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<front>
<journal-meta>
<journal-id journal-id-type="publisher-id">Front. Cell. Infect. Microbiol.</journal-id>
<journal-title>Frontiers in Cellular and Infection Microbiology</journal-title>
<abbrev-journal-title abbrev-type="pubmed">Front. Cell. Infect. Microbiol.</abbrev-journal-title>
<issn pub-type="epub">2235-2988</issn>
<publisher>
<publisher-name>Frontiers Media S.A.</publisher-name>
</publisher>
</journal-meta>
<article-meta>
<article-id pub-id-type="doi">10.3389/fcimb.2024.1377077</article-id>
<article-categories>
<subj-group subj-group-type="heading">
<subject>Cellular and Infection Microbiology</subject>
<subj-group>
<subject>Original Research</subject>
</subj-group>
</subj-group>
</article-categories>
<title-group>
<article-title>
<italic>Helicobacter pylori</italic> glycan biosynthesis modulates host immune cell recognition and response</article-title>
</title-group>
<contrib-group>
<contrib contrib-type="author">
<name>
<surname>Barrett</surname>
<given-names>Katharine A.</given-names>
</name>
<uri xlink:href="https://loop.frontiersin.org/people/2643162"/>
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<role content-type="https://credit.niso.org/contributor-roles/investigation/"/>
<role content-type="https://credit.niso.org/contributor-roles/methodology/"/>
<role content-type="https://credit.niso.org/contributor-roles/validation/"/>
<role content-type="https://credit.niso.org/contributor-roles/visualization/"/>
<role content-type="https://credit.niso.org/contributor-roles/writing-original-draft/"/>
<role content-type="https://credit.niso.org/contributor-roles/writing-review-editing/"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Kassama</surname>
<given-names>Francis Jacob</given-names>
</name>
<uri xlink:href="https://loop.frontiersin.org/people/2643592"/>
<role content-type="https://credit.niso.org/contributor-roles/conceptualization/"/>
<role content-type="https://credit.niso.org/contributor-roles/formal-analysis/"/>
<role content-type="https://credit.niso.org/contributor-roles/investigation/"/>
<role content-type="https://credit.niso.org/contributor-roles/methodology/"/>
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</contrib>
<contrib contrib-type="author">
<name>
<surname>Surks</surname>
<given-names>William</given-names>
</name>
<role content-type="https://credit.niso.org/contributor-roles/formal-analysis/"/>
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</contrib>
<contrib contrib-type="author">
<name>
<surname>Mulholland</surname>
<given-names>Andrew J.</given-names>
</name>
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</contrib>
<contrib contrib-type="author">
<name>
<surname>Moulton</surname>
<given-names>Karen D.</given-names>
</name>
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</contrib>
<contrib contrib-type="author" corresp="yes">
<name>
<surname>Dube</surname>
<given-names>Danielle H.</given-names>
</name>
<xref ref-type="author-notes" rid="fn001">
<sup>*</sup>
</xref>
<uri xlink:href="https://loop.frontiersin.org/people/2641428"/>
<role content-type="https://credit.niso.org/contributor-roles/conceptualization/"/>
<role content-type="https://credit.niso.org/contributor-roles/funding-acquisition/"/>
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</contrib>
</contrib-group>
<aff id="aff1">
<institution>Department of Chemistry &amp; Biochemistry, Bowdoin College</institution>, <addr-line>Brunswick, ME</addr-line>, <country>United States</country>
</aff>
<author-notes>
<fn fn-type="edited-by">
<p>Edited by: Christopher W. Reid, Bryant University, United States</p>
</fn>
<fn fn-type="edited-by">
<p>Reviewed by: Anna Katrina Walduck, Charles Sturt University, Australia</p>
<p>Timothy Cover, Vanderbilt University, United States</p>
<p>Catherine Grimes, University of Delaware, United States</p>
</fn>
<fn fn-type="corresp" id="fn001">
<p>*Correspondence: Danielle H. Dube, <email xlink:href="mailto:ddube@bowdoin.edu">ddube@bowdoin.edu</email>
</p>
</fn>
</author-notes>
<pub-date pub-type="epub">
<day>20</day>
<month>03</month>
<year>2024</year>
</pub-date>
<pub-date pub-type="collection">
<year>2024</year>
</pub-date>
<volume>14</volume>
<elocation-id>1377077</elocation-id>
<history>
<date date-type="received">
<day>26</day>
<month>01</month>
<year>2024</year>
</date>
<date date-type="accepted">
<day>11</day>
<month>03</month>
<year>2024</year>
</date>
</history>
<permissions>
<copyright-statement>Copyright &#xa9; 2024 Barrett, Kassama, Surks, Mulholland, Moulton and Dube</copyright-statement>
<copyright-year>2024</copyright-year>
<copyright-holder>Barrett, Kassama, Surks, Mulholland, Moulton and Dube</copyright-holder>
<license xlink:href="http://creativecommons.org/licenses/by/4.0/">
<p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</p>
</license>
</permissions>
<abstract>
<sec>
<title>Introduction</title>
<p>The pathogenic bacterium <italic>Helicobacter pylori</italic> has evolved glycan-mediated mechanisms to evade host immune defenses. This study tests the hypothesis that genetic disruption of <italic>H. pylori</italic> glycan biosynthesis alters immune recognition and response by human gastric epithelial cells and monocyte-derived dendritic cells.</p>
</sec>
<sec>
<title>Methods</title>
<p>To test this hypothesis, human cell lines were challenged with wildtype <italic>H. pylori</italic> alongside an array of <italic>H. pylori</italic> glycosylation mutants. The relative levels of immune response were measured via immature dendritic cell maturation and cytokine secretion.</p>
</sec>
<sec>
<title>Results</title>
<p>Our findings indicate that disruption of lipopolysaccharide biosynthesis diminishes gastric cytokine production, without disrupting dendritic cell recognition and activation. In contrast, variable immune responses were observed in protein glycosylation mutants which prompted us to test the hypothesis that phase variation plays a role in regulating bacterial cell surface glycosylation and subsequent immune recognition. Lewis antigen presentation does not correlate with extent of immune response, while the extent of lipopolysaccharide O-antigen elaboration does.</p>
</sec>
<sec>
<title>Discussion</title>
<p>The outcomes of this study demonstrate that <italic>H. pylori</italic> glycans modulate the host immune response. This work provides a foundation to pursue immune-based tailoring of bacterial glycans towards modulating immunogenicity of microbial pathogens.</p>
</sec>
</abstract>
<kwd-group>
<kwd>glycan</kwd>
<kwd>immunology</kwd>
<kwd>glycosylation mutant</kwd>
<kwd>phase variation</kwd>
<kwd>
<italic>Helicobacter pylori</italic>
</kwd>
<kwd>metabolic labeling</kwd>
</kwd-group>
<contract-num rid="cn001">R15GM109397, P20GM103423</contract-num>
<contract-num rid="cn002">Beckman Scholar Award</contract-num>
<contract-num rid="cn003">Littlefield Summer Research Fellowship, Fall Research Award</contract-num>
<contract-sponsor id="cn001">National Institutes of Health<named-content content-type="fundref-id">10.13039/100000002</named-content>
</contract-sponsor>
<contract-sponsor id="cn002">Arnold and Mabel Beckman Foundation<named-content content-type="fundref-id">10.13039/100000997</named-content>
</contract-sponsor>
<contract-sponsor id="cn003">Bowdoin College<named-content content-type="fundref-id">10.13039/100015587</named-content>
</contract-sponsor>
