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  • Harris Baker posted an update 3 days, 16 hours ago

    Another approach to targeting virulence is to identify bacterial pathways that are required for cells to survive the harsh, nutrient-limited environment to which they must adapt in an infection, and this is a research area that has recently been explored in A. baumannii. Certainly the physiological state of pathogens is dramatically different when they are residing within a host relative to when they are growing in vitro in a nutrient-replete medium, therefore it is critical to identify screening strategies that more closely mimic the environment these bacteria encounter during an infection. In the case of pathogens that colonize and persist in the pulmonary environment, such as P. aeruginosa, the bacteria are presented with a unique set of physiological challenges that they must tolerate in order to survive. Among these challenges is the metabolic requirement to utilize phosphatidylcholine, which is the major component of lung surfactant and serves as the primary source of carbon in the lung. As lung surfactant is required for pulmonary function, PC is always present in the lung and is available to whatever pathogens choose to inhabit that organ. In the case of P. aeruginosa, it has been shown that this versatile Gram-negative pathogen preferentially migrates towards this phospholipid, having all of the tools necessary to disassemble and transport PC into the cell. After bacterial lipases liberate the long-chain fatty acids from PC, fatty acid transporters in the outer membrane are responsible for importing these lipids into the cell. Once in the cytoplasm, these long chain fatty acids are iteratively reduced by b-oxidation, providing a free acetyl-CoA from each round of this cycle. These two carbon compounds are then shuttled into the TCA cycle, where they are then processed by the bypass pathway known as the glyoxylate shunt. The glyoxylate shunt is composed only of two enzymes, isocitrate lyase and malate synthase, and is fairly well conserved amongst several bacterial species. Importantly, no human ortholog of this pathway has been identified, potentially making it a suitable target for antibacterial therapy. The existence of this TCA bypass is to conserve carbons that are normally expended, in the form of CO2, during the normal progression of the TCA cycle. These conserved carbons serve as substrates for gluconeogenesis, where they are ultimately incorporated into new molecules of glucose. In an environment where carbon is not available in an ample supply, the conservation of metabolized carbon is crucial for cell survival, hence the existence and function of the glyoxylate shunt. Because of the essential nature of this pathway when lipids are provided as the sole carbon source, we sought to exploit this phenotype by screening compounds for antibacterial activity in a defined, minimal medium. Our efforts demonstrate that alternative screening approaches can identify new lead material from existing compound collections that have previously shown no antibacterial activity when screened under traditional conditions. The idea of targeting the glyoxylate shunt for antimicrobial therapy has been described previously. While the most significant consideration and progress in identifying such an inhibitor has been conducted with M. tuberculosis, the orthologous pathway in P. aeruginosa has also been implicated numerous times in various virulence and in vivo survival models. Interestingly, these have included both mammalian and non-mammalian hosts. To confirm these previous findings, we first constructed deletion mutants of the individual ICL- and MSencoding genes, as well as a double knockout to see if any additive effects may exist. As expected, all three mutants were completely unable to grow on M9 Acetate, whereas no growth defects were witnessed, relative to the PAO1 parent strain, when either glucose or succinate was provided as the sole carbon source. Each of these mutants, along with the wild-type strain, was then used to infect mice in both septicemia and pulmonary models of infection. Given that the glyoxylate shunt has been shown to be essential for survival in environments where lipids are the primary source of carbon, it was not surprising that no differences in virulence were seen when these strains were tested in a septicemia model of infection. When the virulence potential of these strains was tested in a pulmonary model of infection, however, we discovered that recovery of the single aceA mutant was significantly reduced, relative to wild-type, at 48 hours post-infection.