<i>Helicobacter pylori</i> modulates host cell responses by CagT4SS-dependent translocation of an intermediate metabolite of LPS inner core heptose biosynthesis

<div><p>Highly virulent <i>Helicobacter pylori</i> cause proinflammatory signaling inducing the transcriptional activation and secretion of cytokines such as IL-8 in epithelial cells. Responsible in part for this signaling is the <i>cag</i> pathogenicity island (<i>cag</i>PAI) that codetermines the risk for pathological sequelae of an <i>H</i>. <i>pylori</i> infection such as gastric cancer. The Cag type IV secretion system (CagT4SS), encoded on the <i>cag</i>PAI, can translocate various molecules into cells, the effector protein CagA, peptidoglycan metabolites and DNA. Although these transported molecules are known to contribute to cellular responses to some extent, a major part of the <i>cag</i>PAI-induced signaling leading to IL-8 secretion remains unexplained. We report here that biosynthesis of heptose-1,7-bisphosphate (HBP), an important intermediate metabolite of LPS inner heptose core, contributes in a major way to the <i>H</i>. <i>pylori cag</i>PAI-dependent induction of proinflammatory signaling and IL-8 secretion in human epithelial cells. Mutants defective in the genes required for synthesis of HBP exhibited a more than 95% reduction of IL-8 induction and impaired CagT4SS-dependent cellular signaling. The loss of HBP biosynthesis did not abolish the ability to translocate CagA. The human cellular adaptor TIFA, which was described before to mediate HBP-dependent activity in other Gram-negative bacteria, was crucial in the <i>cag</i>PAI- and HBP pathway-induced responses by <i>H</i>. <i>pylori</i> in different cell types. The active metabolite was present in <i>H</i>. <i>pylori</i> lysates but not enriched in bacterial supernatants. These novel results advance our mechanistic understanding of <i>H</i>. <i>pylori cag</i>PAI-dependent signaling mediated by intracellular pattern recognition receptors. They will also allow to better dissect immunomodulatory activities by <i>H</i>. <i>pylori</i> and to improve the possibilities of intervention in <i>cagPAI-</i> and inflammation-driven cancerogenesis.</p></div

Results of coincubation of parental, TIFA k/o and TIFA-complemented cell lines with <i>H</i>. <i>pylori</i> and its core heptose LPS biosynthesis mutants and detection of downstream signaling.

<p><b>A)</b> AGS <b>B)</b> HEK293T parental and CRISPR-Cas9 TIFA k/o cells (pool) were transiently transfected with either an empty vector or a vector expressing human TIFA, by lipofectamine 2000 (HEK) or nucleofection (AGS). On the next day, parental, TIFA k/o and TIFA-complemented cells were coincubated with <i>H</i>. <i>pylori</i> of indicated genotypes (mutants indicated by respective gene numbers) at an MOI of 25 for 4 h. Cell supernatants were analyzed for IL-8 secretion by ELISA. Statistical significance of differences was determined using two-tailed, non-paired Student's <i>t</i>-test <b>(</b>**p<0.01, ***p<0.001, ****p<0.0001). Panels<b>C)</b> and <b>D)</b> show qRT-PCR data, for il-8 <b>(C)</b> and cxcl-1 <b>(D)</b> transcript amounts for a panel of bacteria-coincubated AGS parental (wt) and TIFA k/o cells (<i>H</i>. <i>pylori</i> strain N6 and isogenic mutants). HP0527 = <i>cagY</i> inactivation mutant of strain N6, which was used as a control condition of a <i>cag</i>PAI functional-negative mutant in cell coincubations. Panel <b>E)</b> shows a Western blot to detect downstream signaling in AGS and HEK293T cells upon <i>H</i>. <i>pylori</i> coculture, dependent on HP0858 (HldE) activity. Cells were coincubated with <i>H</i>. <i>pylori</i> N6 and isogenic mutants for 4 h. Equal amounts of cellular proteins (20 μg) were separated on SDS gels, blotted and probed with antibodies as indicated to detect the <i>cag</i>PAI-dependent downstream activation of p38, TAK1 and IκBa (as reported in [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006514#ppat.1006514.ref050" target="_blank">50</a>]). Actin was detected as a loading control. LPS core heptose biosynthesis mutants in all panels are designated with gene names according to the nomenclature of strain 26695 as outlined in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006514#ppat.1006514.g001" target="_blank">Fig 1</a>. <b>F), G)</b> results of RT² Profiler RT-PCR arrays in 96 well format (Innate and Adaptive Immune Responses transcript panel selection; Qiagen.com). PCR arrays were performed on cDNA preparations of AGS cells (parental and TIFA knock-out (KO), coincubated with <i>H</i>. <i>pylori</i> N6 and its isogenic HP0858 mutant. Yellow dots represent over-expressed genes for condition on y-axis. Blue dots represent under-expressed genes for condition on y-axis. Panel <b>F)</b> shows the pairwise comparison of amounts of arrayed transcripts between AGS parental cells, coincubated with <i>H</i>. <i>pylori</i> N6 wild type (wt), versus the same cells coincubated with N6 HP0858 mutant. Panel <b>G)</b> depicts the pairwise comparison of transcript amounts between AGS parental cells coincubated with N6 wt and AGS TIFA k/o cells coincubated with the same strain (see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006514#ppat.1006514.s008" target="_blank">S8 Fig</a> for extended results; full results are summarized in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006514#ppat.1006514.s011" target="_blank">S2 Table</a>).</p

