A proteogenomic resource enabling integrated analysis ofListeriagenotype-proteotype-phenotype relationships

Abstract

AbstractListeria monocytogenesis an opportunistic foodborne pathogen responsible for listeriosis, a potentially fatal foodborne disease. Many differentListeriastrains and serotypes exist, but a proteogenomic resource that bridges the gap in our molecular understanding of the relationships between theListeriagenotypes and phenotypes via proteotypes is still missing. Here we devised a next-generation proteogenomics strategy that enables the community to rapidly proteotypeListeriastrains and relate this information back to the genotype. Based on sequencing andde novoassembly of the two most commonly usedListeriamodel strains, EGD-e and ScottA, we established two comprehensiveListeriaproteogenomic databases. A genome comparison established core- and strain-specific genes potentially responsible for virulence differences. Next, we established a DIA/SWATH-based proteotyping strategy, including a new and robust sample preparation workflow, that enables the reproducible, sensitive, and relative quantitative measurement ofListeriaproteotypes. This reusable and publically available DIA/SWATH library covers 70% of open reading frames ofListeriaand represents the most extensive spectral library forListeriaproteotype analysis to date. We used these two new resources to investigate theListeriaproteotype in states mimicking the upper gastrointestinal passage. Exposure ofListeriato bile salts at 37 °C, which simulates conditions encountered in the duodenum, showed significant proteotype perturbations including an increase of FlaA, the structural protein of flagella. Given thatListeriais known to lose its flagella above 30 °C, this was an unexpected finding. The formation of flagella, which might have implications on infectivity, was validated by parallel reaction monitoring and light and scanning electron microscopy.flaAtranscript levels were not significantly different with and without exposure to bile salts at 37 °C, suggesting regulation at the post-transcriptional level. Together, these analyses provide a comprehensive proteogenomic resource and toolbox for theListeriacommunity enabling the analysis ofListeriagenotype-proteotype-phenotype relationships.