Biology subjects

Chacko, F. M.

Publications and source records attributed to Chacko, F. M..

3 recordsLinked to original sources

bioRxiv2025

Acylation of the RTX Toxin MbxA stimulates host membrane disruption through a specific interaction with cholesterol

RTX toxins, characterized by their calcium-binding glycine-rich repeat regions, are known for their ability to disrupt host cell membranes. Although the acylation of specific lysine residues in these toxins is established as crucial for their hemolytic activity, the precise mechanisms underlying this enhancement remain unclear. In this study, we explore the role of acylation in the pore-forming behaviour of the RTX toxin MbxA by comparing the lytic activities of acylated MbxA and its non-acylated form, proMbxA, on in vitro membrane systems, as well as living cells. Our findings demonstrate that a cholesterol specific interaction promotes MbxA- induced membrane disruption in an acylation-dependent manner. Further analysis revealed that acylation is not necessary for initial membrane binding but markedly enhances pore formation. Overall, our results offer valuable insights into the molecular determinants that regulate RTX toxin activity, highlighting a complex interplay between lipid composition (sterols), acylation, and membrane disruption, thereby advancing our understanding of RTX toxin pathogenesis.

biochemistry↗

bioRxiv2025

A starvation-triggered AAA+ ATPase halts chromosome replication progression by disassembling the bacterial DNA sliding clamp

Living cells should coordinate vital events such as DNA replication with the availability of nutrients. For example, when cells encounter starvation, to maintain genomic integrity they should harbour robust mechanisms to stop DNA replication. Mechanisms regulating the progression of DNA replication when bacterial cells encounter starvation remain largely unclear. Herein, we identify the role of IncA -a AAA+ ATPase homologous to the prokaryotic RarA and eukaryotic WRNIP1/Mgs1- in inhibiting the progression of chromosome replication in nutrient-starved stationary phase cells of Caulobacter crescentus. We show that the starvation-induced alarmone (p)ppGpp ensures the confinement of IncA production to the stationary phase cells. At the mechanistic level, IncA directly interacts with the {beta}-sliding clamp protein DnaN and disassembles DnaN from the replisome, thereby stalling the progression of DNA replication. Furthermore, we reveal the requirement of IncAs ATPase activity for disassembling DnaN. Remarkably, we demonstrate that the IncA homolog from E. coli is capable of inhibiting DNA replication in Caulobacter. We propose that IncA homologs serve a stress-dependent role in inhibiting DNA replication across diverse domains of life.

microbiology↗

bioRxiv2022

Heterologously secreted MbxA from Moraxella bovis induces a membrane blebbing response of the human host cell

Many proteins of the Repeats in Toxins (RTX) protein family are toxins of Gram-negative pathogens including hemolysin A (HlyA) of uropathogenic E. coli. RTX proteins are secreted via Type I secretion systems (T1SS) and adopt their native conformation in the Ca2+-rich extracellular environment. Here we employed the E. coli HlyA T1SS as a heterologous surrogate system for the RTX toxin MbxA from the bovine pathogen Moraxella bovis. In E. coli the HlyA system successfully activates the heterologous MbxA substrate by acylation and secretes the precursor proMbxA and active MbxA allowing purification of both species in quantities sufficient for a variety of investigations. The activating E. coli acyltransferase HlyC recognizes the acylation sites in MbxA, but unexpectedly in a different acylation pattern as for its endogenous substrate HlyA. HlyC-activated MbxA shows host species-independent activity including a so-far unknown toxicity against human lymphocytes and epithelial cells. Using live- cell imaging, we show an immediate MbxA-mediated permeabilization and a rapidly developing blebbing of the plasma membrane in epithelial cells, which is associated with immediate cell death.

biochemistry↗