The <i>H</i>. <i>pylori hldE</i> (core LPS heptose biosynthesis) gene cluster, its proposed biosynthetic pathway, and strain characterization of <i>H</i>. <i>pylori hldE</i> gene cluster mutants.

<p><b>A</b>) Heptose phosphate biosynthesis gene cluster in <i>H</i>. <i>pylori</i>. Genes HP0861 through HP0857 of reference strain 26695 probably form an operon (see also [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006514#ppat.1006514.ref110" target="_blank">110</a>]). <b>B</b>) Biosynthesis pathway, LPS intermediates, metabolites and contributing enzymes of the core heptose pathway predicted in <i>H</i>. <i>pylori</i>. In red color, the biosynthesis functions of the bifunctional <i>hldE</i> gene (HP0858) in analogy to its <i>E</i>. <i>coli</i> ortholog are depicted within the pathway. The metabolite HBP, produced through the action of HldE enzyme, is highlighted by a red square. To the right, a schematic structure of <i>H</i>. <i>pylori</i> LPS is shown, indicating its main structural building blocks. The dashed line indicates the potential intermediate LPS structure in the absence of the GmhA/HldE pathway, which consists solely of the lipid A and the keto-deoxy-octonate (KDO) substructures. Gene designations refer to gene names in strain 26695. For references regarding LPS biosynthesis see main text. <b>C</b>) Growth characteristics of <i>H</i>. <i>pylori</i> strain N6 core heptose phosphate biosynthesis mutants (designated by strain 26695 gene numbers) in comparison to the N6 reference strain. Growth curves of single strains were performed in liquid culture for up to 48 h. <b>D</b>), <b>E</b>) Quantification of IL-8 secretion of AGS cells coincubated for 4 h with live <i>H</i>. <i>pylori</i> strains. Hp N6 = strain N6 shown in panels <b>D</b>, <b>F</b>, <b>G</b>; Hp P12 = strain P12 shown in panels <b>E</b>,<b>F</b>,<b>G</b>. <b>F</b>) silver-stained SDS gel of treated lysate (ETL) preparations (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006514#sec007" target="_blank">Methods</a>) from <i>H</i>. <i>pylori</i> and its core heptose biosynthesis mutants. Detected glycans include <i>H</i>. <i>pylori</i> LPS chains as annotated on the right: IC (inner core of LPS), LC (longer LPS chains, including outer core and O-antigen chains) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006514#ppat.1006514.ref016" target="_blank">16</a>] <b>G</b>) Determination of cell adherence of <i>H</i>. <i>pylori</i> wild type strains N6 and P12 and their isogenic core heptose biosynthesis mutants to AGS cells (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006514#sec007" target="_blank">Methods</a>). Strain designations in all figure panels refer to gene names in the <i>gmhA/hldE</i> cluster of strain 26695 as indicated in panel B. comp. = complemented. Statistically significant differences between strains during growth in liquid culture in panel <b>C</b> were calculated by two-way ANOVA, followed by Tukey’s multiple comparison test: **p<0.01, ****p<0.0001, they are depicted next to the graphs for the last time point (48 h); see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006514#ppat.1006514.s010" target="_blank">S1 Table</a> for full results. Statistical significances in <b>D)</b>, <b>E), G)</b> were determined by two-tailed, non-paired Student’s <i>t</i>-test; **p<0.01, ***p<0.001, ****p<0.0001, ns = non-significant.</p

<i>H</i>. <i>pylori</i> IL-8 activation is abolished in AGS TIFA k/o cells, while <i>H</i>. <i>pylori</i> CagA translocation is independent of TIFA expression, IL-8 induction, and core heptose biosynthesis pathway intermediates.

