Biology subjects

Dilawari, R.

Publications and source records attributed to Dilawari, R..

3 recordsLinked to original sources

bioRxiv2025

FLIM Imaging of mCherryTYG Deciphers pH Dynamics and Lifestyles of Salmonella Typhimurium

Intracellular pH regulation is fundamental to bacterial adaptation, virulence, and survival in diverse environments. Salmonella Typhimurium, a key human pathogen, exploits host and environmental pH cues to transition between planktonic, biofilm, and virulence-associated states. However, precise tools to monitor bacterial pH dynamics at subcellular resolution have been limited. Herein, we report the development and application of mCherryTYG, a genetically encoded pH-sensitive fluorophore optimized for fluorescence lifetime imaging (FLIM), enabling robust and high-resolution pH measurements across diverse conditions. mCherryTYG demonstrated exceptional sensitivity across a broad pH range (5.5-8.5) with consistent lifetime responses, and was unaffected by temperature, buffer composition, or ionic strength. Using FLIM, we characterized the pH dynamics of Salmonella across in vitro, host, and biofilm contexts. Under acidic stress in vitro, Salmonella maintained a uniform intracellular pH ([~]6.04), providing clarity on previously debated heterogeneity. In infections of HeLa cells, Salmonella existed in distinct pH environments: acidic vacuolar pH ([~]5.89) and neutral cytoplasmic pH ([~]7.10). During the late infection stage, [~]17% of the bacterial population retained an acidic pH. Biofilm studies revealed stratified pH profiles, with acidic pH near the bottom and neutral pH at the surface, mirroring patterns observed in other pathogens. In heterologous host models, pH gradients shaped bacterial adaptation strategies. In C. elegans, Salmonella experienced a progressive pH gradient from neutral pH ([~]7.10) in the anterior lumen to acidic pH ([~]6.45) in the posterior. Similarly, in zebrafish, Salmonella encountered acidic lysosome-rich enterocytes ([~]5.84) and neutral regions ([~]7.33) in the anterior gut. This study establishes mCherryTYG-FLIM as a transformative tool for studying bacterial pH regulation, revealing pH as a critical modulator of Salmonella lifestyle transitions: virulence, and persistence. Our findings provide new insights into host-microbe interactions and present pH as a promising target for therapeutic interventions against bacterial infections.

microbiology↗

bioRxiv2024

Mycobacterium tuberculosis exploits SIRT2 for iron acquisition to facilitate its intracellular survival

Iron availability is a critical factor for both bacteria and humans, and its availability significantly influences host-pathogen dynamics. As Mtb has coevolved with the human race, Mtb relentlessly tries to exploit iron from the tightly regulated iron machinery of host. Sirtuins are evolutionary conserved NAD+-dependent deacetylases involved in various cellular processes including infection. Notably, the cytosolic protein, Sirtuin 2 regulates cellular iron homeostasis in hepatocytes and after Mtb infection, SIRT2 translocates to the nucleus leading to decreased protective immune response. However, the underlying mechanism as to how Mtb exploits SIRT2 for iron acquisition remains unknown. In the current study, we observe that the decreased bacillary load in SIRT2 inhibited or knock down cells is due to low availability of iron to the bacilli. Inhibition or knockdown of SIRT2 in Mtb infected cells displays differential modulation of iron import and export proteins suggesting ongoing tussle by host to limit the bioavailability of iron to pathogen. More specifically, by flow cytometry analysis, we show significant upregulation of cell surface Apo Tf and GAPDH in infected SIRT2 inhibited macrophages. Thus, in SIRT2 depleted state, we delineate a different mechanism of iron export occurring through Apo Tf and GAPDH during infection in contrast to the classical iron exporter Fpn1. Collectively, our findings showed the importance of SIRT2-mediated iron regulation in Mtb pathogenesis and can encourage designing of novel host-targeted therapeutics.

cell biology↗

bioRxiv2023

RpoS activates Salmonella Typhi biofilms and drives persistence in a Zebrafish model

The development of strategies for targeting the asymptomatic carriage of Salmonella Typhi in chronic typhoid patients has suffered owing to our basic lack of understanding of the molecular mechanisms that enable the formation of S. Typhi biofilms. Traditionally, studies have relied on cholesterol-attached biofilms formed by a closely related serovar, Typhimurium, to mimic multicellular Typhi communities formed on human gallstones. In long-term infections, S. Typhi adopts the biofilm lifestyle to persist in vivo and survive in the carrier state, ultimately leading to the spread of infections via the fecal-oral route of transmission. In the present work, we studied S. Typhi biofilms directly, applied targeted as well as genome-wide genetic approaches to uncover unique biofilm components that do not conform to the CsgD-dependent pathway established in S. Typhimurium. We undertook a genome-wide Tn5 mutation screen in H58, a clinically relevant multidrug resistance strain of S. Typhi, in gallstone-mimicking conditions. We generated New Generation Sequencing libraries based on the ClickSeq technology to identify the key regulators, IraP and RpoS, and the matrix components Sth fimbriae, Vi capsule and lipopolysaccharide. We discovered that the starvation sigma factor, RpoS, was required for the transcriptional activation of matrix-encoding genes in vitro, and for S. Typhi colonization in persistent infections in vivo, using a heterologous fish larval model. An rpoS null mutant failed to colonize the gall bladder in chronic zebrafish infections. Overall, our work uncovered a novel RpoS-driven, CsgD-independent paradigm for the formation of cholesterol-attached Typhi biofilms, and emphasized the role(s) of stress signaling pathways for adaptation in chronic infections. Our identification of the biofilm regulators in S. Typhi paves the way for the development of drugs against typhoid carriage, which will ultimately control the increased incidence of gall bladder cancer in typhoid carriers.

microbiology↗