<counts>
<fig-count count="7"/>
<table-count count="0"/>
<equation-count count="0"/>
<ref-count count="96"/>
<page-count count="15"/>
<word-count count="7740"/>
</counts>
<custom-meta-wrap>
<custom-meta>
<meta-name>section-in-acceptance</meta-name>
<meta-value>Microbes and Innate Immunity</meta-value>
</custom-meta>
</custom-meta-wrap>
</article-meta>
</front>
<body>
<sec id="s1" sec-type="intro">
<label>1</label>
<title>Introduction</title>
<p>Antibiotics are losing their therapeutic efficacy due to the evolution and spread of resistance mechanisms in bacterial pathogens (<xref ref-type="bibr" rid="B85">Thung et&#xa0;al., 2016</xref>). The gram-negative gastric bacterium <italic>Helicobacter pylori</italic> is on the World Health Organization&#x2019;s list of antibiotic-resistant bacteria of highest priority for new antibiotic development (<xref ref-type="bibr" rid="B24">Dong and Graham, 2017</xref>; <xref ref-type="bibr" rid="B87">Tshibangu-Kabamba and Yamaoka, 2021</xref>). <italic>H. pylori</italic> is an opportunistic, gram-negative bacteria present in the gastrointestinal tract of roughly 50% of people worldwide (<xref ref-type="bibr" rid="B26">Dunne et&#xa0;al., 2014</xref>). Despite its relative ubiquity, <italic>H. pylori</italic> only causes severe pathologies in roughly 15% of cases, in which the infection may transform quickly from digestive issues and energy depletion to peptic ulcer disease and gastric carcinomas (<xref ref-type="bibr" rid="B82">Suerbaum and Michetti, 2002</xref>; <xref ref-type="bibr" rid="B15">Chen et&#xa0;al., 2013</xref>; <xref ref-type="bibr" rid="B40">Kim, 2016</xref>; <xref ref-type="bibr" rid="B11">Bravo et&#xa0;al., 2018</xref>; <xref ref-type="bibr" rid="B59">Malfertheiner et al., 2023</xref>). Recent studies suggest links between <italic>H. pylori</italic> pathogenesis and extra-gastric diseases, including cardiovascular, neurologic, and dermatologic conditions (<xref ref-type="bibr" rid="B34">Gravina et&#xa0;al., 2018</xref>). Moreover, low-income communities are at higher risk for infection, as transmission is likely based in contaminated drinking water and ill-resourced sanitation systems (<xref ref-type="bibr" rid="B40">Kim, 2016</xref>; <xref ref-type="bibr" rid="B49">Lim et&#xa0;al., 2013</xref>; <xref ref-type="bibr" rid="B81">Stefano et&#xa0;al., 2018</xref>). <italic>H. pylori</italic> can persistently colonize the human stomach for decades despite development of an immune response. Further, recurrence of infection after initial eradication due to ineffective treatment, dormant state changes, or reinfection poses a threat within the current climate of increased antibiotic resistance (<xref ref-type="bibr" rid="B13">Cellini et&#xa0;al., 2008</xref>; <xref ref-type="bibr" rid="B62">Moya and Crissinger, 2012</xref>; <xref ref-type="bibr" rid="B36">Hu et&#xa0;al., 2017</xref>; <xref ref-type="bibr" rid="B73">Reshetnyak and Reshetnyak, 2017</xref>; <xref ref-type="bibr" rid="B23">Di Fermo et&#xa0;al., 2023</xref>). These factors point to an urgent need to identify novel approaches to mitigate <italic>H. pylori</italic> pathogenesis.</p>
<p>
<italic>H. pylori&#x2019;s</italic> pathogenesis in some environments and dormancy in others suggests it may be possible to modulate the deleterious consequences of infection. In particular, extensive research demonstrates that localized host immune response to <italic>H. pylori</italic> is directly correlated with patient outcome. Upregulation of pro-inflammatory signaling in <italic>H. pylori</italic> infection, such as the secretion of interleukin-8 and other cytokines (<xref ref-type="bibr" rid="B29">El Filaly et&#xa0;al., 2023</xref>), drives the chronic inflammation underpinning gastritis and increases risk for peptic ulcer disease and gastric carcinomas (<xref ref-type="bibr" rid="B74">Robinson et&#xa0;al., 2007</xref>; <xref ref-type="bibr" rid="B43">Lamb and Chen, 2013</xref>; <xref ref-type="bibr" rid="B92">White and Winter, 2015</xref>). Thus, tailoring host immune response has the potential to alter the course of infection, promoting eradication while minimizing downstream consequences of chronic inflammation.</p>
<p>Several factors have been identified that contribute to chronic inflammation and carcinogenesis (<xref ref-type="bibr" rid="B7">Baj et&#xa0;al., 2020</xref>). <italic>H. pylori</italic> produces proteases to degrade mucins protecting epithelial cells and trigger cytokine production by the immune system (<xref ref-type="bibr" rid="B12">Byrd et&#xa0;al., 2000</xref>; <xref ref-type="bibr" rid="B96">Yoshimura et&#xa0;al., 2002</xref>; <xref ref-type="bibr" rid="B69">Posselt et&#xa0;al., 2017</xref>). Moreover, <italic>H. pylori</italic> secretes urease to yield ammonia and neutralize the acidic stomach, and this protein drives inflammation by activating immune cells (<xref ref-type="bibr" rid="B63">Olivera-Severo et&#xa0;al., 2017</xref>). Data indicate that <italic>H. pylori</italic> expression levels of certain adhesins, BabA and SabA (<xref ref-type="bibr" rid="B25">Doohan et&#xa0;al., 2021</xref>), toxins, VacA (<xref ref-type="bibr" rid="B18">Cover and Blanke, 2005</xref>), and other virulence-associated proteins, OipA and DupA, increase pathogenicity of infection (<xref ref-type="bibr" rid="B95">Yamaoka et&#xa0;al., 2002</xref>; <xref ref-type="bibr" rid="B56">Lu et&#xa0;al., 2005</xref>). Notoriously, upregulation of <italic>H. pylori</italic> CagA protein and the type IV secretion system encoded by the <italic>cagA</italic> pathogenicity island leads to injection of CagA, peptidoglycan, and other molecules into host cells, greatly increasing inflammation (<xref ref-type="bibr" rid="B89">Vannini et&#xa0;al., 2014</xref>). Gastric inflammation driven by CagA is so severe that it is correlated with gastric carcinogenesis and CagA is considered oncogenic (<xref ref-type="bibr" rid="B83">Tanaka et&#xa0;al., 2010</xref>; <xref ref-type="bibr" rid="B37">Jim&#xe9;nez-Soto and Haas, 2016</xref>). However, the correlation between these factors and patient pathology is not always linear; other determinants such as host genotype, age of infection, and environmental factors additionally complicate the distribution of inflammation, gastritis, and subsequent pathologies (<xref ref-type="bibr" rid="B31">El-Omar et&#xa0;al., 2003</xref>; <xref ref-type="bibr" rid="B58">MaChado et&#xa0;al., 2003</xref>; <xref ref-type="bibr" rid="B56">Lu et&#xa0;al., 2005</xref>). Regardless of its source, inflammation levels and the unique immune response induced by an <italic>H. pylori</italic> infection determine disease outcome (<xref ref-type="bibr" rid="B74">Robinson et&#xa0;al., 2007</xref>; <xref ref-type="bibr" rid="B92">White and Winter, 2015</xref>; <xref ref-type="bibr" rid="B33">Gobert and Wilson, 2022</xref>). Further investigating pathways by which <italic>H. pylori</italic> evades or stimulates human immune recognition will promote the development of immune-based treatments to bacterial infection.</p>