<p><b>A)</b> qRT-PCR detection of TIFA transcript in AGS wt and AGS TIFA k/o (KO) cells. Cells were mock-incubated or cocultured for 2 h with <i>H</i>. <i>pylori</i> strain N6 wild type (wt), isogenic <i>cagY</i> mutant (HP0527) and heptose pathway <i>hldE</i> (HP0858) or <i>rfaD</i> (HP0859) mutants as indicated. The TIFA transcript amounts are given in % of the mock-coincubated AGS parental cell, which was set to 100%. <b>B)</b> IL-8 induction in AGS CRISPR-Cas9 TIFA knock-out (KO) cells. Indicated <i>H</i>. <i>pylori</i> strains (N6 wt and isogenic core heptose mutants) were coincubated for 4 h at an MOI of 25 bacteria per cell with AGS parental cells, TIFA k/o cell single clone and TIFA k/o cell pool. HP0858 comp. is the complemented strain. Significance of differences (p) between N6 wt coincubated wild type and k/o cells were calculated by Student‘s <i>t</i>-test. <b>C)</b> CagA translocation by <i>H</i>. <i>pylori</i> N6 and isogenic <i>cagY</i> (HP0527; CagT4SS functional negative control) and LPS core heptose biosynthesis mutants in AGS cells and AGS TIFA KO cells. AGS cells were coincubated with <i>H</i>. <i>pylori</i> strain N6 wt or isogenic mutant bacteria for 4 h. 20 μg soluble protein (cleared cell lysates) per lane was separated on an SDS gel and blotted to nitrocellulose membrane. Western blots were incubated with antibodies as indicated. HP signifies the antiserum detection of heat-stable <i>H</i>. <i>pylori</i> surface antigens (Dako Cytomation, for antibodies see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006514#ppat.1006514.s015" target="_blank">S6 Table</a>) and was used as a universal control for amounts of invariable <i>H</i>. <i>pylori</i> proteins in the preparations. Actin detection was used as a loading control for amounts of AGS cell proteins. <b>D</b>) densitometric quantitation of CagA and p-CagA (CagA translocation) of Western blot results shown in panel <b>C</b> (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006514#sec007" target="_blank">Methods</a>). The intensity values were normalized to human actin and to HP invariable protein for each condition and are depicted in % of the positive control (AGS cells coincubated with <i>H</i>. <i>pylori</i> N6 wild type bacteria), which was set to 100%.</p

<i>H</i>. <i>pylori</i> soluble non-proteinaceous metabolite of the HldE-dependent heptose phosphate reaction activates cells when experimentally transfected.

