# Salmonella Typhi serine threonine kinase T4519 induces lysosomal membrane permeabilization by manipulating Toll-like receptor 2-Cystatin B-Cathepsin B-NF-κB-reactive oxygen species pathway and promotes survival within human macrophages

## TL;DR

Salmonella Typhi uses a kinase called T4519 to manipulate host cell pathways, causing lysosomal damage and helping the bacteria survive inside human macrophages.

## Contribution

The study reveals a novel mechanism by which T4519 exploits the host's TLR2-cystatin B-cathepsin B-NF-κB-ROS pathway to promote intracellular survival of S. Typhi.

## Key findings

- T4519 interacts with TLR2 and represses cystatin B, leading to cathepsin B activation.
- Active cathepsin B cleaves IKB-α, enabling NF-κB nuclear translocation and ROS production.
- ROS-induced lysosomal membrane permeabilization supports S. Typhi survival within macrophages.

## Abstract

Intracellular pathogens of Salmonella spp. survive and replicate within the phagosomes, called Salmonella-containing vacuoles (SCVs) inside macrophages by manipulating phagosomal maturation and phagolysosome formation. While controversies exist about the phagosomal traffic of Salmonella Typhimurium, little studies were carried out with the intracellular survival mechanisms of Salmonella Typhi (S. Typhi). We had previously reported that a eukaryote-like serine/threonine kinase of S. Typhi (T4519) contributes to survival within macrophages and activates host pro-inflammatory signaling pathways regulated by NF-κB. However, neither the mechanisms underlying NF-κB activation nor how it contributes to intracellular survival of S. Typhi were studied. Here we show, by using antibody-mediated blocking and gene knockdown studies that T4519 activates Toll-like receptor 2 (TLR2) signals in the human monocyte-derived macrophages. We computationally predicted the NH2-terminal glycine rich repeat domain of T4519 as the TLR2-binding moiety and confirmed the interaction by co-immunoprecipitation experiment. TLR2-T4519 interaction transcriptionally repressed cystatin B, a cathepsin B inhibitor, leading to the activation of cytosolic cathepsin B, leaked from the lysosomes of the infected cells. Through a series of RT-qPCR, western blotting, gene knockdown, flow cytometry and confocal microscopy experiments, we have shown that active cytosolic cathepsin B cleaves IKB-α, resulting in nuclear translocation of NF-κB and transactivation of its target genes, including reactive oxygen species (ROS), which in turn induces lysosomal membrane permeabilization (LMP). TLR2-dependent targeting of the cystatin B-cathepsin B-NF-κB-ROS pathways by T4519, leading to LMP promotes phagosomal survival of S. Typhi. This study describes a unique mechanism of the exploitation of host NF-κB signaling pathways by bacterial pathogens to promote its own persistence within macrophage cells.

Mechanisms behind the persistence of Salmonella Typhi, a facultative, intracellular pathogen within human macrophages remain unknown. The type three secretion system 2 effectors, which play critical roles in macrophage survival of Salmonella Typhimurium are redundant for S. Typhi. We show that T4519, a eukaryote-like serine threonine kinase promotes survival and growth of S. Typhi within human primary macrophages. T4519 binds to the Toll-like receptor 2 expressed by macrophages and represses the expression of cystatin B, leading to activation of cathepsin B. The resultant active cytosolic cathpsin B in S. Typhi-infected macrophages cleaves inhibitor-κB and translocates NF-κB into the nucleus, where it induces reactive oxygen species (ROS) generation. ROS in turn causes lysosomal damage by membrane permeabilization, promoting S. Typhi survival. This is a unique mechanism by which a pathogenic bacterium exploits the protective NF-κB signaling pathways of the host to augment its survival within human macrophages.