Targeting essential proteins in Acinetobacter baumannii: discovery of a lipid metabolism regulator critical for invasive disease

Acinetobacter baumannii is one of the most notorious multi-drug resistant bacteria, classified by both the CDC and WHO as an urgent threat requiring new antibiotics. This hospital-acquired pathogen causes a range of severe infections, including wound and bloodstream infections, pneumonia, and sepsis. It has developed resistance to virtually all antibiotics, leaving few treatment options available. Research is necessary to uncover novel targets and strategies for improving antimicrobial therapy against A. baumannii. My thesis work has addressed the above problems by identifying unappreciated essential genes needed by A. baumannii bacteria for viability. The essential products of such genes could provide leads for expanded antimicrobial targets in the pathogen. To identify and characterize essential genes, I applied two genetic methods, transposon insertion sequencing (Tn-seq) and CRISPR interference (CRISPRi). Using Tn-seq, I analyzed saturated mutant libraries in A. baumannii strain 17978 made with two transposon systems, which yielded 372 essential genes. I developed the CRISPRi platform, which enables highly programmable and inducible gene knockdown in A. baumannii, as a tool to study the essential genes. Using CRISPRi, I confirmed the essentiality of several factors that distinguish A. baumannii, including a unique cell division protein (AdvA); a novel AraC-family transcription factor (ACX60_RS03245); and late steps of the capsular polysaccharide biosynthesis pathway, which are conditionally toxic in the absence of mutations early in the pathway or that activate a stress-response system. I further studied ACX60_RS03245, given the unusual finding of an AraC-family protein being essential for growth in bacteriological medium. I comprehensively characterized the genome-wide transcriptional and DNA-binding profiles of the regulator using RNA-seq and ChIP-seq, respectively. The results revealed that ACX60_RS03245 is a global, direct controller of genes involved in fatty acid metabolism and related pathways. During log phase growth in rich medium lacking abundant fatty acids, ACX60_RS03245 activates genes in fatty acid, biotin, and lipooligosaccharide (LOS) biosynthesis, and it represses the glyoxylate pathway. ACX60_RS03245-deficient cells showed a number of phenotypes consistent with defects in de novo fatty acid synthesis, including altered LOS production, smaller cell size, and hypersensitivity to antibiotic inhibition of fatty acid synthesis as well as to agents that are normally antagonized by an intact outer membrane. The essentiality of the protein for growth as well as most of the above phenotypes could be reversed by addition of abundant exogenous fatty acids. Additionally, I showed that exogenous fatty acids signal to ACX60_RS03245, likely through fatty acyl-coA intermediates, to block its activation of de novo fatty acid biosynthesis and derepress the glyoxylate shunt. The protein thus provides A. baumannii with a new strategy to regulate lipid metabolic pathways in response to fatty acid availability in the environment, and we have renamed it AlmC (Acinetobacter Lipid Metabolism Control). Underlining the importance of AlmC for pathogenesis and viability in the host, mutants lacking the protein are unable to cause lethality in mouse sepsis and pneumonia models and show lower bacterial burdens, particularly at invasive sites of infection (bloodstream and spleen). Overall, my research provides new insights into essential targets in A. baumannii. including the discovery and characterization of a novel fatty acid metabolism regulatory strategy. The new control mechanisms have the potential to inform the development of improved therapies targeting fatty acid biosynthesis for combating infections with the pathogen. --Author's abstract