<p><b>A), B), C)</b> HEK293T luciferase reporter cell transfection with treated lysate ETL preparations of <i>H</i>. <i>pylori</i> strains reveal HldE-dependent activation in a transient transfection setting using NF-κB luciferase plasmid (pNFκB-luc). <b>A</b>) transiently-transfected HEK293T luciferase reporter cells were coincubated for 3 h with live <i>H</i>. <i>pylori</i> of either wild type strain N6, its isogenic heptose core pathway mutants or the <i>cag</i>PAI function-negative mutant HP0527, as indicated, or parental strain 88–3887, its isogenic <i>cag</i>PAI deletion mutant (dcagPAI), or <i>cagA</i> mutant, respectively. <b>B</b>) transiently transfected HEK293T luciferase reporter cells were coincubated with ETL preparations of <i>H</i>. <i>pylori</i> strains, heptose pathway mutants, or <i>cag</i>PAI mutants added to the cell medium in the absence of transfection agent for 3 h (see silver-stained gel of ETL preparations in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006514#ppat.1006514.g001" target="_blank">Fig 1</a>); <b>C</b>) in the third assay set-up, transiently transfected HEK293T cells were super-transfected with ETL preparations of <i>H</i>. <i>pylori</i> strains and their isogenic mutants. In B) and C, ultrapure <i>H</i>. <i>pylori</i> N6 wt LPS was also coincubated or transfected as an additional control. Cells in <b>A</b>), <b>B</b>), <b>C</b>) were incubated prior to the luciferase measurement for 3 h. All assay conditions were measured in triplicates. Two-tailed, non-paired Student’s <i>t</i>-test indicates significant differences of *p<0.05, **p<0.01, ***p<0.001, ns = not significant. <b>D</b>) time-dependent activation of HEK-Blue Null1 SEAP reporter cells (Invivogen) after transfection with treated lysate preparations (ETL) from <i>H</i>. <i>pylori</i> N6 and its isogenic core heptose biosynthesis mutants. Cell activation by release of secreted alkaline phosphatase into the medium was monitored over a time course of 25 hours post transfection using HEK-Blue real time detection medium (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006514#sec007" target="_blank">Methods</a>). All conditions were assayed in triplicates. Statistical significance of differences in D) was calculated by Student’s <i>t</i>-test for comparisons between mock and all other conditions (blue symbols) and HPN6 wt and all other conditions (red symbols); ns = non-significant; **p<0.01; ***p<0.001; ****p<0.0001.</p

<i>Helicobacter hepaticus</i> Induces an Inflammatory Response in Primary Human Hepatocytes

<div><p><i>Helicobacter hepaticus</i> can lead to chronic hepatitis and hepatocellular carcinoma in certain strains of mice. Until now the pathogenic role of <i>Helicobacter species</i> on human liver tissue is still not clarified though <i>Helicobacter species</i> identification in human liver cancer was successful in case controlled studies. Therefore we established an <i>in vitro</i> model to investigate the interaction of primary human hepatocytes (PHH) with <i>Helicobacter hepaticus</i>. Successful co-culturing of PHH with <i>Helicobacter hepaticus</i> was confirmed by visualization of motile bacteria by two-photon-microscopy. Isolated human monocytes were stimulated with PHH conditioned media. Changes in mRNA expression of acute phase cytokines and proteins in PHH and stimulated monocytes were determined by Real-time PCR. Furthermore, cytokines and proteins were analyzed in PHH culture supernatants by ELISA. Co-cultivation with <i>Helicobacter hepaticus</i> induced mRNA expression of Interleukin-1 beta (IL-1β), Tumor necrosis factor-alpha, Interleukin-8 (IL-8) and Monocyte chemotactic protein-1 (MCP-1) in PHH (p<0.05) resulting in a corresponding increase of IL-8 and MCP-1 concentrations in PHH supernatants (p<0.05). IL-8 and IL-1β mRNA expression was induced in monocytes stimulated with <i>Helicobacter hepaticus</i> infected PHH conditioned media (p<0.05). An increase of Cyclooxygenase-2 mRNA expression was observed, with a concomitant increase of prostaglandin E2 concentration in PHH supernatants at 24 and 48 h (p<0.05). In contrast, at day 7 of co-culture, no persistent elevation of cytokine mRNA could be detected. High expression of intercellular adhesion molecule-1 on PHH cell membranes after co-culture was shown by two-photon-microscopy and confirmed by flow-cytomety. Finally, expression of Cytochrome P450 3A4 and albumin mRNA were downregulated, indicating an impairment of hepatocyte synthesis function by <i>Helicobacter hepaticus</i> presence. This is the first <i>in vitro</i> model demonstrating a pathogenic effect of a <i>Helicobacter spp</i>. on human liver cells, resulting in an inflammatory response with increased synthesis of inflammatory mediators and consecutive monocyte activation.</p></div

Primary human hepatocyte (PHH) conditioned media infected with <i>H. hepaticus</i> activate monocytes.

<p>Human CD14⁺ monocytes were isolated from peripheral blood of healthy donors and seeded at 10.000 cells/well. Monocytes were stimulated 24 hours with 100 µl of centrifuged PHH conditioned media infected with <i>H. hepaticus</i> at a MOI of 50 (70×10⁶ Hh) or 100 (140×10⁶ Hh) bacteria per hepatocyte for 48 hours (n = 3). Real-time RT PCR of monocytes revealed a dose-dependent increase of the mRNA expression of (A) Interleukin-1 beta (IL-1 beta) and (B) Interleukin-8 (IL-8). Cytokine mRNA is expressed as the fold induction compared to untreated control culture from the same hepatocyte donor for each time point (A-B). (n = 3). * = p<0.05 and ** = p<0.005 versus control.</p

Primary human hepatocytes (PHH) express acute phase cytokines in response to co-cultivation with <i>H. hepaticus</i>.

