The Role of Post-Translational Modifications in Bacterial Responses to Methylglyoxal

Project Summary and Abstract Methylglyoxal (MGO) is a highly reactive, toxic molecule that is produced non-enzymatically during central metabolism by virtually all cells. Some microorganisms, including enteric bacteria such as Escherichia coli, also enzymatically produce MGO during metabolic shifts in order to mitigate phosphorylated sugar toxicity. Although its production serves to protect E. coli, MGO also directly damages cells, in part through targeted modification (glycation) of proteins. In Eukaryotes, glycation has been shown to modulate the enzymatic activity of certain proteins, which in some cases increases cellular protection from MGO-induced stress. Whether glycation serves a similar function in bacteria is not known. It has been shown, however, that E. coli protects itself from MGO via detoxification to lactate, a process that also activates a potassium (K+)/proton (H+) antiporter, leading to cytoplasmic acidification. In addition to these known mechanisms, my preliminary data suggest a role for the Nitrogen-Related Phosphotransferase System (PTSNtr) in protection from MGO exposure. The PTSNtr protein PtsN regulates activity of several K+ transporters in a phosphorylation-dependent manner. My preliminary results show that deleting ptsN confers a survival advantage during MGO exposure, while knocking out PtsO, the protein that phosphorylates PtsN, decreases survival. However, the pathway and underlying mechanism for this behavior are not known. I hypothesize that post-translational modifications (phosphorylation of the PTSNtr and protein glycation) mediate novel mechanisms of MGO protection in E. coli. Aim 1 will delineate the mechanism underlying the MGO survival advantage of a ΔptsN mutant and determine the contribution of PtsN phosphorylation to this phenotype, revealing a new role for this conserved phosphotransferase system. Aim 2 will characterize protein glycation targets and changes in protein expression in response to MGO. This will provide, for the first time, a global view of proteins glycated by MGO in bacteria, the effects of glycation on protection from MGO stress, and the bacterial regulatory response following exposure to MGO. Completion of this project will elucidate a new role of posttranslational modification – both phosphorylation of PtsN and glycation of select proteins – in E. coli survival during MGO stress. It may also reveal novel protective pathways that can be modulated to combat bacterial infection and inflammation and modulate the host microbiome.