Helicobacter pylori CagL Y58/E59 mutation turns-off type IV secretion-dependent delivery of CagA into host cells

Abstract

The type IV secretion system (T4SS) is a major virulence determinant of the gastric pathogen Helicobacter pylori. The CagL protein is a specialized adhesin of the corresponding T4SS pilus, which establishes initial contact with the integrin β1 receptor on host target cells. Recent studies proposed that Y58 and E59 amino acid polymorphisms in CagL increase the virulence of H. pylori strains by enhanced translocation and phosphorylation of the CagA effector protein. These polymorphisms were therefore correlated with an increased risk of gastric cancer development. Here we show that the Y58/E59 motif, which is located in a loop connecting two α-helices, and corresponding polymorphisms could influence the function of CagL. However, expression of isogenic CagL Y58/E59 variants in H. pylori strain 26695 significantly blocked the translocation and phosphorylation of CagA as compared to complemented wild-type CagL. These results suggest that the function of the T4SS for delivery of CagA is turned-off by the Y58/E59 mutation in CagL. This activity appears to be similar to the one recently described for another T4SS pilus protein, CagY, which is also sufficient to cause gain or loss of T4SS function. These data support the hypothesis that certain mutations in CagL or recombination events in CagY may serve as a sort of molecular switch or perhaps rheostat in the T4SS, which could alter the function of the pilus and "tunes" injection of CagA and host pro-inflammatory responses, respectively.

Figure 1. Role of Helicobacter pylori CagL Y58/E59 mutation in type IV secretion-dependent delivery of CagA in host cells.

(A) Cartoon representation of the crystal structure of CagL (molecule A of the six crystallographically independent molecules, PDB accession code 3ZCJ) from strain 26695 . The various α helices (denoted 1–6), R76 and D78 of the RGD-motif and the amino acids N58 und E59 are highlighted. The amino acids from T52-N57 are not resolved and are shown as a dashed line. This region connects the two indicated helices, α1 (D22-S50) and α2 (E59-K101). The PyMOL program was used to generate the structure illustration (The PyMOL Molecular Graphics System, Schrödinger, LLC). (B) Alignment of amino acid sequences of CagL (position 39–78) among strains 26695 , Hp0621, Hp0412, Hp0710, Hp0902, Hp1035, Hp1393 and the 26695 CagL^YE mutant produced here is shown. A variable region among the CagL proteins between positions 58–62 is boxed in yellow and the Y58/E59 polymorphism is highlighted with red. The strains originated from gastritis (GA), duodenal ulcer (DU) and gastric cancer (GC) patients as indicated . (C) AGS gastric epithelial cells were infected with the indicated H. pylori strains and cagL mutants for 8 hours. Resulting protein lysates were probed with the indicated antibodies as described. The experiments were done at pH 7.4, which gave very strong phospho-CagA signals in a recent study . (D) Quantification of CagA phosphorylation signals in panel C using the luminescence image analyzer. The strongest signal in lane 1 was set as 100%. The data are representative from three independent experiments.