Functional plasticity in the type IV secretion system of Helicobacter pylori.

Helicobacter pylori causes clinical disease primarily in those individuals infected with a strain that carries the cytotoxin associated gene pathogenicity island (cagPAI). The cagPAI encodes a type IV secretion system (T4SS) that injects the CagA oncoprotein into epithelial cells and is required for induction of the pro-inflammatory cytokine, interleukin-8 (IL-8). CagY is an essential component of the H. pylori T4SS that has an unusual sequence structure, in which an extraordinary number of direct DNA repeats is predicted to cause rearrangements that invariably yield in-frame insertions or deletions. Here we demonstrate in murine and non-human primate models that immune-driven host selection of rearrangements in CagY is sufficient to cause gain or loss of function in the H. pylori T4SS. We propose that CagY functions as a sort of molecular switch or perhaps a rheostat that alters the function of the T4SS and "tunes" the host inflammatory response so as to maximize persistent infection

Functional plasticity in the type IV secretion system of Helicobacter pylori.

Helicobacter pylori causes clinical disease primarily in those individuals infected with a strain that carries the cytotoxin associated gene pathogenicity island (cagPAI). The cagPAI encodes a type IV secretion system (T4SS) that injects the CagA oncoprotein into epithelial cells and is required for induction of the pro-inflammatory cytokine, interleukin-8 (IL-8). CagY is an essential component of the H. pylori T4SS that has an unusual sequence structure, in which an extraordinary number of direct DNA repeats is predicted to cause rearrangements that invariably yield in-frame insertions or deletions. Here we demonstrate in murine and non-human primate models that immune-driven host selection of rearrangements in CagY is sufficient to cause gain or loss of function in the H. pylori T4SS. We propose that CagY functions as a sort of molecular switch or perhaps a rheostat that alters the function of the T4SS and "tunes" the host inflammatory response so as to maximize persistent infection

Functional plasticity in the type IV secretion system of Helicobacter pylori.

Helicobacter pylori causes clinical disease primarily in those individuals infected with a strain that carries the cytotoxin associated gene pathogenicity island (cagPAI). The cagPAI encodes a type IV secretion system (T4SS) that injects the CagA oncoprotein into epithelial cells and is required for induction of the pro-inflammatory cytokine, interleukin-8 (IL-8). CagY is an essential component of the H. pylori T4SS that has an unusual sequence structure, in which an extraordinary number of direct DNA repeats is predicted to cause rearrangements that invariably yield in-frame insertions or deletions. Here we demonstrate in murine and non-human primate models that immune-driven host selection of rearrangements in CagY is sufficient to cause gain or loss of function in the H. pylori T4SS. We propose that CagY functions as a sort of molecular switch or perhaps a rheostat that alters the function of the T4SS and "tunes" the host inflammatory response so as to maximize persistent infection

Loss of the capacity to induce IL-8 in <i>H. pylori</i> recovered from rhesus monkeys is associated with changes in the gene encoding CagY, an essential protein in the T4SS.

<p>(A–E) <i>H. pylori</i> was isolated from five rhesus macaques up to 14 months after experimental infection with <i>H. pylori</i> WT J166. Individual colonies were co-cultured with AGS cells, and ELISA was used to measure IL-8 levels, which were normalized to the WT J166 positive control. Each data point represents the results from a single colony. The capacity to induce IL-8 decreased over time in colonies recovered from four monkeys (A–D), but was largely unchanged in one (E). PCR-RFLP analysis showed that <i>H. pylori</i> colonies that lost the capacity to induce IL-8 were associated with a change in <i>cagY</i> (open circles), while those that maintained IL-8 induction typically had <i>cagY</i> that was indistinguishable from WT J166 (filled circles). Animal designation is shown in the upper left corner of each panel. (F) Output strains from each monkey were analyzed by <i>cagY</i> PCR-RFLP and compared to WT <i>H. pylori</i> J166 (dark blue) and to one another. Each pie chart represents all colonies recovered from one of the five monkeys (12–24 colonies/monkey); different colors represent different <i>cagY</i> variants.</p

Mouse adapted <i>H. pylori</i> strain SS1 expresses a CagY that is not functional for induction of IL-8 or translocation of CagA.

