Integrating genomic and Tn-Seq data to identify common in vivo fitness mechanisms across multiple bacterial species

Sepsis, a life-threatening organ dysfunction, is due to an unregulated immune response to infection. Bacteremia is a leading cause of sepsis, and members of the Enterobacterales cause nearly half of bacteremia cases annually. Although previous Tn-Seq studies identified novel bacteremia-fitness genes, evidence for common pathways across species is lacking. To identify common fitness pathways in five bacteremia-causing Enterobacterales species, we utilized our pan-genome pipeline to integrate Tn-Seq fitness data with multiple available functional data types. Core genes from species pan-genomes were used to construct a multi-species core pan-genome, producing 2,850 core gene clusters found in four of five species. Integration of Tn-Seq fitness data identified 373 protein clusters conserved in all five species and a fitness gene in at least one of them. A scoring rubric was applied to these clusters, which incorporated Tn-Seq fitness defects, operon localization, and antibiotic susceptibility data, which reduced the number of bacteremia-fitness genes and identified seven common fitness mechanisms. Independent mutational validation of one prioritized fitness gene, tatC, showed reduced fitness in vivo across all species tested and increased susceptibility to ẞ-lactams that was restored following tatC complementation in trans. By integrating known operon structures and antibiotic susceptibility with Tn-Seq fitness data, common genes within the core pan-genome emerged and revealed mechanisms essential for survival in the mammalian bloodstream. Our prediction and validation of tatC as a common bacteremia fitness factor supports the utility of this bioinformatic approach. This study represents a major step forward in prioritizing novel targets for therapy against sepsis infections.