Towards a functional understanding of genotypic and phenotypic diversity across the Salmonella species

Project Summary Despite the discovery of antibiotics nearly a century ago, infectious diarrheal diseases remain a global health threat. One of the most prevalent diarrheal pathogens is Salmonella enterica, which infects over 100 million people annually. This pathogen exhibits remarkable genetic diversity, with over 500,000 sequenced isolates on NCBI displaying dramatic differences in host-range and disease manifestations. For example, non-typhoidal Salmonella serovars (e.g Typhimurium, Enteriditis) cause self-limiting gastroenteritis in many hosts. In contrast, human-restricted typhoidal serovars (e.g. Typhi, Paratyphi A) trigger enteric fever, a deadly systemic disease with a mortality rate of up to 30%. While bioinformatic studies have computationally identify genetic differences encoded across various Salmonella serovars, a comprehensive, functional approach towards characterizing the genetic landscape in this pathogen has remained largely lacking. To address this gap, during my time in the Monack group I have optimized random barcoded transposon sequencing (Rb-Tn-seq) in genetically diverse Salmonella isolates, enabling me to rapidly identify fitness effects under dozens of host-associated stressors. In this proposal, I will use my established Salmonella Rb-Tn-seq pipeline to systematically understand how evolutionary pressures from both the human host and diverse bacteriophages contribute to the vast genetic diversity in this pathogen. In Aim 1, I will expand upon my Rb-Tn-seq experiments by studying two uncharacterized genes with serovar-specific fitness effects during host-associated stress: RS_03310, a putative transcription factor associated with amino acid metabolism in Typhi and Paratyphi A, and BT_120, an unstudied plasmid-encoded gene contributing to human macrophage infection in Typhimurium. In Aims 2 & 3, I will apply this pipeline to a new direction- understanding how phage predation has contributed to Salmonella genome evolution. To this end, I have received a panel of clinical Salmonella isolates and diverse phage isolated from Malawi, Africa, where these Salmonella infections are endemic. In Aim 2, I will study how isolate-specific surface features affect phage-bacterial interactions in the context of host-associated stress. In Aim 3, I will use computational tools (MGEfinder) and functional genomic experiments to identify mobile genetic elements (MGE) protecting against phage-associated stress. Using undomesticated clinical strains will greatly expand our phenotypic understanding of the Salmonella genome. Furthermore, the genes and pathways identified in this study may serve as novel therapeutic targets for Salmonella-triggered illnesses. My mentoring team includes Denise Monack, a leading expert in the Salmonella field, as well as experts in phage (Dr. Jay Hinton, Dr. Alex Gao) and MGE biology (Dr. Ami Bhatt). This training will prepare me for a career as an independent researcher systematically studying genetic diversity and evolution across the Salmonella species.