Measurement of Internal pH in Helicobacter pylori by Using Green Fluorescent Protein Fluorimetry.

Helicobacter pylori is an organism known to colonize the normal human stomach. Previous studies have shown that the bacterium does this by elevating its periplasmic pH via the hydrolysis of urea. However, the value of the periplasmic pH was calculated indirectly from the proton motive force equation. To measure the periplasmic pH directly in H. pylori, we fused enhanced green fluorescent protein (EGFP) to the predicted twin-arginine signal peptides of HydA and KapA from H. pylori and TorA from Escherichia coli The fusion proteins were expressed in the H. pylori genome under the control of the cagA promoter. Confocal microscopic and cell fractionation/immunoblotting analyses detected TorA-EGFP in the periplasm and KapA-EGFP in both the periplasm and cytoplasm, while the mature form of HydA-EGFP was seen at low levels in the periplasm, with major cytoplasmic retention of the precursor form. With H. pylori expressing TorA-EGFP, we established a system to directly measure periplasmic pH based on the pH-sensitive fluorimetry of EGFP. These measurements demonstrated that the addition of 5 mM urea has little effect on the periplasmic pH at a medium pH higher than pH 6.5 but rapidly increases the periplasmic pH to pH 6.1 at an acidic medium pH (pH 5.0), corresponding to the opening of the proton-gated channel, UreI, and confirming the basis of gastric colonization. Measurements of the periplasmic pH in an HP0244 (FlgS)-deficient mutant of H. pylori expressing TorA-EGFP revealed a significant loss of the urea-dependent increase in the periplasmic pH at an acidic medium pH, providing additional evidence that FlgS is responsible for recruitment of urease to the inner membrane in association with UreI.IMPORTANCEHelicobacter pylori has been identified as the major cause of chronic superficial gastritis and peptic ulcer disease. In addition, persistent infection with H. pylori, which, if untreated, lasts for the lifetime of an infected individual, predisposes one to gastric malignancies, such as adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma. A unique feature of the neutralophilic bacterium H. pylori is its ability to survive in the extremely acidic environment of the stomach through its acid acclimation mechanism. The presented results on measurements of periplasmic pH in H. pylori based on fluorimetry of fully active green fluorescent protein fusion proteins exported with the twin-arginine translocase system provide a reliable and rapid tool for the investigation of acid acclimation in H. pylori.

A novel method for assessment of local pH in periplasmic space and of cell surface potential in yeast.

Yeast cells exhibit a negative surface potential due to negative charges at the cell membrane surface. Consequently, local concentrations of cations at the periplasmic membrane surface may be significantly increased compared to their bulk environment. However, in cell suspensions only bulk concentrations of cations can be measured directly. Here we present a novel method enabling the assessment of local pH at the periplasmic membrane surface which can be directly related to the underlying cell surface potential. In this proof of concept study using Saccharomyces cerevisiae cells with episomally expressed pH reporter, pHluorin, intracellular acidification induced by the addition of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) was measured using synchronously scanned fluorescence spectroscopy (SSF). The analysis of titration curves revealed that the pH at the periplasmic surface of S. cerevisiae cells was about two units lower than the pH of bulk medium. This pH difference was significantly decreased by increasing the ionic strength of the bulk medium. The cell surface potential was estimated to amount to -130 mV. Comparable results were obtained also with another protonophore, pentachlorophenol (PCP).