corn Root Colonization

The reciprocal interaction between rhizosphere bacteria and their plant hosts results in a complex battery of genetic and physiological responses. In this study, we used insertion sequencing (INSeq) to reveal the genetic determinants responsible for the fitness of Pseudomonas aeruginosa PGPR2 during root colonization. We generated a random transposon mutant library of Pseudomonas aeruginosa PGPR2 comprising 39,500 unique insertions and identified genes required for growth in culture and on corn roots. A total of 108 genes were identified as contributing to the fitness of strain PGPR2 on roots. The importance in root colonization of four genes identified in the TnSeq screen was verified by constructing deletion mutants in the genes and testing them for the ability to colonize corn roots singly or in competition with the wild type. All four mutants were affected in corn root colonization, displaying 5-to 100-fold reductions in populations in single inoculations, and all were outcompeted by the wild type by almost 100-fold after seven days on corn roots in mixed inoculations of the wild type and mutant. The genes identified in the screen had homology to genes involved in amino acid catabolism, stress adaptation, detoxification, signal transduction, and transport. INSeq technology proved a successful tool to identify fitness factors in P. aeruginosa PGPR2 for root colonization.

genomics↗