Defining macrophage defense during Klebsiella pneumoniae bacteremia

Sepsis is defined as organ dysfunction resulting from dysregulated host response and is a both a leading cause of global mortality and the most expensive hospital treated condition. Gram-negative species underly about half of all bloodstream infections, thus bacteremia pathology is critical to understand. K. pneumoniae (Kp) is the second leading cause of Gram-negative bacteremia and a leading cause of health-care associated infections. The pathogenesis of Gram-negative bacteremia involves three phases: 1) initial site infection, 2) dissemination to the bloodstream, and 3) survival in blood filtering organs. Kp fitness in the lung, a common initial site of infection, has been studied but last phase of Kp-bacteremia is largely unexplored. There is a fundamental gap in our understanding of how Kp perpetuates infection in the blood and filtering organs. Macrophages have differing interactions with Kp across organs that correlates to overall Kp abundance within tissues during infection. However, the relationship between macrophages and Kp is poorly understood. The objective of this proposal is to define mechanisms of Kp restriction across distinct macrophage subsets relevant to bacteremia. The central hypothesis is that tissue-resident macrophages provide distinct niches that restrict or permit Kp in a manner dependent on bacterial initiation of cell death. I will test this hypothesis through three specific aims: 1) define splenic macrophage restriction of Kp during bacteremia, 2) define host pathways of Kp restriction by macrophages, and 3) discover Kp mechanisms of resistance to macrophage- mediated killing through initiation of cell death pathways. This work is innovative as it will harness CRISPR- genomic screening to study Kp pathogenesis and use existing transposon sequencing data to identify bacterial factors that are required for resistance to macrophage killing. Findings from these approaches will be applied to splenic macrophages, an immune cell subset rarely studied in Gram-negative disease. The research in this proposal will support sustained positive impact through identifying both host and pathogen factors required for unique tissue-specific responses across sites in bacteremia. This proposal is supported by a comprehensive training plan which will support the development of technical and conceptual skills to investigate host-pathogen interactions across tissue-resident immune cells during Gram-negative bacteremia. The skills gained with this training plan could be applied to multiple organ sites and Gram-negative pathogens. This training will occur at the University of Michigan with a team of mentors dedicated to the success of this proposal. The candidate's background in innate immunology and bacteriology will be unified by mentors with expertise in host-pathogen interactions, macrophage biology, CRISPR-genomics, bacterial genetics, and spleen biology. Together, the expertise of the mentors and the research in this proposal will facilitate independence of the candidate. This research will reveal tissue-specific interactions between macrophages and Kp during bacteremia.