<p>The glycocalyx, which serves as the interface for host-bacterial interactions, may be key to understanding and ultimately modulating <italic>H. pylori</italic>&#x2019;s immunogenicity. <italic>H. pylori</italic> adorns its cell envelope with carbohydrates termed glycans (<xref ref-type="bibr" rid="B86">Tra and Dube, 2014</xref>). Broadly, bacterial glycans play roles in cell adhesion, host mimicry, and immune evasion (<xref ref-type="bibr" rid="B86">Tra and Dube, 2014</xref>; <xref ref-type="bibr" rid="B70">Prado Acosta and Lepenies, 2019</xref>; <xref ref-type="bibr" rid="B1">Alemka et&#xa0;al., 2013</xref>; <xref ref-type="bibr" rid="B93">Williams et&#xa0;al., 2020</xref>; <xref ref-type="bibr" rid="B61">Moulton et&#xa0;al., 2020</xref>). <italic>H. pylori</italic> installs Lewis antigen tetrasaccharides at the terminus of lipopolysaccharide (LPS) in its outer membrane; these epitopes are commonly found on human blood cells and assist the pathogen in evading the host immune response (<xref ref-type="bibr" rid="B90">Wang et&#xa0;al., 2000</xref>; <xref ref-type="bibr" rid="B78">Simoons-Smit et&#xa0;al., 1996</xref>; <xref ref-type="bibr" rid="B72">Rad et&#xa0;al., 2002</xref>). Furthermore, <italic>H. pylori</italic> adhesin protein glycosylation is essential to their function of binding to host gastric epithelial cells (<xref ref-type="bibr" rid="B14">Champasa et&#xa0;al., 2013</xref>; <xref ref-type="bibr" rid="B84">Teng et&#xa0;al., 2022</xref>). Given previous demonstrations of how bacterial glycans mediate host immune recognition and response, from host recognition of lipopolysaccharide and peptidoglycan by pattern recognition receptors (e.g., Toll-like receptors) to trigger an immune response to bacteria using glycan mimicry to engage immune tolerance receptors (e.g., DC-SIGN) (<xref ref-type="bibr" rid="B1">Alemka et&#xa0;al., 2013</xref>; <xref ref-type="bibr" rid="B79">Smedley et&#xa0;al., 2005</xref>), we hypothesized that <italic>H. pylori</italic>&#x2019;s cell surface glycans play a key role in modulating host immune responses by stimulating chronic inflammatory signals and obstructing detection by professional antigen-presenting cells. To assess this hypothesis, we aimed to characterize human immune cell responses to <italic>H. pylori</italic> strains bearing mutations at distinct steps along the glycan biosynthesis pathway.</p>
<p>Herein we characterize the immune response elicited by five glycosylation mutant strains upon coculture with two models of the human gastric immune microenvironment (<xref ref-type="fig" rid="f1">
<bold>Figure&#xa0;1A</bold>
</xref>). We evaluated gastric cytokine production and immune cell maturation when stimulated by each glycosylation mutant relative to wildtype <italic>H. pylori</italic>. Our results demonstrate that strains bearing truncated LPS induced dampened pro-inflammatory cytokine production despite stimulating equivalent levels of immature dendritic cell (iDC) activation compared to wildtype <italic>H. pylori</italic>. Moreover, protein glycosylation mutants induced variable immune responses that did not appear to correlate with phase-variable Lewis Y epitope expression. These results suggest that LPS presentation plays a role in host immune recognition of <italic>H. pylori</italic>, and that the precise role of glycoproteins in driving immune recognition remains unclear. This study demonstrates the need and feasibility for future investigation that probes the relationship between glycocalyx structure and gastric immune response to pathogenic microbes.</p>
<fig id="f1" position="float">
<label>Figure&#xa0;1</label>
<caption>
<p>Glycan biosynthesis mutants were screened for immune stimulatory properties. <bold>(A)</bold> Schematic of experimental flow of approach to assessing glycan assembly mutations on host immune recognition and response to <italic>H. pylori</italic>. <bold>(B)</bold> Schematic of putative glycosylation biosynthetic pathway including five genes targeted for mutation and their roles in glycoprotein and lipopolysaccharide assembly. GP, glycoprotein; GT, glycosyltransferase; LPS, lipopolysaccharides; OST, oligosaccharyltransferase; UndPP, undecaprenyl diphosphate. Images were created using <uri xlink:href="https://www.Biorender.com">BioRender</uri>.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fcimb-14-1377077-g001.tif"/>
</fig>
</sec>
<sec id="s2" sec-type="materials|methods">
<label>2</label>
<title>Materials and methods</title>
<sec id="s2_1">
<label>2.1</label>
<title>
<italic>H. pylori</italic> strains and bacterial growth.</title>
<p>Wildtype G27 <italic>H. pylori</italic> were grown on horse blood agar plates (HBA) made from 4% Columbia agar base, 5% horse blood, 10 &#xb5;g/mL of vancomycin, 5 &#xb5;g/mL cefsulodin, 5 &#x3bc;g/mL trimethoprim, and 8 &#x3bc;g/mL amphotericin B. Bacteria were grown at 37&#xb0;C in 14% CO<sub>2</sub>. <italic>H. pylori</italic> glycosylation mutants (&#x394;579, &#x394;580, &#x394;1179, &#x394;wzk, &#x394;waaL) bearing a chloramphenicol acetyl transferase cassette were grown on HBA plates supplemented with 34 &#xb5;g/mL of chloramphenicol. Different freezer lots were used in each experiment. Prior to each experiment, wildtype and mutant bacteria were grown for 4-5 days until a full lawn of bacterial growth was observed and diluted to a concentration of 1.0-1.4 x 10 (<xref ref-type="bibr" rid="B15">Chen et&#xa0;al., 2013</xref>) cells/mL for cocultures with adenocarcinoma-derived gastric epithelial cells (AGS) or 4.7 x 10 (<xref ref-type="bibr" rid="B40">Kim, 2016</xref>) cells/mL for dendritic cell coculture.</p>
</sec>
<sec id="s2_2">
<label>2.2</label>
<title>Bioinformatics analysis to identify a putative oligosaccharyltransferase gene</title>
<p>With the goal of identifying a putative oligosaccharyltransferase gene involved in <italic>H. pylori&#x2019;s</italic> general protein glycosylation system, a bioinformatics analysis of <italic>H. pylori</italic> G27 was conducted. Briefly, whole genome alignment was performed using Mauve (version 1.1.3) whole genome alignment on Geneious Prime 2020 (<xref ref-type="bibr" rid="B21">Darling et&#xa0;al., 2004</xref>) to identify genes encoding glycosyltransferases that were conserved across other <italic>H. pylori</italic> genomes (G27, 26695, J99, and P12). The G27 genome was analyzed for open reading frames using Glimmer (<xref ref-type="bibr" rid="B22">Delcher et&#xa0;al., 2007</xref>) on the KBase Server (<xref ref-type="bibr" rid="B5">Arkin et&#xa0;al., 2018</xref>) with the RAST (RASTtk &#x2013; v1.073) pipeline (<xref ref-type="bibr" rid="B6">Aziz et&#xa0;al., 2008</xref>), which was preloaded onto the KBase server with default parameters as &#x201c;Annotate Microbial Genome.&#x201d; Genetic analysis of the G27 genomes was performed using Geneious Prime 2020, and the comparison of genes was done using BLAST (<xref ref-type="bibr" rid="B2">Altschul et&#xa0;al., 1990</xref>). Domains were identified using HMMER (<xref ref-type="bibr" rid="B27">Eddy, 2011</xref>) search against the G27 genomes using the PFam HMM 32.0 database (<xref ref-type="bibr" rid="B30">El-Gebali et&#xa0;al., 2019</xref>). For open reading frames that did not have previous biochemical characterization, homology was analyzed using the PHYRE2 recognition server (<xref ref-type="bibr" rid="B39">Kelley et&#xa0;al., 2015</xref>). This approach led to the identification of <italic>HpG27_1179</italic> as a putative oligosaccharyltransferase that may play a role in <italic>H. pylori</italic>&#x2019;s general protein glycosylation system.</p>
</sec>
<sec id="s2_3">
<label>2.3</label>
<title>Construction of glycosylation mutants and analysis of glycoprotein biosynthesis phenotypes</title>
