Functional proteomics identify mannitol metabolism in serum resistance and therapeutic implications in Vibrio alginolyticus.

Serum resistance is recognized as one of the most important pathogenic traits of bacterial pathogens, and no control measure is available. Based on our previous discovery that pathogenic Escherichia coli represses glycine, serine, and threonine metabolism to confer serum resistance and that the reactivation of this pathway by exogenous glycine could restore serum sensitivity, we further investigate the mechanism underlying the action of glycine in Vibrio alginolyticus. Thus, V. alginolyticus is treated with glycine, and the proteomic change is profiled with tandem mass tag-based quantitative proteomics. Compared to the control group, glycine treatment influences the expression of a total of 291 proteins. Among them, a trap-type mannitol/chloroaromatic compound transport system with periplasmic component, encoded by N646_0992, is the most significantly increased protein. In combination with the pathway enrichment analysis showing the altered fructose and mannitol metabolism, mannitol has emerged as a possible metabolite in enhancing the serum killing activity. To demonstrate this, exogenous mannitol reduces bacterial viability. This synergistic effect is further confirmed in a V. alginolyticus-Danio rerio infection model. Furthermore, the mechanism underlying mannitol-enabled serum killing is dependent on glycolysis and the pyruvate cycle that increases the deposition of complement components C3b and C5b-9 on the bacterial surface, whereas inhibiting glycolysis or the pyruvate cycle significantly weakened the synergistic effects and complement deposition. These data together suggest that mannitol is a potent metabolite in reversing the serum resistance of V. alginolyticus and has promising use in aquaculture.

Exogenous glycine promotes oxidation of glutathione and restores sensitivity of bacterial pathogens to serum-induced cell death.

Pathogenic strains of bacteria are often highly adept at evading serum-induced cell death, which is an essential complement-mediated component of the innate immune response. This phenomenon, known as serum-resistance, is poorly understood, and as a result, no effective clinical tools are available to restore serum-sensitivity to pathogenic bacteria. Here, we provide evidence that exogenous glycine reverses defects in glycine, serine and threonine metabolism associated with serum resistance, restores susceptibility to serum-induced cell death, and alters redox balance and glutathione (GSH) metabolism. More specifically, in Vibrio alginolyticus and Escherichia coli, exogenous glycine promotes oxidation of GSH to GSH disulfide (GSSG), disrupts redox balance, increases oxidative stress and reduces membrane integrity, leading to increased binding of complement. Antioxidant or ROS scavenging agents abrogate this effect and agents that generate or potentiate oxidation stimulate serum-mediated cell death. Analysis of several clinical isolates of E. coli demonstrates that glutathione metabolism is repressed in serum-resistant bacteria. These data suggest a novel mechanism underlying serum-resistance in pathogenic bacteria, characterized by an induced shift in the GSH/GSSG ratio impacting redox balance. The results could potentially lead to novel approaches to manage infections caused by serum-resistant bacteria both in aquaculture and human health.

Serine Metabolism Tunes Immune Responses To Promote Oreochromis niloticus Survival upon Edwardsiella tarda Infection.

Overactive immune response is a critical factor triggering host death upon bacterial infection. However, the mechanism behind the regulation of excessive immune responses is still largely unknown, and the corresponding control and preventive measures are still to be explored. In this study, we find that Nile tilapia, Oreochromis niloticus, that died from Edwardsiella tarda infection had higher levels of immune responses than those that survived. Such immune responses are strongly associated with metabolism that was altered at 6 h postinfection. By gas chromatography-mass spectrometry-based metabolome profiling, we identify glycine, serine, and threonine metabolism as the top three of the most impacted pathways, which were not properly activated in the fish that died. Serine is one of the crucial biomarkers. Exogenous serine can promote O. niloticus survival both as a prophylactic and therapeutic upon E. tarda infection. Our further analysis revealed exogenous serine flux into the glycine, serine, and threonine metabolism and, more importantly, the glutathione metabolism via glycine. The increased glutathione synthesis could downregulate reactive oxygen species. Therefore, these data together suggest that metabolic modulation of immune responses is a potential preventive strategy to control overactive immune responses. IMPORTANCE Bacterial virulence factors are not the only factors responsible for host death. Overactive immune responses, such as cytokine storm, contribute to tissue injury that results in organ failure and ultimately the death of the host. Despite the recent development of anti-inflammation strategies, the way to tune immune responses to an appropriate level is still lacking. We propose that metabolic modulation is a promising approach in tuning immune responses. We find that the metabolomic shift at as early as 6 h postinfection can be predictive of the consequences of infection. Serine is a crucial biomarker whose administration can promote host survival upon bacterial infection either in a prophylactic or therapeutic way. Further analysis demonstrated that exogenous serine promotes the synthesis of glutathione, which downregulates reactive oxygen species to dampen immune responses. Our study exemplifies that the metabolite(s) is a potential therapeutic reagent for overactive immune response during bacterial infection.