Phase variation in the Helicobacter pylori phospholipase A gene and its role in acid adaptation

Abstract

Previously, we have shown that Helicobacter pylori can spontaneously and reversibly change its membrane lipid composition, producing variants with low or high content of lysophospholipids. The "lyso" variant contains a high percentage of lysophospholipids, adheres better to epithelial cells, and releases more proteins such as urease and VacA, compared to the "normal" variant, which has a low content of lysophospholipids. Prolonged growth of the normal variant at pH 3.5, but not under neutral conditions, leads to enrichment of lyso variant colonies, suggesting that the colony switch is relevant to acid adaptation. In this study we show that the change in membrane lipid composition is due to phase variation in the pldA gene. A change in the (C) tract length of this gene results in reversible frameshifts, translation of a full-length or truncated pldA, and the production of active or inactive outer membrane phospholipase A (OMPLA). The role of OMPLA in determining the colony morphology was confirmed by the construction of an OMPLA-negative mutant. Furthermore, variants with an active OMPLA were able to survive acidic conditions better than variants with the inactive form. This explains why the lyso variant is selected at low pH. Our studies demonstrate that phase variation in the pldA gene, resulting in an active form of OMPLA, is important for survival under acidic conditions. We also demonstrated the active OMPLA genotype in fresh isolates of H. pylori from patients referred to gastroscopy for dyspepsia.

FIG. 1

Structures of phospholipids. I, phosphatidylethanolamine; II, lysophosphatidylethanolamine made by the action of PLA.

FIG. 2

AFLP pattern of H. pylori phase variants compared to type strain NCTC 11637, confirming that the variants originate from one H. pylori clone.

FIG. 3

SDS-PAGE and silver staining of H. pylori phase variants. Lane 1, NCTC 11637; lane 2, 17L; lane 3, 17S; lane 4, 17L_r.

FIG. 4

Partial sequences. (A) Partial sequence of the pldA gene from H. pylori phase variants 17L/17S_r and 17S/S_i. The change in ORF caused by the slipped-strand mispairing results in the translation of a truncated or full-length pldA. (B) Partial sequence of the pldA gene from H. pylori isolates 12/LH, 23/LH, and 25/LH compared to the colony phase variant 17S, showing that the gene is “on” in all isolates.

FIG. 5

Survival of H. pylori phase variants after one passage on blood agar plates at pH 3.5 incubated under microaerobic conditions at 37°C for 16 h. The bacteria were harvested at pH 3.5, plated onto blood agar plates (pH 7.4), and then counted after reincubation.