Deciphering Metabolic Adaptations in Salmonella Enterica Serovar Typhimurium 14028S During Batch Growth and Gentamicin Exposure Using Absolute Quantitative Proteomics

Abstract

Understanding the mechanisms underlying antibiotic-mediated bacterial cell death is crucial for the development of novel strategies to control bacterial infections. Although the primary targets of antibiotics are well known, the actual molecular events leading to bacterial growth inhibition and cell death remain unclear. Recent studies have suggested that the interaction of antibiotics with their primary targets in bacteria results in harmful events, such as futile cycles, activation of toxic pathways, and accumulation of harmful by-products that cause bacterial growth inhibition and death. However, these events remain only partially understood and evidence suggests an intricate interplay between metabolic activities of the bacterium and the perturbations caused by antibiotics. To unravel this interplay, global analyses of bacterial responses to antibiotic treatment are required. In this thesis, we performed absolute quantification of the proteome in the food-borne pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) using data-independent acquisition (DIA) mass spectrometry (MS)-based approach. We first mapped the metabolic activities of S. Typhimurium cells throughout batch growth (early, mid, and late logarithmic and early stationary phase) in order to establish a baseline of metabolic states during regular unperturbed growth. Our data show that the logarithmic growth is not uniform due to metabolic shift as a result of continuous depletion of the medium. For instance, we observed a reduced abundance of proteins involved in translation within the logarithmic phases (mid and late logarithmic) corresponding to a reduced growth rate. Moreover, the proteomic profiles suggested a switch from glycolysis to gluconeogenesis from the mid-logarithmic phase (4.5 h) onwards, likely the result of glucose depletion in the medium. We also observed an increase in proteins involved in amino acid metabolism (Asp, Gly, Ser, Thr) beyond the mid-logarithmic phase. Although these amino acids could fuel the TCA cycle to provide precursors for gluconeogenesis, the proteome data does not conclusively indicate either anabolic or catabolic pathways. In presence of gentamicin, we observed an increase in the abundance of ribosomal proteins, suggesting that S. Typhimurium might compensate for ribosomal inhibition by synthesizing additional ribosomes. Moreover, we observed an increased abundance of proteins involved in multiple metabolic pathways such as fatty acid metabolism and oxidative phosphorylation during gentamicin exposure, suggesting that the ribosomal inhibition by gentamicin can result in pleiotropic metabolic effects. Furthermore, we demonstrated that the addition of glucose improves the ability of S. Typhimurium to grow in presence of gentamicin. Proteomic profiling revealed that after supplementation of glucose the abundance of proteins involved in glycolytic and fermentative pathways increased, whereas proteins of the TCA cycle were reduced in abundance. Previous studies suggested that bacterial susceptibility towards other ribosome-targeting antibiotics may be related to the TCA cycle. Based on our data, we speculate that if enough energy can be generated from glycolytic pathways, bypassing the TCA cycle could support S. Typhimurium survival in the presence of gentamicin.