<p>Strains &#x394;579 and &#x394;580 were previously described (<xref ref-type="bibr" rid="B61">Moulton et&#xa0;al., 2020</xref>), and &#x394;1179, &#x394;wzk and &#x394;waaL were generated for this study. Gibson assembly was used to generate linear DNA fragments for insertional inactivation of target genes with a chloramphenicol acetyl transferase cassette. The resulting linear DNA was transformed into wildtype strain G27 through natural transformation using the patch method, and gene interruption was autonomously completed by homologous recombination. The successful insertion of the resistance cassette in mutant strains selected on chloramphenicol/HBA agar was confirmed by polymerase chain reaction (PCR) analysis of genomic DNA from selected mutants. A previously reported metabolic glycan labeling strategy using peracetylated <italic>N</italic>-azidoacetylgluocsamine (Ac<sub>4</sub>GlcNAz) and bioorthogonal chemistry with Phos-FLAG was used to detect glycoprotein biosynthesis (<xref ref-type="bibr" rid="B61">Moulton et&#xa0;al., 2020</xref>) in wildtype <italic>H. pylori</italic> and newly constructed glycosylation mutants &#x394;1179, &#x394;wzk and &#x394;waaL.</p>
</sec>
<sec id="s2_4">
<label>2.4</label>
<title>Tissue cell culture</title>
<p>Tissue culture reagents, tissue culture plates, and ELISA kits were purchased from ThermoFisher Scientific (Waltham, MA), MilliporeSigma (Burlington, MA), R&amp;D Systems (Minneapolis, MN), and USA Scientific (Ocala, FL). AGS cells (ATCC Number: CRL-1739) were grown in Ham&#x2019;s F-12 Glutamax media and passaged upon reaching 80-90% confluency. THP-1 immature dendritic cells (iDCs; ATCC Number: TIB-202) were diluted 1:3 &#x2013; 1:5 with fresh media (RPMI 1640 with glutamine) when cell concentrations exceeded 1 x 10 (<xref ref-type="bibr" rid="B11">Bravo et&#xa0;al., 2018</xref>) cells/mL to prevent overcrowding. Prior to coculture, THP-1 monocytes were differentiated into immature dendritic cells via human recombinant cytokines IL-4 (1500 IU/mL) and GM-CSF (1500 IU/mL) before seeding at a concentration of 2 x 10 (<xref ref-type="bibr" rid="B82">Suerbaum and Michetti, 2002</xref>) cells per well in a 6-well plate for coculture. AGS cells were seeded at 5 x 10 (<xref ref-type="bibr" rid="B82">Suerbaum and Michetti, 2002</xref>) cells/mL in a 6-well plate before coculture.</p>
</sec>
<sec id="s2_5">
<label>2.5</label>
<title>Coculture</title>
<p>Wildtype or mutant <italic>H. pylori</italic> were added in 1 mL of tissue culture media to seeded 6-well plates at an MOI of 1:100 for immature dendritic cell challenge or 1:200 for gastric cell culture. Cocultures were incubated for 3-24 hours at 37&#xb0;C with 5% CO<sub>2</sub> before harvesting.</p>
</sec>
<sec id="s2_6">
<label>2.6</label>
<title>ELISA assay</title>
<p>After 3-hour cocultures for AGS cells or 24-hour cocultures for immature dendritic cells, culture medium was collected for analyses. Human Cytokine DuoSet Enzyme Linked Immunoassay (ELISA) kits (R&amp;D Systems) were utilized to detect relative concentrations of CXCL-8 (IL-8), IL-10, TNF- <inline-formula>
<mml:math display="inline" id="im1">
<mml:mtext>&#x3b1;</mml:mtext>
</mml:math>
</inline-formula>IL-6, or IL-1 <inline-formula>
<mml:math display="inline" id="im2">
<mml:mrow>
<mml:mtext>&#x3b2;&#xa0;</mml:mtext>
</mml:mrow>
</mml:math>
</inline-formula> conditioned media samples. All cytokine concentration data were determined based on an 8-point standard curve created using recombinant human cytokine standards included in each DuoSet kit.</p>
</sec>
<sec id="s2_7">
<label>2.7</label>
<title>Flow cytometry analysis of immature dendritic cells</title>
<p>After coculture with wildtype and mutant bacteria, immature dendritic cells were harvested and resuspended in RPMI media with 10% FBS containing FITC-conjugated CD80 and PE-conjugated CD86 goat anti-human antibodies for 30 min on ice (BD Biosciences, Franklin Lakes, NJ). Cells were then washed in PBS and analyzed by flow cytometry using a BD Accuri C6<sup>+</sup> instrument (BD Biosciences, San Jose, California), with 10,000 live cells gated for each replicate. Dendritic cells expressing CD80 and CD86 were gated and subsequently counted using FlowJo software (TreeStar, Ashland, OR).</p>
</sec>
<sec id="s2_8">
<label>2.8</label>
<title>Lewis Y Western blot</title>
<p>In parallel to dendritic cell challenge, a same-day plating of the same lot of bacterial cells was used to analyze Lewis Y produced by bacterial cells. Cells were resuspended in <italic>H. pylori</italic> lysis buffer with protease inhibitor [20 mM Tris-HCl, pH 7.4, 1% Igepal, 150 mM NaCl, 1 mM EDTA, Protease inhibitor (MilliporeSigma)] and were frozen for 30 minutes to lyse cells. The protein concentration of lysates was determined using a Lowry assay, and lysate concentrations were standardized to 2.5 mg/ml. Samples were electrophoresed via SDS-PAGE on a Mini-PROTEAN TGX Stain-Free Precast 12% acrylamide gel with 4% stacking layer (Bio-Rad). Coomassie stain was used to evaluate protein loading. For Lewis Y detection, electrophoresed samples were transferred to nitrocellulose for western blot analysis. The nitrocellulose membrane with transferred samples was probed with anti-Lewis Y antibody (Abcam, Waltham, MA) followed by anti-mouse IgM HRP (Southern Biotech, Birmingham, AL), then treated with luminol/peroxidase reagent and visualized using a Syngene G box (Cambridge, UK).</p>
</sec>
<sec id="s2_9">
<label>2.9</label>
<title>LPS expression profiling</title>
<p>In parallel to dendritic cell challenge, a same-day plating of the same lot of bacterial cells was used to analyze LPS produced by bacterial cells. Bacteria were lysed and diluted in LPS lysis buffer [10% SDS, 4% &#x3b2;-mercaptoethanol, 0.06 mg/mL bromophenol blue, 10% glycerol, 75% 1M Tris-HCl (pH 6.8)] and incubated at 100&#xb0;C for 10 minutes before cooling to room temperature and treating with Proteinase K (New England Biolabs) at 55&#xb0;C overnight. To visualize LPS, samples were subsequently electrophoresed alongside a 250 &#xb5;g/mL <italic>E. coli</italic> LPS (serotype 055:B5) as control via SDS-PAGE using 15% Tris-HCl SDS-PAGE gels. Following electrophoresis, gels were stained using the ProQ Emerald 300 Lipopolysaccharide Gel Stain Kit (Thermo Fisher Scientific) according to manufacturer&#x2019;s instructions. Gels were visualized with a UVP BioDoc-It Imaging System (Upland, CA) to determine LPS expression at time of coculture.</p>
</sec>
</sec>
<sec id="s3" sec-type="results">
<label>3</label>
<title>Results</title>
<sec id="s3_1">
<label>3.1</label>
<title>LPS elaboration increases pro-inflammatory gastric signaling</title>
