Biology subjects

Rajendhran, J.

Publications and source records attributed to Rajendhran, J..

4 recordsLinked to original sources

bioRxiv2023

Massively parallel mutant selection identifies genetic determinants of Pseudomonas aeruginosa colonization of Drosophila melanogaster

Pseudomonas aeruginosa is recognized for its ability to colonize diverse habitats and cause disease in a variety of hosts, including plants, invertebrates, and mammals. Understanding how this bacterium is able to occupy wide-ranging niches is important for deciphering its ecology. We used transposon sequencing (Tn-Seq, also known as INSeq) to identify genes in P. aeruginosa that contribute to fitness during colonization of Drosophila melanogaster. Our results reveal a suite of critical factors, including those that contribute to polysaccharide production, DNA repair, metabolism, and respiration. Comparison of candidate genes with fitness determinants discovered in previous studies of P. aeruginosa identified several genes required for colonization and virulence determinants that are conserved across hosts and tissues. This analysis provides evidence for both the conservation of function of several genes across systems, as well as host-specific functions. These findings, which represent the first use of transposon sequencing of a gut pathogen in Drosophila, demonstrate the power of Tn-Seq in the fly model system and advance existing knowledge of intestinal pathogenesis by D. melanogaster, revealing bacterial colonization determinants that contribute to a comprehensive portrait of P. aeruginosa lifestyles across habitats. ImportanceDrosophila melanogaster is a powerful model for understanding host-pathogen interactions. Research with this system has yielded notable insights into mechanisms of host immunity and defense, many of which emerged from analysis of bacterial mutants defective for well-characterized virulence factors. These foundational studies - and advances in high-throughput sequencing of transposon mutants - support unbiased screens of bacterial mutants in the fly. To investigate mechanisms of host-pathogen interplay and exploit the tractability of this model host, we used a high-throughput, genome-wide mutant analysis to find genes that enable a pathogen, P. aeruginosa, to colonize the fly. Our analysis reveals critical mediators of P. aeruginosa establishment in its host, some of which are required across fly and mouse systems. These findings demonstrate the utility of massively parallel mutant analysis and provide a platform for aligning the fly toolkit with comprehensive bacterial genomics.

microbiology↗

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↗