196,092 research outputs found
CagY Is an Immune-Sensitive Regulator of the Helicobacter pylori Type IV Secretion System
No full textBACKGROUND & AIMS: Peptic ulcer disease and gastric cancer are caused most often by Helicobacter pylori strains that harbor the cag pathogenicity island, which encodes a type IV secretion system (T4SS) that injects the CagA oncoprotein into host cells. cagY is an essential gene in the T4SS and has an unusual DNA repeat structure that predicts in-frame insertions and deletions. These cagY recombination events typically lead to a reduction in T4SS function in mouse and primate models. We examined the role of the immune response in cagY-dependent modulation of T4SS function.
METHODS: H pylori T4SS function was assessed by measuring CagA translocation and the capacity to induce interleukin (IL)8 in gastric epithelial cells. cagY recombination was determined by changes in polymerase chain reaction restriction fragment-length polymorphisms. T4SS function and cagY in H pylori from C57BL/6 mice were compared with strains recovered from Rag1-/- mice, T- and B-cell-deficient mice, mice with deletion of the interferon gamma receptor (IFNGR) or IL10, and Rag1-/- mice that received adoptive transfer of control or Ifng-/- CD4+ T cells. To assess relevance to human beings, T4SS function and cagY recombination were assessed in strains obtained sequentially from a patient after 7.4 years of infection.
RESULTS: H pylori infection of T-cell-deficient and Ifngr1-/- mice, and transfer of CD4+ T cells to Rag1-/- mice, showed that cagY-mediated loss of T4SS function requires a T-helper 1-mediated immune response. Loss of T4SS function and cagY recombination were more pronounced in Il10-/- mice, and in control mice infected with H pylori that expressed a more inflammatory form of cagY. Complementation analysis of H pylori strains isolated from a patient over time showed changes in T4SS function that were dependent on recombination in cagY.
CONCLUSIONS: Analysis of H pylori strains from mice and from a chronically infected patient showed that CagY functions as an immune-sensitive regulator of T4SS function. We propose that this is a bacterial adaptation to maximize persistent infection and transmission to a new host under conditions of a robust inflammatory response
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.
No full text<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
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.
No full text<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
Toll-like Receptor 5 Activation by the CagY Repeat Domains of Helicobacter pylori.
No full textHelicobacter pylori (Hp) is an important human pathogen associated with gastric inflammation and neoplasia. It is commonly believed that this bacterium avoids major immune recognition by Toll-like receptors (TLRs) because of low intrinsic activity of its flagellin and lipopolysaccharides (LPS). In particular, TLR5 specifically detects flagellins in various bacterial pathogens, while Hp evolved mutations in flagellin to evade detection through TLR5. Cancerogenic Hp strains encode a type IV secretion system (T4SS). The T4SS core component and pilus-associated protein CagY, a large VirB10 ortholog, drives effector molecule translocation. Here, we identify CagY as a flagellin-independent TLR5 agonist. We detect five TLR5 interaction sites, promoting binding of CagY-positive Hp to TLR5-expressing cells, TLR5 stimulation, and intracellular signal transduction. Consequently, CagY constitutes a remarkable VirB10 member detected by TLR5, driving crucial innate immune responses by this human pathogen
CagY decorates the <i>H. pylori</i> bacterial surface but is not associated with T4SS pili.
No full text<p><i>H. pylori</i> was co-cultured with AGS gastric cells at an MOI of 100∶1, incubated with antibodies to the CagY MRR or CagA, and imaged by FEG-SEM in the environmental mode. CagY was detected on the bacterial surface of the WT strain but was not associated with pili. CagA was detected both on the bacterial surface and in close approximation to the tips of the pili of the WT strain. There was markedly reduced CagA labeling on the surface of ▵<i>cagY</i> mutant strain compared to the WT strain. No staining was seen when primary antibody was omitted. Pili are sometimes not as well visualized and more often appear broken in these images compared to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003189#ppat-1003189-g007" target="_blank">Figure 7</a> due to the lack of metal coating and more frequent washes. Magnification bars indicate 500 nm.</p
<i>cagY</i> variants that fail to induce IL-8 and translocate CagA do not induce expression of NF-κB.
No full text<p>(A) Co-culture of <i>H. pylori</i> with AGS cells stably transformed with a reporter plasmid demonstrated that activation of NF-κB was seen in WT J166 but not in a strain with a deletion of the <i>cag</i>PAI (▵PAI). Reintroduction of J166 <i>cagY</i> into a <i>cagY</i> deletion mutant restored NF-κB activation. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003189#s1" target="_blank">Introduction</a> of <i>cagY</i> from monkey (B) or mouse (C) output strains showed that increased NF-κB activation compared to ▵<i>cagY</i> (▵Y) or ▵PAI was seen only in strains bearing a <i>cagY</i> allele that was competent for induction of IL-8 and translocation of CagA (rOut3, mOut3, mOut4). ***<i>P</i><0.001.</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.
