A glutamate decarboxylase system protects Listeria monocytogenes in gastric fluid

We observed that glutamate greatly enhances the survival of Listeria monocy togenes in gastric fluid, a phenomenon that is directly linked to glutamate decarboxylase activity (GAD). Glutamate-mediated acid tolerance has been a ssociated in other intestinal genera with the GAD system, in which glutamat e is internalized and converted to gamma -aminobutyrate (consuming an intra cellular proton) that is subsequently exchanged for another extracellular g lutamate via a membrane-located antiporter. Molecular analysis of L. monocy togenes LO28 revealed the presence of two glutamate decarboxylase homologue s, designated gadA and gadB, that are differentially expressed. The gadB ge ne is co-transcribed in tandem with an upstream gene, gadC, which encodes a potential glutamate/gamma -aminobutyrate antiporter. Expression of this tr anscript is upregulated in response to mild acid stress (pH 5.5). In contra st, expression of the monocistronic gadA message was weaker and was not ind uced by mild acid treatment. Non-polar deletion mutations resulted in a dra matic decrease in the level of GAD activity and a concomitant decrease in a cid resistance in the order LO28 > Delta gadA > Delta gadB = Delta gadC > D elta gadAB for both stationary and logarithmic phase cultures. The exquisit e sensitivity of the Delta gadAB mutant to ex vivo porcine and synthetic hu man gastric fluid demonstrates a clear role for the GAD system in facilitat ing survival of the organism in the stomach after ingestion and in other lo w-pH environments. Furthermore, variations in levels of GAD activity betwee n different strains of L. monocytogenes correlate significantly with levels of tolerance to gastric fluid. Sensitive strains, which include the sequen ced L. monocytogenes EGD, exhibit reduced levels of GAD activity. It is cle ar from this study that expression of GAD by L. monocytogenes strains is an absolute requirement for survival in the stomach environment.