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Alanine Enhances Aminoglycosides-Induced ROS Production by Metabolic Regulation

Jinzhou Ye

Metabolite-enabled killing of antibiotic-resistant pathogens by antibiotics is an attractive strategy to manage antibiotic resistance. Our previous study demonstrated that alanine or/and glucose increased the killing efficacy of kanamycin on antibiotic-resistant bacteria, whose action is through up-regulating TCA cycle, increasing proton motive force and enhancing antibiotic uptake. Despite the fact that alanine altered several metabolic pathways, other mechanisms could be potentially involved in alanine-mediated kanamycin killing of bacteria which remain to be explored. In the present study, we adopted proteomic approach to analyze the proteome changes induced by exogenous alanine. Our results revealed that the expression of three outer membrane proteins was altered and the deletion of nagE and fadL decreased the intracellular kanamycin concentration, implying their possible roles in mediating kanamycin transport. More importantly, the integrated analysis of proteomic and metabolomic data pointed out that alanine metabolism could connect to riboflavin metabolism that provides the source for reactive oxygen species (ROS) production. Functional studies confirmed that alanine treatment together with kanamycin could promote ROS production that in turn potentiates the killing of antibiotic-resistant bacteria. Further investigation showed that alanine repressed the transcription of antioxidant-encoding genes, and alanine metabolism to riboflavin metabolism connected with riboflavin metabolism through TCA cycle, glucogenesis pathway and pentose phosphate pathway. Our results suggest a novel mechanism by which alanine facilitates kanamycin killing of antibiotic-resistant bacteria via promoting ROS production. The widespread of antibiotic-resistant bacteria is a growing problem, imposing catastrophic threat to people in every country throughout the world. The control of antibiotic-resistant bacteria becomes an urgent issue to the society. Although governmental interventions are undertaken to control the use of antibiotics in clinics and poultry industry, the approach to eliminate the existing antibiotic-resistant  bacteria is still limited. Developing novel classes of antibiotics as well as antibiotic derivatives through chemical modifications to provide antibiotic derivatives are the main strategies by pharmaceutical companies and health care

