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

• Published in: Journal of bacteriology 2018-07, Vol.200 (14)
• Main Authors: Wen, Yi, Scott, David R, Vagin, Olga, Tokhtaeva, Elmira, Marcus, Elizabeth A, Sachs, George
• Format: Article
• Language: English
• Subjects: Acclimation; Acclimatization; Acids; Adenocarcinoma; Arginine; Bacteria; Bacteriology; Chronic infection; Colonization; Cytoplasm; Deficient mutant; E coli; Fluorescence; Fluorimetry; Fractionation; Fusion protein; Gastritis; Genomes; Green fluorescent protein; Helicobacter pylori; Immunoblotting; Lymphoma; Malt; Mucosa; Mucosal-associated lymphoid tissue; Mutation; Peptides; Periplasm; pH effects; Proteins; Protonmotive force; Signal peptides; Stomach; Translocase; Twin-arginine translocase; Urea; Urease
• ISSN: 0021-9193; 1098-5530
• DOI: 10.1128/JB.00178-18

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 , we fused enhanced green fluorescent protein (EGFP) to the predicted twin-arginine signal peptides of HydA and KapA from and TorA from The fusion proteins were expressed in the genome under the control of the 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 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 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. has been identified as the major cause of chronic superficial gastritis and peptic ulcer disease. In addition, persistent infection with , 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 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 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 .