<p>An important indicator of immune response and downstream clinical pathology is the secretion of interleukin 8 (IL-8 or CXCL-8) in the gastric microenvironment. CXCL-8 is a pro-inflammatory cytokine that is upregulated to promote an inflammatory response and recruit neutrophils to the site of infection (<xref ref-type="bibr" rid="B35">Harada et&#xa0;al., 1994</xref>; <xref ref-type="bibr" rid="B10">Bickel, 1993</xref>; <xref ref-type="bibr" rid="B71">Qi et&#xa0;al., 2020</xref>). Briefly, gastric epithelial cells express Toll-like receptors (TLRs) on their surfaces that bind to pathogen-associated molecular patterns (PAMP) including bacterial glycans, leading to secretion of CXCL-8 when challenged by bacterial pathogens (<xref ref-type="bibr" rid="B45">Lepper et&#xa0;al., 2005</xref>; <xref ref-type="bibr" rid="B80">Smith, 2014</xref>; <xref ref-type="bibr" rid="B66">Pachathundikandi et&#xa0;al., 2015</xref>; <xref ref-type="bibr" rid="B65">Pachathundikandi and Backert, 2016</xref>). Additionally, <italic>H. pylori</italic> can stimulate gastric epithelial cell CXCL-8 production via a Cag type IV secretion system (Cag-T4SS) dependent mechanism, involving ADP-heptose injection into cells, PAMP recognition intracellularly, and NF-kB activation (<xref ref-type="bibr" rid="B32">Faass et&#xa0;al., 2021</xref>; <xref ref-type="bibr" rid="B68">Pfannkuch et&#xa0;al., 2019</xref>). In <italic>H. pylori</italic> infection, CXCL-8 overexpression is directly linked to gastritis and cancer propagation (<xref ref-type="bibr" rid="B91">Waugh and Wilson, 2008</xref>; <xref ref-type="bibr" rid="B54">Liu et&#xa0;al., 2016</xref>). Given the importance of CXCL-8 in <italic>H. pylori</italic> pathogenesis, we first sought to determine the effect of modulating <italic>H. pylori</italic>&#x2019;s glycocalyx on relative CXCL-8 response from gastric cells.</p>
<p>For these experiments, we were particularly interested in the roles of <italic>H. pylori</italic> glycoproteins and LPS in host recognition and response. Thus, we turned to glycosylation genes that play a role in <italic>H. pylori</italic>&#x2019;s general O-linked protein glycosylation system and/or LPS biosynthesis (<xref ref-type="fig" rid="f1">
<bold>Figure&#xa0;1B</bold>
</xref>) (<xref ref-type="bibr" rid="B61">Moulton et&#xa0;al., 2020</xref>; <xref ref-type="bibr" rid="B84">Teng et&#xa0;al., 2022</xref>; <xref ref-type="bibr" rid="B48">Li et&#xa0;al., 2019</xref>; <xref ref-type="bibr" rid="B47">Li et&#xa0;al., 2016</xref>). Briefly, previous work in our laboratory and others measured glycoprotein and LPS biosynthesis phenotypes in G27 <italic>H. pylori</italic> mutants bearing insertionally inactivated glycosylation genes (<xref ref-type="bibr" rid="B61">Moulton et&#xa0;al., 2020</xref>; <xref ref-type="bibr" rid="B48">Li et&#xa0;al., 2019</xref>). These studies identified genes involved in <italic>H. pylori</italic>&#x2019;s glycoprotein and LPS biosynthesis, as well as provided evidence for initially overlapping glycoprotein and LPS biosynthesis pathways that bifurcate at a later stage (<xref ref-type="fig" rid="f1">
<bold>Figure&#xa0;1B</bold>
</xref>). Both pathways appear to begin with glycosyltransferase-catalyzed addition of monosaccharides one-at-a-time onto an undecaprenyl-phosphate lipid-carrier to produce an elaborated lipid-linked glycan on the cytosolic face of the membrane. The elaborated glycan is then flipped across the membrane via the flippase Wzk, then transferred en bloc onto lipid A by the ligase WaaL to produce LPS or onto target proteins (by waaL, or the putative oligosaccharyltransferase (OST) encoded by <italic>HpG27_1179</italic>) to produce glycoproteins. The elaborated glycan on glycoproteins appears to undergo further tailoring by glycosyltransferases encoded by <italic>HpG27_579</italic> and <italic>HpG27_580</italic> (see <xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Table S1</bold>
</xref> for <italic>H. pylori</italic> 26695 orthologs) (<xref ref-type="bibr" rid="B61">Moulton et&#xa0;al., 2020</xref>). Previous studies demonstrated <italic>H. pylori</italic> glycosylation mutants &#x394;579 and &#x394;580 are defective in glycoprotein biosynthesis (<xref ref-type="bibr" rid="B61">Moulton et&#xa0;al., 2020</xref>) and established that &#x394;wzk and &#x394;waaL synthesize core lipid A that lacks O-antigen (<xref ref-type="bibr" rid="B48">Li et&#xa0;al., 2019</xref>). To further probe this glycoprotein biosynthesis model, we insertionally inactivated <italic>wzk</italic>, <italic>waaL</italic>, and <italic>HpG27_1179</italic> with the chloramphenicol acetyltransferase cassette and probed glycoprotein biosynthesis in the mutant strains. Using an established metabolic glycan labeling based screen (<xref ref-type="bibr" rid="B61">Moulton et&#xa0;al., 2020</xref>), we determined that &#x394;1179, &#x394;wzk, and &#x394;waaL have defects in glycoprotein biosynthesis (<xref ref-type="fig" rid="f2">
<bold>Figure&#xa0;2</bold>
</xref>), thus implicating <italic>1179</italic>, <italic>wzk</italic>, and <italic>waaL</italic> in glycoprotein biosynthesis. Armed with this modest panel of five <italic>H. pylori</italic> glycan biosynthesis mutants (&#x394;579, &#x394;580, &#x394;wzk, &#x394;waaL, &#x394;1179; <xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Table S1</bold>
</xref>), we assessed immunogenicity of these strains relative to wildtype bacteria.</p>
<fig id="f2" position="float">
<label>Figure&#xa0;2</label>
<caption>
<p>Metabolic labeling revealed that &#x394;1179, &#x394;waaL, and &#x394;wzk exhibit glycoprotein biosynthesis defects. <bold>(A)</bold> Western blot analysis with anti-FLAG antibody revealed robust glycoprotein biosynthesis in wildtype (WT) <italic>H. pylori</italic> treated with Ac<sub>4</sub>GlcNAz (Az) and no apparent azide-dependent signal in the negative control treated with Ac<sub>4</sub>GlcNAc (no Az). Glycosylation mutants &#x394;<italic>1179</italic>, &#x394;<italic>waaL</italic>, and &#x394;<italic>wzk</italic> showed markedly reduced glycoprotein biosynthesis relative to WT when metabolically labeled with Ac<sub>4</sub>GlcNAz. <bold>(B)</bold> Coomassie staining of electrophoresed samples from metabolic glycan labeling experiment revealed that Western samples contained equivalent amounts of protein.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fcimb-14-1377077-g002.tif"/>
</fig>
<p>To assess relative pro-inflammatory response elicited from mutant strains versus wildtype <italic>H. pylori</italic>, gastric epithelial cells were cocultured with the bacterial strains for 3 hours. The supernatants were subsequently collected to measure CXCL-8 secretion levels via an enzyme-linked immunosorbent assay (ELISA) (<xref ref-type="fig" rid="f3">
<bold>Figure&#xa0;3A</bold>
</xref>). As a negative control, supernatants were collected from gastric cells cultured in media alone. Supernatants from gastric cells cocultured with wildtype (WT) bacteria acted as a positive control. As expected based on the literature, all wildtype bacteria treatments significantly increased gastric cell secretion of CXCL-8 compared to control cells with no bacterial challenge (<xref ref-type="fig" rid="f3">
<bold>Figure&#xa0;3</bold>
</xref>) (<xref ref-type="bibr" rid="B29">El Filaly et&#xa0;al., 2023</xref>; <xref ref-type="bibr" rid="B33">Gobert and Wilson, 2022</xref>; <xref ref-type="bibr" rid="B64">Outlioua et&#xa0;al., 2020</xref>). The glycoprotein mutant with elaborated LPS structures, &#x394;579, induced consistently increased CXCL-8 secretion from gastric cells compared to wildtype bacteria (<xref ref-type="fig" rid="f3">
<bold>Figure&#xa0;3B</bold>
</xref>), whereas the LPS mutant &#x394;wzk led to consistently decreased CXCL-8 secretion (<xref ref-type="fig" rid="f3">
<bold>Figure&#xa0;3C</bold>
</xref>). These data are in line with pro-inflammatory properties of fully elaborated LPS engaging TLR4 that may be tempered by interfering with LPS biosynthesis. Diminished inflammatory signaling with LPS biosynthesis interference also aligns with the established mechanism of CXCL-8 induction from Cag T4SS dependent LPS metabolite delivery (<xref ref-type="bibr" rid="B32">Faass et&#xa0;al., 2021</xref>; <xref ref-type="bibr" rid="B19">Cover et&#xa0;al., 2020</xref>). By contrast, the glycoprotein mutants, &#x394;580 and &#x394;1179, and the LPS mutant, &#x394;waaL elicited variable CXCL-8 levels that were sometimes significantly higher than wildtype (<xref ref-type="fig" rid="f3">
<bold>Figures&#xa0;3D, F, H</bold>
</xref>), and sometimes significantly lower than wildtype and even close to control levels (<xref ref-type="fig" rid="f3">
<bold>Figures&#xa0;3E, G, I</bold>
</xref>). Though we were initially surprised by the variability observed in CXCL-8 secretion across biological replicate experiments for &#x394;580, &#x394;1179, and &#x394;waaL treatments, we conducted a large number of independent experiments to confirm these results. For these mutants, at least three independent replicates led to an increase in CXCL-8 secretion levels relative to wildtype bacteria, and at least three additional biological replicates led to a decrease in cytokine secretion levels relative to wildtype bacteria. The inconsistencies in CXCL-8 response to mutants with similar phenotypic truncations suggests additional factors are at play in modulating CXCL-8 secretion.</p>
<fig id="f3" position="float">
<label>Figure&#xa0;3</label>
<caption>
<p>CXCL-8 secretion varied with glycan alterations. <bold>(A)</bold> Experimental workflow for assessing pro-inflammatory cytokine secretion of glycosylation mutants compared to wildtype <italic>H. pylori</italic>. Image was created using BioRender. <bold>(B)</bold> The glycoprotein mutant with elaborated LPS structures, &#x394;579, induced increased CXCL-8 secretion from gastric epithelial cells relative to wildtype. <bold>(C)</bold> The LPS mutant, &#x394;wzk, induced decreased CXCL-8 secretion from gastric epithelial cells relative to wildtype. <bold>(D&#x2013;I)</bold> Variable impact on CXCL-8 secretion was observed with glycoprotein mutants &#x394;580 and &#x394;1179, and LPS mutant, &#x394;waaL, compared to wildtype depending on the coculture experiment. Error bars reflect technical replicates. Tukey&#x2019;s multiple comparison test one-way ANOVA was used. (**P&lt; 0.01, ***P&lt; 0.001, ****P&lt; 0.0001, ns, not significant). Data are representative of independent replicate experiments (n &gt; 3) that exhibited the same findings.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fcimb-14-1377077-g003.tif"/>
</fig>
<p>To pinpoint immunostimulatory activity of the glycocalyx irrespective of bacterial fitness, we utilized heat-killed bacteria in coculture with gastric epithelial cells. Strikingly, there was no upregulation of CXCL-8 secretion from gastric cells challenged with heat-killed <italic>H. pylori</italic> (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figure S1</bold>
</xref>). Instead, negative control levels of CXCL-8, on par with the addition of no bacteria, were released by gastric cells treated with heat-killed wildtype <italic>H. pylori</italic> or heat-killed &#x394;580 cells (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figure S1</bold>
</xref>). These results indicate that active infection of bacteria and the presentation of an integrated glycocalyx may be essential to immune-stimulatory responses.</p>
</sec>
<sec id="s3_2">
<label>3.2</label>
<title>Dendritic cells activate against <italic>H. pylori</italic> glycosylation mutants</title>
<p>Dendritic cells play a central role in the formation of a cell-mediated, adaptive immune response. These cells balance immune tolerance through binding regulatory T cells with immune reaction through phagocytosis and antigen-presentation to CD4 and CD8 T cells (<xref ref-type="bibr" rid="B17">Coombes and Powrie, 2008</xref>). <italic>H. pylori</italic> induces strong activation and maturation of human dendritic cells at levels comparable to the LPS of <italic>E. coli</italic> (<xref ref-type="bibr" rid="B42">Kranzer et&#xa0;al., 2004</xref>). However, it is not clear how <italic>H. pylori</italic> modulates regulation of dendritic cell maturation, nor how this modulation may affect the role of dendritic cells in discerning pathogens from commensal gut microbes. Some studies suggest that relatively low immunostimulatory activity of <italic>H. pylori</italic> LPS contributes to <italic>H. pylori</italic>&#x2019;s apparent immune evasion (<xref ref-type="bibr" rid="B67">P&#xe9;rez-P&#xe9;rez et&#xa0;al., 1995</xref>), while other studies suggest a shift in downstream pathways promotes regulatory T cell binding and immune tolerance to the pathogen (<xref ref-type="bibr" rid="B38">Kao et&#xa0;al., 2010</xref>).</p>
<p>In either case, we aimed to complement our innate immune response findings from AGS cells with a more direct measure of adaptive immune modulation via dendritic cell maturation. As professional antigen-presenting cells, dendritic cells traverse the gastric epithelial layer, surveying the gastrointestinal tract until met with a pathogen. Upon contact and recognition, immature dendritic cells are activated, altering their morphology to activate both CD4 and CD8 T cells for a robust adaptive immune response (<xref ref-type="bibr" rid="B52">Liu and Cao, 2015</xref>). As part of this transformation, they upregulate cell-surface coregulatory receptors CD80 and CD86, which bind CD28 on CD4<sup>+</sup> and CD8<sup>+</sup> T cells. These biomarkers may be detected as a proxy for immune cell activation, as they are expressed at low levels on immature dendritic cells (<xref ref-type="bibr" rid="B41">Kim and Kim, 2019</xref>). The percentage of iDC activation reflects the extent of adaptive immune response. High CD80 and CD86 expression levels generated by wildtype <italic>H. pylori</italic> correspond to higher recognition and increased downstream adaptive immune responses, including cytotoxic T cell development.</p>
<p>To test for an effect of glycan truncation on immune cell activation, immature dendritic cells were challenged with wildtype <italic>H. pylori</italic> and glycosylation mutants, then their relative immune response was determined via flow cytometry analysis. After 24 hours in coculture with bacteria, iDCs were isolated, probed with anti-CD80 and anti-CD86 antibodies, and analyzed by flow cytometry to measure fluorescence (<xref ref-type="fig" rid="f4">
<bold>Figure&#xa0;4A</bold>
</xref>). Higher fluorescence indicates higher biomarker expression levels, corresponding to an increased immune response by iDCs. iDCs cultured with no bacteria served as a negative control. Flow cytometry analysis revealed that dendritic cells challenged with bacteria exhibited significantly increased CD80 and CD86 biomarker expression relative to unchallenged dendritic cells (<xref ref-type="fig" rid="f4">
<bold>Figure&#xa0;4</bold>
</xref>; <xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figure S2</bold>
</xref>). Flow cytometry histograms of iDCs challenged with wildtype <italic>H. pylori</italic> overlaid closely with iDCs challenged with glycosylation mutants (<xref ref-type="fig" rid="f4">
<bold>Figure&#xa0;4</bold>
</xref>). There were no significant differences in CD80 and CD86 levels on iDCs, or with percent of cells with high expression levels, following challenge with wildtype <italic>H. pylori</italic> versus glycosylation mutants (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figure S2</bold>
</xref>). These findings indicate an upregulation of cell-mediated immune response to <italic>H. pylori</italic> glycosylation mutants tested that was on par with activation stimulated by wildtype <italic>H. pylori</italic> (<xref ref-type="fig" rid="f4">
<bold>Figures&#xa0;4B, C</bold>
</xref>; <xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figure S2</bold>
</xref>).</p>
<fig id="f4" position="float">
<label>Figure&#xa0;4</label>
<caption>
<p>Immature dendritic cells became activated when challenged with wildtype <italic>H. pylori</italic> and glycosylation mutants. <bold>(A)</bold> Experimental workflow for assessing iDC activation upon challenge with glycosylation mutant bacteria compared to wildtype. Image was created using BioRender. <bold>(B, C)</bold> Flow cytometry histograms revealed CD80 and CD86 expression of iDCs was increased upon challenge of cells with glycosylation mutants and wildtype <italic>H. pylori</italic> relative to iDCs treated with no bacterial (CTRL). Data are representative of independent replicate experiments (n &gt; 3).</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fcimb-14-1377077-g004.tif"/>
</fig>
<p>We next measured levels of cytokine secretion by dendritic cells in response to 24-hour challenge with wildtype <italic>H. pylori</italic> versus glycosylation mutants (<xref ref-type="fig" rid="f5">
<bold>Figure&#xa0;5A</bold>
</xref>). Though iDCs exposed to wildtype and mutant <italic>H. pylori</italic> strains became activated to a similar extent, they secreted markedly different cytokine levels in response to wildtype versus glycosylation mutants (<xref ref-type="fig" rid="f5">
<bold>Figure&#xa0;5</bold>
</xref>). Secretion of the pro-inflammatory cytokines TNF- &#x3b1;, IL-1&#x3b2; and IL-6, as well as the anti-inflammatory cytokine IL-10, by iDCs revealed similar relative patterns of cytokine secretion levels in response to challenge across mutant samples in a single experiment (<xref ref-type="fig" rid="f5">
<bold>Figures&#xa0;5B&#x2013;E, G, I</bold>
</xref>). In all experiments, the LPS mutants &#x394;waaL and &#x394;wzk both induced lower IL-6 secretion from dendritic cells relative to wildtype challenge (<xref ref-type="fig" rid="f5">
<bold>Figures&#xa0;5C, G</bold>
</xref>), with levels secreted by &#x394;waaL on par to wildtype-induced secretion for TNF- &#x3b1; (<xref ref-type="fig" rid="f5">
<bold>Figures&#xa0;5B, F</bold>
</xref>) and IL-1&#x3b2; (<xref ref-type="fig" rid="f5">
<bold>Figures&#xa0;5D, H</bold>
</xref>). In contrast, the glycoprotein mutant &#x394;579 significantly increased TNF- &#x3b1; and IL-6 secretion compared to wildtype coculture (<xref ref-type="fig" rid="f5">
<bold>Figures&#xa0;5B, C</bold>
</xref>). The trends suggest diminished secretion of pro-inflammatory cytokines in the absence of LPS, which mirror the findings from gastric epithelial pro-inflammatory cytokine modulation. In contrast, anti-inflammatory cytokine IL-10 secretion levels were not altered in a consistent manner upon treatment with glycosylation mutants relative to wildtype challenge (<xref ref-type="fig" rid="f5">
<bold>Figures&#xa0;5E, I</bold>
</xref>).</p>
<fig id="f5" position="float">
<label>Figure&#xa0;5</label>
<caption>
<p>Immature dendritic cell cytokine secretion changed when challenged by <italic>H. pylori</italic> glycosylation mutants. <bold>(A)</bold> Experimental workflow used to assess TNF-&#x3b1;, IL-6, IL-1&#x3b2;, and IL-10 secretion from iDCs following challenge for 24-hours with glycosylation mutants or wildtype bacteria. Image was created using BioRender. Datasets from two different iDC challenge experiments are shown, with <bold>(B&#x2013;E)</bold> collected in one experiment and <bold>(F&#x2013;I)</bold> collected in a second experiment. &#x394;580.1 and &#x394; 580.2, as well as &#x394;1179.1 and &#x394;1179.2, represent different freeze lots of the same strain. Error bars reflect technical replicates. Tukey&#x2019;s multiple comparison test one-way ANOVA was used. (*P&lt; 0.05, **P&lt; 0.01, ***P&lt; 0.001, ****P&lt; 0.0001, ns, not significant).</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fcimb-14-1377077-g005.tif"/>
</fig>
<p>Taken together, the results of these studies indicate that dendritic cells are activated by <italic>H. pylori</italic> glycosylation mutants, and the extent of cytokine secretion varies based on the glycocalyx expression profile. As with the gastric cell results, pro-inflammatory cytokine responses are somewhat diminished in coculture with bacteria bearing truncated LPS (&#x394;waaL and &#x394;wzk) and enhanced in coculture with mutants displaying fully elaborated LPS but reduced glycoprotein (&#x394;579). Notably, all mutant <italic>H. pylori</italic> bearing an impaired glycocalyx induce immature dendritic cell immune recognition and activation on par with the wildtype pathogen. Alterations in iDC cytokine secretion, in particular levels of IL-6 and IL-10, induced by glycosylation mutants suggest that glycan structure may modulate downstream polarization and the downstream CD4 response.</p>
<p>As with variable CXCL-8 secretion observed in challenge experiments with AGS cells (<xref ref-type="fig" rid="f3">
<bold>Figures&#xa0;3D&#x2013;I</bold>
</xref>), we observed high variability in relative levels of dendritic cell cytokine secretion across biological replicates (<xref ref-type="fig" rid="f5">
<bold>Figure&#xa0;5</bold>
</xref>). Challenge of iDCs with different lots of &#x394;1179 (&#x394;1179.1 and &#x394;1179.2) in the same experiment led to large differences in IL-6 and IL-10 secretion for these lots relative to one another (<xref ref-type="fig" rid="f5">
<bold>Figures&#xa0;5G, I</bold>
</xref>) and across experiments relative to wildtype bacteria (<xref ref-type="fig" rid="f5">
<bold>Figure&#xa0;5</bold>
</xref>). More subtly, challenge with different lots of &#x394;580 (&#x394;580.1 and &#x394;580.2) in the same experiment led to significantly different IL-6 secretion from iDCs for those lots relative to one another (<xref ref-type="fig" rid="f5">
<bold>Figure&#xa0;5C</bold>
</xref>). Further, &#x394;580 elicited variable cytokine secretion across experiments, with no clear trends in cytokine secretion relative to wildtype bacterial challenge (<xref ref-type="fig" rid="f5">
<bold>Figure&#xa0;5</bold>
</xref>). Variation across replicates in both the gastric cell and dendritic cell models could be due to phase variation in expression of glycan epitopes, heterogeneity in the mutant population, variations in virulence factors such as Cag T4SS activity, or technical problems. Given the well-established literature of phase variable glycocalyx construction in <italic>H. pylori</italic>, we reasoned that further assessment of mutant glycocalyx expression at the time of coculture was warranted.</p>
</sec>
<sec id="s3_3">
<label>3.3</label>
<title>Immune response correlates with extent of LPS O-antigen elaboration</title>
<p>The variable cytokine secretion induced by glycosylation mutants in both the gastric and dendritic cell cocultures (<xref ref-type="fig" rid="f3">
<bold>Figures&#xa0;3D&#x2013;I, 5</bold>
