Functional γ-aminobutyrate shunt in Listeria monocytogenes: role in acid tolerance and succinate biosynthesis

Listeria monocytogenes, the causative agent of human listeriosis, is known for its ability to withstand severe environmental stresses. The glutamate decarboxylase (GAD) system is one of the principal systems utilized by the bacterium to cope with acid stress, a reaction that produces γ-aminobutyrate (GABA) from glutamate. Recently, we have shown that GABA can accumulate intracellularly under acidic conditions, even under conditions where no extracellular glutamate-GABA exchange is detectable. The GABA shunt, a pathway that metabolizes GABA to succinate, has been described for several other bacterial genera, and the present study sought to determine whether L. monocytogenes has this metabolic capacity, which, if present, could provide a possible route for succinate biosynthesis in L. monocytogenes. Using crude protein extracts from L. monocytogenes EGD-e, we show that this strain exhibits activity for the two main enzyme reactions in the GABA shunt, GABA aminotransferase (GABA-AT) and succinic semialdehyde dehydrogenase (SSDH). Two genes were identified as candidates for encoding these enzyme activities, argD (GABA-AT) and lmo0913 (SSDH). Crude protein extracts prepared from a mutant lacking a functional argD gene significantly reduced GABA-AT activity, while an lmo0913 mutant lost all detectable SSDH activity. The deletion of lmo0913 increased the acid tolerance of EGD-e and showed an increased accumulation of intracellular GABA, suggesting that this pathway plays a significant role in the survival of this pathogen under acidic conditions. This is the first report of such a pathway in the genus Listeria, which highlights an important link between metabolism and acid tolerance and also presents a possible compensatory pathway to partially overcome the incomplete tricarboxylic acid cycle of Listeria.

Role of glutamate metabolism in bacterial responses towards acid and other stresses

Glutamate plays a central role in a wide range of metabolic processes in bacterial cells. This review focuses on the involvement of glutamate in bacterial stress responses. In particular it reviews the role of glutamate metabolism in response against acid stress and other stresses. The glutamate decarboxylase (GAD) system has been implicated in acid tolerance in several bacterial genera. This system facilitates intracellular pH homeostasis by consuming protons in a decarboxylation reaction that produces γ-aminobutyrate (GABA) from glutamate. An antiporter system is usually present to couple the uptake of glutamate to the efflux of GABA. Recent insights into the functioning of this system will be discussed. Finally the intracellular fate of GABA will also be discussed. Many bacteria are capable of metabolising GABA to succinate via the GABA shunt pathway. The role and regulation of this pathway will be addressed in the review. © 2012 The Authors Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.

Anti-platelet effect of aspirin is substantially reduced after administration of heparin during carotid endarterectomy

OBJECTIVES: Aspirin therapy is usually continued throughout the perioperative period to reduce the risk for thromboembolic stroke and myocardial infarction after carotid endarterectomy (CEA). Aspirin irreversibly binds cyclooxygenase-1, thereby reducing platelet aggregation for the lifetime of each platelet. However, recent research from this unit has shown that aggregation in response to arachidonic acid increases significantly, but transiently, during CEA, which suggests that the anti-platelet effect of aspirin is temporarily reversed. The purpose of the current study was to determine when this phenomenon occurs and to identify the possible mechanisms involved. METHODS: Platelet aggregation was measured in platelet-rich plasma from 41 patients undergoing CEA who were stabilized with 150 mg of aspirin daily. Blood was taken at 8 time points: before anesthesia, after anesthesia, before heparinization, 3 minutes after heparinization, 3 minutes after shunt insertion, 10 minutes after flow restoration, 4 hours postoperatively, and 24 hours postoperatively. Platelet aggregation was also measured at similar times in a group of 18 patients undergoing peripheral angioplasty without general anesthesia. RESULTS: All patient platelets were effectively inhibited by aspirin at the start of the operation. There was a significant intraoperative increase in platelet response to arachidonic acid in both groups of patients, which occurred within 3 minutes of administration of unfractionated heparin. In the CEA group this resulted in a greater than 10-fold increase in mean aggregation, to 5 mmol/L of arachidonic acid (5 mmol/L), rising from 3.9% +/- 2.2% preoperatively to 45.1% +/- 29.3% after administration of heparin ( P <.0001). This increased aggregation persisted into the early postoperative period, but by 24 hours post operation aggregation had returned to near preoperative values. Aggregation in response to other platelet agonists (adenosine diphosphate, thrombin receptor agonist peptide) showed only a small increase at the same time, which could be accounted for by a parallel increase in the level of spontaneous aggregation. CONCLUSION: Administration of heparin significantly increases platelet aggregation in response to arachidonic acid, despite adequate inhibition by aspirin administered preoperatively. This apparent reversal in anti-platelet activity persisted into the immediate early postoperative period, and could explain why a small proportion of patients are at increased risk for acute cardiovascular events after major vascular surgery, despite aspirin therapy.