PROJECT SUMMARY Bloodstream infections (BSI) represent a major public health burden and are associated with high rates of mortality. These infections are especially problematic for individuals in healthcare settings where risk factors for infection are increased and antibiotic resistant organisms are frequently encountered. The Gram-negative bacterial pathogen Serratia marcescens is among the ten most common causes of all bloodstream infections, but the virulence factors that drive S. marcescens infection are largely uncharacterized. We have recently determined that fitness of S. marcescens in the mammalian bloodstream is dependent on the production of a polysaccharide capsule. Survival of S. marcescens in a murine bacteremia model is capsule-dependent as is resistance to the bactericidal activity of human serum. Despite the importance of S. marcescens capsule, a comprehensive genetic assessment of capsule production has not been performed for this organism. The majority of genes responsible for capsule production are clustered in a single chromosomal locus that includes a mixture of conserved genes, encoding functions such as polysaccharide transport, as well as accessory genes that are type-specific. Despite the substantial species-level variation within the capsule biosynthetic locus, we have determined that there is a high prevalence of two specific capsule types among S. marcescens bacteremia isolates. Furthermore, BSI-associated capsule types harbor accessory capsule genes that are absent from other isolates. The overarching goal of this proposal is to define the genetic variability of the capsule locus for S. marcescens strains isolated from patients with BSI and determine the role of variable capsule genes during infection. This investigation will focus on two specific aims: 1) Define the genetic variability of the S. marcescens capsule biosynthesis locus and identify capsule types associated with BSI. 2) Determine the contribution of variable BSI-associated capsule genes to S. marcescens virulence. Upon completion of these aims, we will have isolated and sequenced S. marcescens strains originating from BSI, determined the polysaccharide structure of BSI-associated capsule types, and determined the contribution of variable capsule accessory genes to bloodstream infection. This work will have a substantial impact on the current state of knowledge regarding S. marcescens pathogenesis and has the potential to inform future anti-capsule-based therapies to combat BSI.