<p>PHH were co-cultured with <i>H. hepaticus ATCC 51449</i> at a MOI of 50 (70×10⁶ Hh) or 100 (140×10⁶ Hh) bacteria per hepatocyte (n = 3). Real-time RT PCR of hepatocytes revealed increased mRNA expression of the acute phase cytokines Monocyte chemotactic protein 1 (MCP-1) (A), Interleukin 8 (IL-8) (B), Tumor necrosis factor alpha (TNF-alpha) (C) and Interleukin 1 beta (IL-1 beta) (D). Cytokine mRNA is expressed as the fold induction compared to untreated control culture from the same hepatocyte donor for each time point (A-D). Up-regulation of acute phase cytokine mRNA in hepatocytes resulted in an increased IL-8 (E) and MCP-1 (F) concentration in the supernatant of infected PHH as measured by ELISA. Data shown as mean±SEM, n = 3. * = p<0.05 versus control.</p

<i>H. hepaticus</i> ATCC 51449 induces an early transient COX-2 expression, resulting in an increased PGE2-synthesis by primary human hepatocytes (PHH).

<p>PHH were co-cultured with <i>H. hepaticus</i> at a MOI of 50 (70×10⁶ Hh) or 100 (140×10⁶ Hh) bacteria per hepatocyte (n = 3). (A) Real-time RT PCR of hepatocytes revealed significantly increased mRNA expression of COX-2 6 hours after infection. COX-2 mRNA is expressed as the fold induction compared to untreated control culture from the same hepatocyte donor for each time point. (B) Induction of COX-2 led to an increased synthesis of PGE2 by PHH as measured in the supernatant of co-cultured hepatocytes by ELISA with maximum concentration at 24 hours after infection with <i>H. hepaticus</i> at a MOI of 100 cells per hepatocyte (n = 3). * = p<0.05 versus control.</p

ICAM-1 mRNA expression and ICAM-1 incorporation into primary human hepatocytes (PHH) cell membranes are increased after <i>H. hepaticus</i> exposure.

<p>PHH were co-cultured with <i>H. hepaticus</i> at a MOI of 50 (70×10⁶ Hh) or 100 (140×10⁶ Hh) bacteria per hepatocyte (n = 3). (A) Real-time RT PCR of hepatocytes showed increased mRNA expression of ICAM-1 2 and 6 hours after infection. ICAM-1 mRNA expression is displayed as x-fold induction compared to untreated control cultures from the same hepatocyte donor for each time point. (B) PHH were co-cultured with <i>H. hepaticus</i> for 48 and 96 hours at a MOI of 100 per hepatocyte. Subsequently, hepatocytes were harvested, stained with an AlexaFluor488-labeled anti-human ICAM-1 antibody, and analyzed by flow cytometry. Flow cytometry results confirmed an increased incorporation of ICAM-1 into cell membranes of PHH under co-culture conditions with <i>H. hepaticus</i>. * = p<0.05 versus the control culture. (C) After 48 hours of PHH culture in the absence or presence of <i>H. hepaticus</i> at a MOI of 100 per hepatocyte, cell surface staining for ICAM-1 was performed with an AlexaFluor488-labeled anti-human ICAM-1 antibody (green). <i>H. hepaticus</i> and hepatocyte nuclei were stained with DAPI (white). Representative snapshots are shown taken from two-photon microscopy time lapse recordings of PHH cultures in the absence (control, upper row) or presence (with bacteria, lower row) of <i>H. hepaticus</i> (n = 3). Aquisition settings (TiSa laser tuned to 760 nm, 485 nm long pass filter, no bandpass filters) and snapshot image processing were identical for all recordings. While surface ICAM-1 staining was virtually absent in control cultures (open red arrow heads), PHH cultivated in the presence of <i>H. hepaticus</i> displayed a marked ICAM-1 expression on the cell surface (filled red arrow heads).</p