Genetic Vulnerabilities Exposed by Intrinsic Beta-Lactamase Overexpression in Acinteobacter Baumannii

ABSTRACT Multidrug resistant (MDR) Acinetobacter baumannii is a significant global health problem responsible for approximately 2 million infections and 450,000 deaths annually worldwide. In fact, A. baumannii's most severe infections, ventilator-associated pneumonia and septicemia, result in morbidity and mortality rates of 30- 50% with few, if any, therapeutic treatment options available. This burden has caused the Centers for Disease Control (CDC) and the World Health Organization (WHO) to designate MDR A. baumannii as critical priority pathogen for new antimicrobial development. In response, researchers are pushing for a deeper understanding of the mechanisms responsible for cellular adaptation, antibiotic resistance and pathogenesis. One major antibiotic resistance mechanism is the expression of β-lactamases. In fact, stable overexpression of at least one β-lactamase in A. baumannii is reported in 70-98.5% of modern clinical isolates. One of these β-lactamases, AmpC, is universally present in the chromosome of A. baumannii suggesting it may have yet-to-be-identified cellular functions beyond antibiotic resistance. However, the cellular impact associated with β-lactamase overexpression is not well understood and often conflicting results are reported across different bacterial species. Some studies have implied links between antibiotic resistance, energetic cost and bacterial fitness, whereas other publications provide evidence that β-lactamases may maintain residual peptidoglycan crosslinking activity and contribute to cell shape maintenance. Based on current knowledge in other bacteria, I hypothesize that overexpression of AmpC β-lactamase in A. baumannii results in collateral changes that create new cellular vulnerabilities. These vulnerabilities will result in certain genes now becoming essential in AmpC overexpressing A. baumannii, but not in wild-type bacteria. Therefore, the identified conditionally essential genes may represent novel antimicrobial targets which we can exploit for finding new antimicrobial compounds and will limit off-target effects on the host microbiome. Additionally, AmpC is highly conserved in Gram-negative Enterobacteriaceae suggesting that results from this proposal may reveal broad-spectrum antimicrobial targets and putative housekeeping functions for other MDR pathogens, such as Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae. To test this hypothesis, I propose to: (i) identify conditional essential genes in response to AmpC β- lactamase overexpression in A. baumannii cells in vitro in human serum and in vivo in a murine pneumonia model to identify conserved conditional essential genes and genes unique to the host infection required for AmpC overexpression adaptation and (ii) identify small molecule inhibitors of gene products that are conditionally essential in AmpC overexpressing A. baumannii using both whole cell and CRISPRi targeted methods to find compounds that inhibit AmpC overexpressing A. baumannii but not wild-type A. baumannii. The overarching goals of this CDA-2 proposal are to: (i) illuminate the consequences of AmpC β- lactamase overexpression on A. baumannii physiology and viability in physiological relevant conditions, (ii) strengthen my professional and career development skills to enhance my standing in the scientific community, and (iii) provide the foundation for independent merit review and extramural funding applications. Achievement of these goals will fortify my career goal of becoming an independent investigator with joint appointments as a VA Research Scientist and a Research Assistant Professor at the Emory University School of Medicine. Moreover, the scope of this project has been designed with significant input from my mentor and scientific advisory committee to combine my current skills with the unique expertise and resources at the Atlanta VAMC as well as Emory University School of Medicine to advance my path toward becoming an independent researcher.