systems to eliminate resistance. However, the approach is difficult given the fact that the industry’s search of novel chemical agents acting on new biological targets is proven to be non-productive. Another major strategy is the non-antibiotic approach including antibacterial vaccines, phage therapy, immunostimulants, adjuvants, anti-virulence therapies, probiotics and their combinations. Unfortunately, the development of non-antibiotic approaches lagged behind the expectation, and meet limited success. The major challenge to kill antibiotic-resistant bacteria is the limited concentration of antibiotics that can be achieved inside bacterial cells, which is likely due to the elevated efflux or reduced influx of the antibiotics. Novel approaches thus are required to overcome this limitation to increase the intracellular antibiotic concentration to a certain threshold so that the resistant bugs can be killed. However, several lines of evidences demonstrated that microbial environment confounds antibiotic efficacy through metabolic processes. Metabolites like indole, produced by a subpopulation of bacteria but shared by all enabled the whole population to defend against antibiotic stress. Gas is another type of cytoprotective agent that protects bacteria against a wide range of antibiotics, e.g., nitric oxide alleviates antibiotic-induced ROS in bacteria thus prevent cell death. The micro-environment of bacteria community is thus determining antibiotic susceptibility, which provides the basis to engineer bacterial metabolic pathways to combat antibiotic resistance. Metabolites have been proved to be a useful way. The treatment of persisters, the highly antibiotic-tolerant subpopulation of the bacteria, with glucose, mannitol or fructose would greatly enhance the killing of persisters by aminoglycosides. Moreover, several recent studies highlight the importance of TCA cycle in fighting against multidrug resistant bacteria. The promotion of tricarboxylic cycle (TCA cycle) through exogenous alanine, glucose and fructose could greatly enhance the killing efficacy of kanamycin on different types of multidrug-resistant bacteria like Vibrio parahaemolyticus, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus, persisters, and in vivo biofilm infections. The underlying mechanism involves the metabolites in promoting TCA cycle, increasing the generation of NADH, the substrates for proton motive force (PMF) production. The increased PMF ultimately increased intracellular concentration of kanamycin through enhanced antibiotic uptake. Thus, these studies highlighted the role of activation of TCA cycle in killing antibiotic-resistant bacteria by aminoglycosides A later study further demonstrated that the tuning of TCA cycle could influence the antibiotic susceptibility of Pseudomonas aeroginosa to antibiotics. Thus, the combinatorial use of metabolite and antibiotics has promising potential in eliminating the antibiotic-resistant bacteria by “reusing” old antibiotics. The metabolic mechanism of alanine, glucose and fructose in potentiating kanamycin to kill antibiotic-resistant bacteria is well elucidated in our previous studies. However, whether other mechanisms have been involved in alanine and antibiotic-triggered cell death is still unexplored. In this study, we adopted proteomic approach to investigate the global proteome change in response to exogenous alanine. We found that exogenous alanine affects the expression of three outer membrane proteins. Furthermore, the integrated analysis of proteomic and metabolomics data directs our attention to ROS that can be synergistically produced by the combination of alanine and kanamycin. This study thus gains new insights on mechanisms of alanine-enabled killing of antibiotic-resistant bacteria by kanamycin.In our previous report, we found that exogenous alanine reprogrammed the metabolome of Edwardsiella tarda EIB202, featured with twelve altered metabolic pathways. Although the metabolomic data provided profound insights into how alanine modulates the metabolome of target cell and causes the death of multidrug-resistant bacteria by kanamycin, other biological processes that are involved might be neglected during metabolomics analysis. Thus, we implemented proteomic approach to further investigate the proteome change that is associated with exogenous alanine. We continued using the wild-type multidrug-resistant E. tarda strain EIB202, and treated EIB202 with the dose of alanine (40 mM) we previously adopted. After treatment, the whole cells were lysed, and total proteins were purified, labeled with iTRAQ and analyzed with LC-MS/MS. A total of 1972 protein were identified, where 40 proteins were differentially expressed as compared to the control group treated with saline buffer (fold of average change larger than 1.5 and p < 0.05 in both biological replicates is considered as differentially expressed proteins) . Among the differential proteins, the expression levels of 22 proteins were increased while 19 proteins were decreased.

Metabolite-enabled killing of antibiotic-resistant pathogens by antibiotics is an attractive strategy to manage antibiotic resistance. Our previous study demonstrated that alanine or/and glucose increased the killing efficacy of kanamycin on antibiotic-resistant bacteria, whose action is through up-regulating TCA cycle, increasing proton motive force and enhancing antibiotic uptake. Despite the fact that alanine altered several metabolic pathways, other mechanisms could be potentially involved in alanine-mediated kanamycin killing of bacteria which remain to be explored. In the present study, we adopted proteomic approach to analyze the proteome changes induced by exogenous alanine. Our results revealed that the expression of three outer membrane proteins was altered and the deletion of nagE and fadL decreased the intracellular kanamycin concentration, implying their possible roles in mediating kanamycin transport. More importantly, the integrated analysis of proteomic and metabolomic data pointed out that alanine metabolism could connect to riboflavin metabolism that provides the source for reactive oxygen species (ROS) production. Functional studies confirmed that alanine treatment together with kanamycin could promote ROS production that in turn potentiates the killing of antibiotic-resistant bacteria. Further investigation showed that alanine repressed the transcription of antioxidant-encoding genes, and alanine metabolism to riboflavin metabolism connected with riboflavin metabolism through TCA cycle, glucogenesis pathway and pentose phosphate pathway. Our results suggest a novel mechanism by which alanine facilitates kanamycin killing of antibiotic-resistant bacteria via promoting ROS production. The widespread of antibiotic-resistant bacteria is a growing problem, imposing catastrophic threat to people in every country throughout the world. The control of antibiotic-resistant bacteria becomes an urgent issue to the society. Although governmental interventions are undertaken to control the use of antibiotics in clinics and poultry industry, the approach to eliminate the existing antibiotic-resistant  bacteria is still limited. Developing novel classes of antibiotics as well as antibiotic derivatives through chemical modifications to provide antibiotic derivatives are the main strategies by pharmaceutical companies and health care

