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

Barrozo RM, Cooke CL, Hansen LM, Lam AM, Gaddy JA, Johnson EM, Cariaga TA, Suarez G, Peek RM, Cover TL, Solnick JV - PLoS Pathog. (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Center for Comparative Medicine, University of California Davis, Davis, California, United States of America.

ABSTRACT
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.

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Related in: MedlinePlus

Recombination in cagY during infection of rhesus monkeys is sufficient to reduce the capacity of H. pylori to induce IL-8 and translocate CagA.Deletion of cagY (▵Y) from WT H. pylori J166 significantly reduced its capacity to induce IL-8 (mean ± SEM of 3 replicates), which was recovered when the chromosomal WT cagY 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 cagY alleles (rOut1, rOut2) lost the capacity to induce IL-8 (gray bars) and translocate CagA, although they expressed CagY. Replacement of ▵cagY with cagY 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 cagY from an output strain (rOut3) that expressed a unique cagY 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 cagY deletion mutant. **P<0.01; ***P<0.001.
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ppat-1003189-g002: Recombination in cagY during infection of rhesus monkeys is sufficient to reduce the capacity of H. pylori to induce IL-8 and translocate CagA.Deletion of cagY (▵Y) from WT H. pylori J166 significantly reduced its capacity to induce IL-8 (mean ± SEM of 3 replicates), which was recovered when the chromosomal WT cagY 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 cagY alleles (rOut1, rOut2) lost the capacity to induce IL-8 (gray bars) and translocate CagA, although they expressed CagY. Replacement of ▵cagY with cagY 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 cagY from an output strain (rOut3) that expressed a unique cagY 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 cagY deletion mutant. **P<0.01; ***P<0.001.

Mentions: Recombination in cagY might be associated with changes in IL-8, but not mechanistically linked to the function of the cagPAI. Therefore, we used contraselection [26], [27] to replace the cagY in WT J166 with the cagY gene from rOut1 or rOut2, each of which induced low IL-8 and had a unique cagY RFLP pattern. The cagY gene from streptomycin resistant J166 was deleted by homologous recombination with the cat-rpsL cassette (chloramphenicol resistant, dominant streptomycin susceptible), and then transformed with chromosomal DNA from either WT J166 (restoring the WT cagY allele) or one of the two rhesus output strains. Transformants that were chloramphenicol susceptible and streptomycin resistant (due to loss of the cassette), and had the appropriate cagY gene by PCR-RFLP and confirmed by full-length DNA sequence analysis, were then tested for induction of IL-8 and translocation of CagA. As expected, deletion of cagY in J166 markedly reduced IL-8 induction, and replacement of the WT cagY allele restored expression of CagY and induction of IL-8 (Figure 2). In contrast, replacement with cagY from rOut1 and rOut2, which induced low IL-8, did not restore IL-8 induction, even though the CagY protein was expressed. Although it was uncommon, we also identified a rhesus output strain (rOut3) that induced IL-8 at a level similar to WT J166, but had a unique cagY allele. As expected, replacement of the WT cagY allele with cagY from rOut3 maintained the capacity to induce IL-8. Only those strains that induced IL-8 were also capable of inducing CagA translocation and phosphorylation. These results demonstrate that recombination in cagY is sufficient to alter the functionality of the T4SS encoded by the cagPAI.


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

Barrozo RM, Cooke CL, Hansen LM, Lam AM, Gaddy JA, Johnson EM, Cariaga TA, Suarez G, Peek RM, Cover TL, Solnick JV - PLoS Pathog. (2013)

Recombination in cagY during infection of rhesus monkeys is sufficient to reduce the capacity of H. pylori to induce IL-8 and translocate CagA.Deletion of cagY (▵Y) from WT H. pylori J166 significantly reduced its capacity to induce IL-8 (mean ± SEM of 3 replicates), which was recovered when the chromosomal WT cagY 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 cagY alleles (rOut1, rOut2) lost the capacity to induce IL-8 (gray bars) and translocate CagA, although they expressed CagY. Replacement of ▵cagY with cagY 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 cagY from an output strain (rOut3) that expressed a unique cagY 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 cagY deletion mutant. **P<0.01; ***P<0.001.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585145&req=5

ppat-1003189-g002: Recombination in cagY during infection of rhesus monkeys is sufficient to reduce the capacity of H. pylori to induce IL-8 and translocate CagA.Deletion of cagY (▵Y) from WT H. pylori J166 significantly reduced its capacity to induce IL-8 (mean ± SEM of 3 replicates), which was recovered when the chromosomal WT cagY 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 cagY alleles (rOut1, rOut2) lost the capacity to induce IL-8 (gray bars) and translocate CagA, although they expressed CagY. Replacement of ▵cagY with cagY 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 cagY from an output strain (rOut3) that expressed a unique cagY 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 cagY deletion mutant. **P<0.01; ***P<0.001.
Mentions: Recombination in cagY might be associated with changes in IL-8, but not mechanistically linked to the function of the cagPAI. Therefore, we used contraselection [26], [27] to replace the cagY in WT J166 with the cagY gene from rOut1 or rOut2, each of which induced low IL-8 and had a unique cagY RFLP pattern. The cagY gene from streptomycin resistant J166 was deleted by homologous recombination with the cat-rpsL cassette (chloramphenicol resistant, dominant streptomycin susceptible), and then transformed with chromosomal DNA from either WT J166 (restoring the WT cagY allele) or one of the two rhesus output strains. Transformants that were chloramphenicol susceptible and streptomycin resistant (due to loss of the cassette), and had the appropriate cagY gene by PCR-RFLP and confirmed by full-length DNA sequence analysis, were then tested for induction of IL-8 and translocation of CagA. As expected, deletion of cagY in J166 markedly reduced IL-8 induction, and replacement of the WT cagY allele restored expression of CagY and induction of IL-8 (Figure 2). In contrast, replacement with cagY from rOut1 and rOut2, which induced low IL-8, did not restore IL-8 induction, even though the CagY protein was expressed. Although it was uncommon, we also identified a rhesus output strain (rOut3) that induced IL-8 at a level similar to WT J166, but had a unique cagY allele. As expected, replacement of the WT cagY allele with cagY from rOut3 maintained the capacity to induce IL-8. Only those strains that induced IL-8 were also capable of inducing CagA translocation and phosphorylation. These results demonstrate that recombination in cagY is sufficient to alter the functionality of the T4SS encoded by the cagPAI.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Center for Comparative Medicine, University of California Davis, Davis, California, United States of America.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus