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. This finding did not seem to be a consequence of initial in vivo survival defects of the aceA mutant, however, as Astemizole bacterial burdens of all strains tested were equivalent at 2 hours post-infection. The,50-fold decrease in virulence of an ICL-deficient strain of P. aeruginosa is consistent with data previously shown in other infection models. Contrary to the aceA mutant, however, the glcB mutant did not show any attenuation in this model, with similar bacterial recovery seen between this strain and the wild-type PAO1. To our knowledge, this is the first report of an MS-deficient strain of P. aeruginosa being tested in a pulmonary model of infection, although such mutants have been shown to be involved in virulence in an alfalfa seedling model. We have speculated that the ICL-mediated production of succinate from isocitrate is sufficient to promote in vivo survival, AIDA despite the accumulation of glyoxylate. Perhaps most interestingly, we found the double aceA glcB mutant to be severely attenuated in this model, much more so than the single aceA mutant, with no recoverable colonies at 48 hours despite equal lung burdens at 2 hours. These data suggest that no bypass mechanisms can circumvent the loss of both enzymes in the glyoxylate shunt. Taken together, these in vivo results demonstrate that glyoxylate shunt inhibitors that have the greatest potential to serve as therapeutic agents against P. aeruginosa need to inhibit both enzymes in the pathway. One potential caveat to this approach, however, is the likelihood that glyoxylate shunt inhibitors would only be efficacious in the pulmonary environment, as evidenced by the lack of virulence differentiation between wild-type PAO1 and its isogenic glyoxylate shunt mutants when mice were challenged in a septicemia model of infection.