No full text<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
CagY-dependent regulation of type IV secretion in<i>Helicobacter pylori</i>is associated with alterations in integrin binding
No full textABSTRACTStrains ofHelicobacter pylorithat cause ulcer or gastric cancer typically express a type IV secretion system (T4SS) encoded by thecagpathogenicity island (PAI). CagY is an ortholog of VirB10 that, unlike other VirB10 orthologs, has a large middle repeat region (MRR) with extensive repetitive sequence motifs, which undergo CD4+ T cell-dependent recombination during infection of mice. Recombination in the CagY MRR reduces T4SS function, diminishes the host inflammatory response, and enables the bacteria to colonize at a higher density. Since CagY is known to bind human α5β1integrin, we tested the hypothesis that recombination in the CagY MRR regulates T4SS function by modulating binding to α5β1integrin. Using a cell-free microfluidic assay, we found thatH. pyloribinding to α5β1integrin under shear flow is dependent on the CagY MRR, but independent of the presence of the T4SS pili, which are only formed whenH. pyloriis in contact with host cells. Similarly, expression of CagY in the absence of other T4SS genes was necessary and sufficient for whole bacterial cell binding to α5β1integrin. Bacteria with variantcagYalleles that reduced T4SS function showed comparable reduction in binding to α5β1integrin, though CagY was still expressed on the bacterial surface. We speculate thatcagY-dependent modulation ofH. pyloriT4SS function is mediated by alterations in binding to α5β1integrin, which in turn regulates the host inflammatory response so as to maximize persistent infection.IMPORTANCEInfection withH. pylorican cause peptic ulcers, and is the most important risk factor for gastric cancer, the third most common cause of cancer death worldwide. The majorH. pylorivirulence factor that determines whether infection causes disease or asymptomatic colonization is the type IV secretion system (T4SS), a sort of molecular syringe that injects bacterial products into gastric epithelial cells and alters host cell physiology. We previously showed that recombination in CagY, an essential T4SS component, modulates the function of the T4SS. Here we found that these recombination events produce parallel changes in specific binding to α5β1integrin, a host cell receptor that is essential for T4SS-dependent translocation of bacterial effectors. We propose that CagY-dependent binding to α5β1integrin acts like a molecular rheostat that alters T4SS function and modulates the host immune response to promote persistent infection.</jats:sec
CagY-Dependent Regulation of Type IV Secretion in Helicobacter pylori Is Associated with Alterations in Integrin Binding
No full textABSTRACT
Strains of
Helicobacter pylori
that cause ulcer or gastric cancer typically express a type IV secretion system (T4SS) encoded by the
cag
pathogenicity island (
cag
PAI). CagY is an ortholog of VirB10 that, unlike other VirB10 orthologs, has a large middle repeat region (MRR) with extensive repetitive sequence motifs, which undergo CD4
+
T cell-dependent recombination during infection of mice. Recombination in the CagY MRR reduces T4SS function, diminishes the host inflammatory response, and enables the bacteria to colonize at a higher density. Since CagY is known to bind human α
5
β
1
integrin, we tested the hypothesis that recombination in the CagY MRR regulates T4SS function by modulating binding to α
5
β
1
integrin. Using a cell-free microfluidic assay, we found that
H. pylori
binding to α
5
β
1
integrin under shear flow is dependent on the CagY MRR, but independent of the presence of the T4SS pili, which are only formed when
H. pylori
is in contact with host cells. Similarly, expression of CagY in the absence of other T4SS genes was necessary and sufficient for whole bacterial cell binding to α
5
β
1
integrin. Bacteria with variant
cagY
alleles that reduced T4SS function showed comparable reduction in binding to α
5
β
1
integrin, although CagY was still expressed on the bacterial surface. We speculate that
cagY-
dependent modulation of
H. pylori
T4SS function is mediated by alterations in binding to α
5
β
1
integrin, which in turn regulates the host inflammatory response so as to maximize persistent infection.
IMPORTANCE
Infection with
H. pylori
can cause peptic ulcers and is the most important risk factor for gastric cancer, the third most common cause of cancer death worldwide. The major
H. pylori
virulence factor that determines whether infection causes disease or asymptomatic colonization is the type IV secretion system (T4SS), a sort of molecular syringe that injects bacterial products into gastric epithelial cells and alters host cell physiology. We previously showed that recombination in CagY, an essential T4SS component, modulates the function of the T4SS. Here we found that these recombination events produce parallel changes in specific binding to α
5
β
1
integrin, a host cell receptor that is essential for T4SS-dependent translocation of bacterial effectors. We propose that CagY-dependent binding to α
5
β
1
integrin acts like a molecular rheostat that alters T4SS function and modulates the host immune response to promote persistent infection.
</jats:p
Statistical evoked potential detection with number of degrees of freedom estimated from EEG autocorrelation function
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