(28ac) Aspa Mediated Persistence State in Escherichia coli

Bacterial persisters are phenotypic variants that can survive high doses of antibiotics, which causes challenges in treating chronic infections. In our study, we conducted a screening of an E. coli K-12 MG1655 library with promoters fused to a fast-folding green fluorescent protein (GFP) gene and observed a downregulation in the expression level of aspA in E. coli cells in response to gentamicin (an aminoglycoside). AspA catalyzes the decomposition of L-aspartate into fumarate and ammonia, which are important in carbon and nitrogen metabolism. Deletion of aspA (ΔaspA) significantly increased the tolerance of E. coli cells to aminoglycoside antibiotics (Figure 1). By examining the effects of the reaction products (fumarate and ammonia), we discovered that the increased tolerance was due to perturbation of fumarate metabolism, and the survival rate was drastically reduced when fumarate was added to bacterial cultures. Because fumarate is crucial in energy metabolism, we tested a variety of knockout strains in which genes associated with the TCA cycle and the electron transport chain were deleted. The tolerance of these strains to gentamicin was found to be increased. Aminoglycosides inhibit ribosomes, leading to the production of truncated, toxic peptides, and proton motive force (PMF) is thought to be necessary for aminoglycoside uptake. The link between energy metabolism and protein synthesis should be the main factor that mediates the increased tolerance of the knockout strains; and we aim to characterize the molecular mechanisms underlying this link in this study. Altogether, our study, which highlights a distinct and novel aminoglycoside tolerance mechanism, further enhances our knowledge of persister cell physiology.

Figure 1. Persister levels of WT and ΔaspA strains treated with aminoglycosides (gentamicin, kanamycin, streptomycin, amikacin).