<p>(A) <i>H. pylori</i> was isolated from C57BL/6 WT or RAG1−/− mice (N = 3–6/time point) 8 weeks after experimental infection with <i>H. pylori</i> PMSS1. Individual colonies (3–6/mouse) were co-cultured with AGS cells, and ELISA was used to measure IL-8 levels, which were normalized to the PMSS1 positive control (line = mean). Each data point represents the results from a single colony. Induction of IL-8 in colonies isolated from WT mice was significantly lower than in RAG1−/− mice, and was associated with changes in <i>cagY</i> PCR-RFLP (open circles). (B) <i>cagY</i> in <i>H. pylori</i> strain SS1 is larger than that in the progenitor strain PMSS1, and has a different fingerprint on PCR-RFLP. (C) Deletion of <i>cagY</i> from WT <i>H. pylori</i> PMSS1 reduced the induction of IL-8 and eliminated translocation of CagA, which were recovered when the WT PMSS1 <i>cagY</i> gene was restored (▵Y[PMSS1]. However, replacement of the PMSS1 <i>cagY</i> gene with that from <i>H. pylori</i> SS1 (▵Y [SS1]) showed reduced levels of IL-8 and no CagA translocation. (D) WT <i>H. pylori</i> SS1 showed little induction of IL-8 and no CagA translocation, and it was unaffected by deletion of <i>cagY</i> or restoration of the WT SS1 <i>cagY</i> allele. However, replacement of the WT SS1 <i>cagY</i> allele with that from PMSS1 markedly increased IL-8 induction and CagA translocation, though not to the level of PMSS1. All assays represent the mean ±SEM of 3 replicates. **<i>P</i><0.01; ***<i>P</i><0.001.</p

Loss of the capacity to induce IL-8 and change <i>cagY</i> during infection of mice is dependent on an intact host immune system.

<p><i>H. pylori</i> was isolated from C57BL/6 WT (A) or RAG2−/− (B) mice (N = 3–6/time point) up to 16 weeks after experimental infection with <i>H. pylori</i> WT J166. Individual colonies (3–6/mouse) were co-cultured with AGS cells, and ELISA was used to measure IL-8 levels, which were normalized to the WT J166 positive control (line = mean). Each data point represents the results from a single colony. Induction of IL-8 in colonies isolated from WT mice was significantly lower than in RAG2−/− mice at 12 and 16 weeks PI (<i>P</i><0.01). Changes in <i>cagY</i> (open circles) were detected by PCR-RFLP in 28 of 70 colonies from WT mice but in 0 of 64 colonies from RAG2−/− mice (Fishers exact test, <i>P</i><0.0001). Output strains from WT C57BL/6 mice were analyzed by <i>cagY</i> PCR-RFLP and compared to WT <i>H. pylori</i> J166 (dark blue) and to one another (C). Each pie chart represents the unique <i>cagY</i> RFLP patterns identified in a single mouse from 2 to 16 weeks PI, and is positioned according to the mean IL-8 induction by colonies recovered from that mouse.</p

Changes in the motif structure of the CagY middle repeat region that alter the function of the <i>cag</i>PAI do not affect expression of T4SS pili on the bacterial surface.

<p><i>H. pylori</i> was co-cultured with AGS gastric cells at an MOI of 100∶1 and imaged by FEG-SEM. T4SS pilus structures were readily apparent in the WT <i>H. pylori</i> J166 but not in the <i>cag</i>PAI deletion mutant (J166▵<i>cag</i>PAI). T4SS pili were also observed in <i>H. pylori</i> J166 in which the WT <i>cagY</i> allele was replaced with that from output strains with a functional (rOut3, mOut3) or a non-functional (rOut2 mOut2) <i>cag</i>PAI. Pili were also seen in <i>H. pylori</i> strains J166 and 26695 with deletions in <i>cagY</i>. Magnification bars indicate 500 nm.</p

Recombination in <i>cagY</i> during infection of rhesus monkeys is sufficient to reduce the capacity of <i>H. pylori</i> to induce IL-8 and translocate CagA.

