Biology subjects

Karash, S.

Publications and source records attributed to Karash, S..

4 recordsLinked to original sources

bioRxiv2026

Mining the Pseudomonas aeruginosa genome for regulators of type III secretion system gene expression using FT-Tn-seq

The Pseudomonas aeruginosa type III secretion system (T3SS) is an important virulence determinant used to mute and/or impair host immune defenses. Traditional transposon mutagenesis screens have been instrumental in identifying genes required for T3SS gene expression but our understanding of how those genes orchestrate regulatory control remains incomplete. A comprehensive inventory of the genes involved in the control of T3SS gene expression would help define regulatory mechanisms and provide additional targets for anti-virulence therapeutics. Here, we designed a fluorescently tagged transposon mutagenesis screen (FT-Tn-seq) and identified over 70 new genes important for T3SS gene expression. A role for 31 of those genes was validated using a secondary screen. Several candidate genes were selected for further studies. We demonstrate that deletion of shaC, which encodes a sodium-proton antiporter subunit, results in a significant defect in T3SS gene expression. That defect results from a combined effect on exsA transcription and ExsA translation. Deletion of wspF, a cyclic-di-GMP phosphodiesterase, results in a significant reduction of T3SS expression in strain PAK, but not in PA103. The effect on the T3SS is likely due to elevated levels of c-di-GMP in PAK. Finally, we identified a new sRNA (ivy) that participates in the control of the T3SS. Expression of ivy inhibits T3SS gene expression and requires the RNA-chaperone Hfq for inhibitory activity. Findings from this study will open new research avenues to better understand regulation of T3SS gene expression in P. aeruginosa and develop T3SS-specific therapeutic interventions.

microbiology↗

bioRxiv2021

Genome-wide characterization of Salmonella Typhimurium genes required for the fitness under iron restriction

Iron is a crucial element for bacterial survival and virulence. During Salmonella infection, the host utilizes a variety of mechanisms to starve the pathogen from iron. However, Salmonella activates distinctive defense mechanisms to acquire iron and survive in iron-restricted host environments. Yet, the comprehensive set of the conditionally essential genes that underpin Salmonella survival under iron-restricted niches have not been fully explored. Here, we employed transposon sequencing (Tn-seq) method for high-resolution elucidation of the genes in Salmonella Typhimurium (S. Typhimurium) 14028s strain required for the growth under the in vitro conditions with 4 different levels of iron restriction achieved by iron chelator 2,2'-Dipyridine (Dip): mild (100 and 250), moderate (250) and severe iron restriction (400 M). We found that the fitness of the mutants reduced significantly for 28 genes, suggesting the importance of these genes for the growth under iron restriction. These genes include sufABCDSE, iron transport fepD, siderophore tonB, sigma factor E ropE, phosphate transport pstAB, and zinc exporter zntA. The siderophore gene tonB was required in mild and moderate iron-restricted conditions, but they became dispensable in severe iron-restricted conditions. Remarkably, rpoE was required in moderate and severe iron restrictions, leading to complete attenuation of the mutant under these conditions. We also identified 30 genes for which the deletion of the genes resulted in increased fitness under iron-restricted conditions. The findings broaden our knowledge of how S. Typhimurium survives in iron-deficient environments, which could be utilized for the development of new therapeutic strategies targeting the pathways vital for iron metabolism, trafficking, and scavenging.

microbiology↗

bioRxiv2017

Reactive Oxygen Species-Dependent Essential Genes in Salmonella Typhimurium

BackgroundThe molecular mechanisms underlying bacterial cell death due to stresses or bactericidal antibiotics are complex and remain puzzling. Previously, it was shown that iron is required for effective killing of bacterial cells by numerous bactericidal antibiotics. Here, our high-resolution Tn-seq analysis demonstrated that transposon mutants of S. Typhimurium with insertions in essential genes escaped immediate killing or growth inhibition under iron-restricted conditions for approximately one-third of all essential genes.\n\nResultsWe grouped essential genes into two categories, iron-dependent and iron-independent essential genes. The iron-dependency of the iron-dependent essential genes was further validated by the fact that the relative abundance of these essential gene mutants increased further with more severe iron restrictions. Our unexpected observation can be explained well by the recently proposed common killing mechanisms of bactericidal antibiotics via production of reactive oxygen species (ROS). In this model iron restriction would inhibit production of ROS, leading to reduced killing activity following blocking of an essential function. Interestingly, the targets of most antibiotics currently in use clinically, whether bacteriostatic or bactericidal, are iron-dependent essential genes.\n\nConclusionsOur result suggests that targeting iron-independent essential genes may be a better strategy for future antibiotic development, because blocking these genes would lead to immediate cell death regardless of iron concentration. On the contrary, blocking iron-dependent pathways under iron limited in vivo environment could lead to reduced killing action, which might increase drug-resistance by mutagenic action of sublethal concentrations of ROS. This work expands our knowledge on the role of iron to a broader range of essential pathways, and provides novel insights for development of more effective antibiotics.

microbiology↗

bioRxiv2017

A Comprehensive Assessment of the Genetic Determinants in Salmonella Typhimurium for Resistance to Hydrogen Peroxide

Salmonella is an intracellular pathogen that infects a wide range of hosts and can survive in macrophages. An essential mechanism uses by the macrophages to eradicate Salmonella is production of reactive oxygen species. Here, we used proteogenomics to determine the candidate genes and proteins that have a role in resistance of S. Typhimurium to H2O2. For Tn-seq, a highly saturated Tn5 insertion library was grown in vitro under either 2.5 (H2O2L) or 3.5 mM H2O2 (H2O2H). We identified two sets of overlapping genes that are required for resistance of S. Typhimurium to H2O2L and H2O2H, and the results were validated via phenotypic evaluation of 50 selected mutants. The enriched pathways for resistance to H2O2 included DNA repair, aromatic amino acid biosynthesis (aroBK), Fe-S cluster biosynthesis, iron homeostasis and a putative iron transporter system (ybbKLM), flagellar genes (fliBC), H2O2 scavenging enzymes, and DNA adenine methylase. Proteomics revealed that the majority of essential proteins, including ribosomal proteins, were downregulated upon exposure to H2O2. A subset of proteins identified by Tn-seq were analyzed by targeted proteomics, and 70% of them were upregulated upon exposure to H2O2. The identified candidate genes will deepen our understanding about mechanisms of S. Typhimurium survival in macrophages, and can be exploited to develop new antimicrobial drugs.

microbiology↗