systems to eliminate resistance. However, the approach is difficult given the fact that the industry’s search of novel chemical agents acting on new biological targets is proven to be non-productive. Another major strategy is the non-antibiotic approach including antibacterial vaccines, phage therapy, immunostimulants, adjuvants, anti-virulence therapies, probiotics and their combinations. Unfortunately, the development of non-antibiotic approaches lagged behind the expectation, and meet limited success. The major challenge to kill antibiotic-resistant bacteria is the limited concentration of antibiotics that can be achieved inside bacterial cells, which is likely due to the elevated efflux or reduced influx of the antibiotics. Novel approaches thus are required to overcome this limitation to increase the intracellular antibiotic concentration to a certain threshold so that the resistant bugs can be killed. However, several lines of evidences demonstrated that microbial environment confounds antibiotic efficacy through metabolic processes. Metabolites like indole, produced by a subpopulation of bacteria but shared by all enabled the whole population to defend against antibiotic stress. Gas is another type of cytoprotective agent that protects bacteria against a wide range of antibiotics, e.g., nitric oxide alleviates antibiotic-induced ROS in bacteria thus prevent cell death. The micro-environment of bacteria community is thus determining antibiotic susceptibility, which provides the basis to engineer bacterial metabolic pathways to combat antibiotic resistance. Metabolites have been proved to be a useful way. The treatment of persisters, the highly antibiotic-tolerant subpopulation of the bacteria, with glucose, mannitol or fructose would greatly enhance the killing of persisters by aminoglycosides. Moreover, several recent studies highlight the importance of TCA cycle in fighting against multidrug resistant bacteria. The promotion of tricarboxylic cycle (TCA cycle) through exogenous alanine, glucose and fructose could greatly enhance the killing efficacy of kanamycin on different types of multidrug-resistant bacteria like Vibrio parahaemolyticus, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus, persisters, and in vivo biofilm infections. The underlying mechanism involves the metabolites in promoting TCA cycle, increasing the generation of NADH, the substrates for proton motive force (PMF) production. The increased PMF ultimately increased intracellular concentration of kanamycin through enhanced antibiotic uptake. Thus, these studies highlighted the role of activation of TCA cycle in killing antibiotic-resistant bacteria by aminoglycosides A later study further demonstrated that the tuning of TCA cycle could influence the antibiotic susceptibility of Pseudomonas aeroginosa to antibiotics. Thus, the combinatorial use of metabolite and antibiotics has promising potential in eliminating the antibiotic-resistant bacteria by “reusing” old antibiotics. The metabolic mechanism of alanine, glucose and fructose in potentiating kanamycin to kill antibiotic-resistant bacteria is well elucidated in our previous studies. However, whether other mechanisms have been involved in alanine and antibiotic-triggered cell death is still unexplored. In this study, we adopted proteomic approach to investigate the global proteome change in response to exogenous alanine. We found that exogenous alanine affects the expression of three outer membrane proteins. Furthermore, the integrated analysis of proteomic and metabolomics data directs our attention to ROS that can be synergistically produced by the combination of alanine and kanamycin. This study thus gains new insights on mechanisms of alanine-enabled killing of antibiotic-resistant bacteria by kanamycin. In our previous report, we found that exogenous alanine reprogrammed the metabolome of Edwardsiella tarda EIB202, featured with twelve altered metabolic pathways. Although the metabolomic data provided profound insights into how alanine modulates the metabolome of target cell and causes the death of multidrug-resistant bacteria by kanamycin, other biological processes that are involved might be neglected during metabolomics analysis. Thus, we implemented proteomic approach to further investigate the proteome change that is associated with exogenous alanine. We continued using the wild-type multidrug-resistant E. tarda strain EIB202, and treated EIB202 with the dose of alanine (40 mM) we previously adopted. After treatment, the whole cells were lysed, and total proteins were purified, labeled with iTRAQ and analyzed with LC-MS/MS. A total of 1972 protein were identified, where 40 proteins were differentially expressed as compared to the control group treated with saline buffer (fold of average change larger than 1.5 and p < 0.05 in both biological replicates is considered as differentially expressed proteins) . Among the differential proteins, the expression levels of 22 proteins were increased while 19 proteins were decreased.