Substrate selectivity in glutamate-dependent acid resistance in enteric bacteria

Abstract

The bacterial antiporter GadC plays a central role in the glutamate (Glu)-dependent acid resistance system, which protects enteric bacteria against the extreme acidity of the human stomach. Upon acid shock, GadC imports Glu into the cytoplasm, where Glu decarboxylases consume a cytoplasmic proton, which ends up as a "virtual" proton in the decarboxylated product γ-aminobutyric acid (GABA) and is then exported via GadC. It was therefore proposed that GadC counters intracellular acidification by continually pumping out virtual protons. This scenario, however, is oversimplified. In gastric environments, GadC encounters substrates in multiple carboxyl protonation forms (outside: Glu(-), Glu(0), Glu(+); inside: GABA(0), GABA(+)). Of the six possible combinations of antiport partners, Glu(+)/GABA(0) results in proton influx, Glu(0)/GABA(0) and Glu(+)/GABA(+) are proton neutral, and Glu(-)/GABA(0), Glu(-)/GABA(+), or Glu(0)/GABA(+) lead to proton extrusion. Which of these exchanges does GadC catalyze? To attack this problem, we developed an oriented GadC liposome system holding a three-unit inward pH gradient to mimic the conditions facing bacteria in the stomach. By assessing the electrogenicity of substrate transport, we demonstrate that GadC selectively exchanges Glu(-) or Glu(0) with GABA(+), resulting in effective proton extrusion of >0.9 H(+) per turnover to counter proton invasion into acid-challenged bacteria. We further show that GadC selects among protonated substrates using a charge-based mechanism, rather than directly recognizing the protonation status of the carboxyl groups. This result paves the way for future work to identify the molecular basis of GadC's substrate selectivity.

Fig. 1.

GadC in the gastric environment. GadC switches between outward- and inward-open conformations to import extracellular Glu while expelling cytoplasmic GABA, which carries the virtual proton in a C–H bond (black circle). The protonable carboxyl groups of Glu and GABA are highlighted.

Fig. 2.

Sidedness of GadC established by asymmetric pH. Icons represent the two orientations of GadC incorporated into liposomes, with the N- and C termini, which mark the transporter’s intracellular side, represented as wiggly lines. (A) The ³H-Glu_ex/GABA_in exchange using I21C-GadC liposomes, with no treatments (filled square), MTSET in outside to silence inside-out GadC (open circle), or MTSET in both sides to react with all proteins (open square), in the presence of a three-unit inward pH gradient. (B) Effect of extracellular pH on ³H-Glu_ex/GABA_in exchange. The extraliposomal pH of I21C-containing liposomes was varied from 2.2–7, with internal pH fixed at 5.0. Uptake of ³H-Glu_ex was recorded 4 min after initiation of transport. (C) Effect of cytoplasmic pH on ³H-Glu_ex/GABA_in exchange. I21C liposomes, oriented outside-out (with external pH 2.2), were allowed to uptake Glu for 4 min in various inside pH ranging from 2.2 to 5.

Fig. 3.

Substrate selectivity of GadC. WT GadC proteoliposomes (inside pH 5.0, outside pH 2.2) and a 1,000-fold outward K⁺ gradient, were used to quantify (A) ³H-Glu_ex/GABA_in and (B) ³H-Gln_ex/GABA_in exchanges, in the presence (open squares) or absence (filled squares) of Vln.

Fig. 4.

Substrate selectivity of GadC. (A) ³H-Gln_ex, pH 2.2/Gln_in, pH 5.0 and (B) ³H-GABA_ex, pH 3.0/GABA_in, pH 5.0 exchanges were examined in WT GadC liposomes holding a 1,000-fold outward K⁺ gradient, in the presence (open squares) or absence (filled squares) of Vln.