<p>Deletion of <i>cagY</i> (▵Y) from WT <i>H. pylori</i> J166 significantly reduced its capacity to induce IL-8 (mean ± SEM of 3 replicates), which was recovered when the chromosomal WT <i>cagY</i> allele was restored (▵Y [J166]) by complementation (black bars). Immunoblot showed that only the WT or ▵Y [J166] expressed CagY protein (α-CagY) and translocated CagA that was tyrosine phosphorylated (α-PY99). Two rhesus output strains with unique <i>cagY</i> alleles (rOut1, rOut2) lost the capacity to induce IL-8 (gray bars) and translocate CagA, although they expressed CagY. Replacement of ▵<i>cagY</i> with <i>cagY</i> from rOut1 (▵Y [rOut1]) or rOut2 (▵Y [rOut2]) recapitulated their failure to induce IL-8 induction (white bars) and translocate phosphorylated CagA. Similarly, complementation with <i>cagY</i> from an output strain (rOut3) that expressed a unique <i>cagY</i> but maintained the capacity to induce IL-8 (gray bar) and translocate CagA, also phenocopied its IL-8 induction and translocation of CagA. All strains expressed CagA (α-CagA), though only those that induced IL-8 had the capacity to translocate CagA that was tyrosine phosphorylated. Multiple bands in the CagY immunoblot could represent different transcription or translation products, or even protein fragments, but they are CagY-specific since they are absent in the <i>cagY</i> deletion mutant. **<i>P</i><0.01; ***<i>P</i><0.001.</p

<i>H. pylori</i> colonization of rhesus monkeys and mice is associated with changes in the motif structure of the CagY middle repeat region.

<p>As in strains J99 and 26695 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003189#ppat.1003189-Delahay1" target="_blank">[19]</a>, the predicted amino acid sequence of CagY in WT <i>H. pylori</i> J166 is organized into a 5′ repeat region (residues 9–398), a 3′ region orthologous to VirB10 (residues 1784–2028), and a middle repeat region (residues 715–1512) that is composed of a series of B motifs (yellow) that bracket one to four A motifs (orange). Passage of WT J166 in rhesus monkeys and in mice results in some strains that lose one or more A or B motifs, which is sometimes sufficient to reduce the capacity to induce IL-8 (rOut1 and rOut2; mOut1 and mOut2) and other times is not (rOut3; mOut3 and mOut4).</p

Recombination in <i>cagY</i> during infection of mice is sufficient to reduce the capacity of <i>H. pylori</i> to induce IL-8 and translocate CagA.

<p>Deletion of <i>cagY</i> (▵Y) from WT <i>H. pylori</i> J166 significantly reduced its capacity to induce IL-8 (mean ± SEM of 3 replicates), which was recovered when the chromosomal WT <i>cagY</i> allele was restored (▵Y [J166]) by complementation (black bars). Two output strains from C57BL/6 mice with unique <i>cagY</i> alleles (mOut1, mOut2) lost the capacity to induce IL-8 (gray bars) and translocate CagA, although they expressed CagY (α-CagY). Complementation of ▵<i>cagY</i> with <i>cagY</i> from mOut1 (▵Y [mOut1]) or mOut2 (▵Y [mOut2]) recapitulated their lack of IL-8 induction (white bars) and translocation of phosphorylated CagA (α-PY99). Similarly, replacement with <i>cagY</i> from two output strains (mOut3, mOut4) that expressed a unique <i>cagY</i> but maintained the capacity to induce IL-8 (gray bars) and translocate CagA, also phenocopied their IL-8 induction and translocation of CagA. All strains expressed CagA (α-CagA), though only those that induced IL-8 had the capacity to translocate CagA that was tyrosine phosphorylated. Multiple bands in the CagY immunoblot could represent different transcription or translation products, or even protein fragments, but they are CagY-specific since they are absent in the <i>cagY</i> deletion mutant. **<i>P</i><0.01; ***<i>P</i><0.001.</p