Project Summary Klebsiella pneumoniae is one of the most common nosocomial pathogens and is typically associated with urinary tract infections (UTIs), pneumonia, and septicemia. Two lineages of K. pneumoniae have emerged from classical strains that challenge the successful treatment of these infections. One lineage includes hypervirulent strains causing invasive community-acquired infections and the other encompasses carbapenem-resistant (CR) isolates, primarily causing multi-drug resistant UTIs; the transfer of CR to hypervirulent strains is a real and imminent threat as evidenced by the emergence of such strains in Asia. Infections caused by either lineage have greater morbidity and mortality. Hypervirulent strains often exhibit a hypermucoviscous (hmv) phenotype evident when bacterial colonies are pulled off a surface form a long, adherent string. Typically, hmv K. pneumoniae expresses K1 or K2 capsule type and if capsule is ablated the strain becomes non-mucoid and avirulent. The supposition that capsule and hypermucoidy are inextricably linked to hypervirulence is tenuous as UTI isolates are often non-mucoid, encapsulated and infectious; moreover, some reports have identified strains that are encapsulated but avirulent, non-encapsulated but hypermucoid, or hypervirulent but non-mucoid. These observations identify a major gap in our understanding of the molecular mechanisms that control hypermucoviscosity and how it influences K. pneumoniae pathogenesis. Preliminary data suggests that hmv strains phenotypically switch to non-mucoid when grown in human urine, without loss of capsule. We have capitalized on this groundbreaking phenotype to identify genes that, when disrupted, restore hmv in urine or suppress hmv under standard culture conditions. This proposal will use these mutants as a tool to identify capsule-independent mechanisms that control mucoidy and delineate how hmv distinctly influences K. pneumoniae uropathogenesis versus invasive disease. We hypothesize that capsule and mucoidy are linked, but non-synonymous, and that each distinctly influences K. pneumoniae via different infectious routes. Experiments proposed in Aim 1 will investigate the roles of capsule and mucoidy during primary pneumonia and dissemination, identifying capsule-dependent and -independent genes that control mucoidy and their individual contribution to invasive infection. Studies in Aim 2 will evaluate the functional role of mucoidy and capsule during UTI using a model hmv K. pneumoniae strain and two clinical UTI isolates. The successful execution of this proposal will deepen our understanding of the cellular factors that drive mucoidy and capsule, and dissect their contribution to invasive infections versus UTIs. The identification of these cellular factors may provide novel targets for therapeutics and diagnostics, improving our ability to diagnose and treat the most challenging K. pneumoniae infections.