In Vivo Dual RNA-Seq identifies a novel AB Toxin-like as a key effector of Leptospira interrogans barrier disruption (or host colonization)

The ability to evade host immune responses and disrupt epithelial barriers is essential for microbial pathogenesis, yet the underlying mechanisms of these processes in extracellular pathogens remain poorly understood. Through in vivo dual RNA-Seq profiling, we reveal how Leptospira interrogans compromises cell barrier integrity and disseminates within its host. Unlike most pathogens that trigger a rapid immune response, L. interrogans employs a stealth strategy, delaying host recognition even as it establishes tissue colonization. Through our analysis, we identify a novel family of bacterial effectors, the Virulence-Modifying (VM) proteins, as key mediators of barrier disruption. These AB toxin-like proteins, also identified in other extracellular pathogens, induce intracellular calcium influx in epithelial cells, activating calmodulin and myosin light chain kinase to disrupt tight junctions. Mutants deficient in VM proteins show a loss of virulence in animals, underscoring their essential role in pathogenicity. This study uncovers a unique strategy by which an extracellular pathogen exploits host calcium signaling to breach epithelial barriers through a novel class of AB toxin-like proteins. These findings offer new possibilities for therapeutic targeting and our understanding of microbial pathogenesis. Overall design: Here, we developed a dedicated protocol for dual RNA-Seq profiling of L. interrogans-infected hamsters, a model of human acute leptospirosis, to elucidate how this pathogen spreads and compromises cell barrier integrity. Golden Syrian hamsters were infected with 108 L. interrogans by intraperitoneal route.