</xref>) prompted us to probe whether phase variable expression of glycan epitopes might be eliciting inconsistent immune responses across experiments. Phase variation is a characteristic of many prokaryotic microbes in which they employ selective gene expression for proteins from generation to generation. In essence, phase variation is a reversible switch between an &#x201c;on&#x201d; or &#x201c;off&#x201d; expression phase, which manipulates the level of expression for one or more proteins in microbes of a clonal population (<xref ref-type="bibr" rid="B88">van der Woude and B&#xe4;umler, 2004</xref>). Phase variation is well established in <italic>H. pylori</italic> and related pathogens. Expression of Lewis blood-group antigens on LPS may vary within a single strain of <italic>H. pylori</italic> as a result of high-frequency on/off switching of fucosyltransferase genes involved in LPS biosynthesis (<xref ref-type="bibr" rid="B90">Wang et&#xa0;al., 2000</xref>; <xref ref-type="bibr" rid="B88">van der Woude and B&#xe4;umler, 2004</xref>; <xref ref-type="bibr" rid="B8">Bergman et&#xa0;al., 2006</xref>). We hypothesized that the variable pro-inflammatory cytokine secretion of host cells in response to challenge by &#x394;580, &#x394;1179, and &#x394;waaL could reflect the structure of the glycocalyx at the time of challenge.</p>
<p>Lewis Y is an established phase-variable epitope whose level of expression is linked to a strand slippage event that controls fucosyltransferase activity in <italic>H. pylori</italic>. Curious about the expression of the Lewis Y tetrasaccharide in glycosylation mutants used in immune challenge experiments, we determined relative expression of the Lewis Y antigen of mutants used in dendritic cell challenge experiments (<xref ref-type="fig" rid="f5">
<bold>Figure&#xa0;5</bold>
</xref>). Western blot analysis revealed Lewis Y expression varied dramatically across <italic>H. pylori</italic> strains despite equivalent protein levels (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figure S3A</bold>
</xref>). Two strains, &#x394;579 and &#x394;580.2, exhibited robust Lewis Y expression (<xref ref-type="fig" rid="f6">
<bold>Figure&#xa0;6A</bold>
</xref>). Conversely, &#x394;580.1, &#x394;1179, &#x394;waaL, and wildtype cells displayed minimal Lewis Y expression (<xref ref-type="fig" rid="f6">
<bold>Figure&#xa0;6A</bold>
</xref>). These results are consistent with variable Lewis Y expression on LPS structures of wildtype <italic>H. pylori</italic> (<xref ref-type="bibr" rid="B78">Simoons-Smit et&#xa0;al., 1996</xref>; <xref ref-type="bibr" rid="B8">Bergman et&#xa0;al., 2006</xref>).</p>
<fig id="f6" position="float">
<label>Figure&#xa0;6</label>
<caption>
<p>The glycocalyx was probed to assess how Lewis Y expression and LPS elaboration correlate to cytokine secretion by immune cells. <bold>(A)</bold> Western blot analysis reveals robust Lewis Y expression in &#x394;579 and &#x394;580.2. The strain samples used in these experiments correspond to those used in iDC challenge experiments for the dataset shown in <xref ref-type="fig" rid="f5">
<bold>Figure&#xa0;5</bold>
</xref>. <bold>(B, C)</bold> Analysis of LPS by gel electrophoresis reveals that &#x394;wzk and &#x394;waaL synthesize only truncated, low molecular weight LPS relative to wildtype <italic>H. pylori</italic>, whereas &#x394;579 appears to synthesize LPS with somewhat more prominent bands at higher molecular weights (e.g., &gt;29kDa) than wildtype <italic>H. pylori</italic>. <bold>(B)</bold> The strain samples used in these experiments correspond to those used in dendritic cell challenge experiments for the dataset shown in <xref ref-type="fig" rid="f5">
<bold>Figure&#xa0;5</bold>
</xref>. &#x394;580.1 and &#x394; 580.2 represent different freeze lots of the same strain. <bold>(C)</bold> Analysis of LPS by gel electrophoresis for strain samples used in dendritic cell challenge experiments for the dataset shown in <xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figure S3</bold>
</xref>. &#x394;1179.1 and &#x394;1179.2 represent different freeze lots of the same strain and appear to have comparable LPS fingerprints.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fcimb-14-1377077-g006.tif"/>
</fig>
<p>Notably, two freeze lots of the &#x394;580 glycoprotein mutant, &#x394;580.1 and &#x394;580.2, demonstrated distinctly different expression levels of Lewis Y (<xref ref-type="fig" rid="f6">
<bold>Figure&#xa0;6A</bold>
</xref>). Such variation between lots of the same glycan mutant is consistent with previous evidence that the <italic>H. pylori</italic> glycocalyx displays antigenic variation within a single strain or clinical isolate (<xref ref-type="bibr" rid="B94">Wirth et&#xa0;al., 1999</xref>). For example, wildtype <italic>H. pylori</italic>, even the same strain, exhibits phase-variable Lewis Y expression (<xref ref-type="bibr" rid="B94">Wirth et&#xa0;al., 1999</xref>). Thus, the difference in Lewis Y expression in &#x394;580.1 and &#x394;580.2 is likely due to phase variation. Further, these data are concordant with the possibility of phase variable epitopes influencing immune recognition and response. However, when we scrutinized the correlation between Lewis Y expression and relative cytokine secretion from the same samples in our dendritic cell model systems, we observed no clear trend. In particular, &#x394;579 and &#x394;1179 exhibited disparate Lewis Y expression (<xref ref-type="fig" rid="f6">
<bold>Figure&#xa0;6A</bold>
</xref>) yet both induced heightened levels of pro-inflammatory cytokine secretion in immature dendritic cell challenge experiments (<xref ref-type="fig" rid="f5">
<bold>Figure&#xa0;5</bold>
</xref>). Similarly, drastically different Lewis Y expression from &#x394;580.1 and &#x394;580.2 (<xref ref-type="fig" rid="f6">
<bold>Figure&#xa0;6A</bold>
</xref>) yielded similar IL-1&#x3b2; and IL-10 secretion levels relative to each other and wildtype bacteria (<xref ref-type="fig" rid="f5">
<bold>Figure&#xa0;5</bold>
</xref>). These results suggest that Lewis Y expression is not a principal factor driving pro-inflammatory cytokine secretion from dendritic cells. It is likely that some other epitope is more important.</p>
<p>LPS structures similarly undergo phase variation in <italic>H. pylori</italic>&#x2019;s glycocalyx, where the expression of the glycosyltransferase enzymes responsible for O-antigen elaboration may be turned on or off (<xref ref-type="bibr" rid="B4">Appelmelk and Vandenbrouck-Grauls, 2003</xref>; <xref ref-type="bibr" rid="B57">Luk&#xe1;&#x10d;ov&#xe1; et&#xa0;al., 2008</xref>). Understanding that LPS is a mediator of <italic>H. pylori&#x2019;</italic>s interactions in its environment, we sought to investigate the extent of elaboration of LPS produced by mutant and wildtype bacteria at the time of immune challenge as an additional parameter to contextualize immune response with glycan architecture. We hypothesized that the extent of LPS elaboration at the time of coculture would correlate with level of immune response. Crude LPS was isolated from bacterial samples used in the dendritic cell experiment in <xref ref-type="fig" rid="f5">
<bold>Figures&#xa0;5B&#x2013;E</bold>
</xref> and <xref ref-type="fig" rid="f5">
<bold>5F&#x2013;I</bold>
</xref>, and compared to literature fingerprints for <italic>H. pylori</italic> LPS (<xref ref-type="bibr" rid="B46">Li and Benghezal, 2017</xref>). Briefly, bacterial lysates were analyzed by Coomassie stain to confirm equivalent protein loading (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figure S3</bold>
</xref>), then treated with proteinase K to yield crude LPS. LPS samples were electrophoresed and their molecular weight distribution was visualized alongside an <italic>Escherichia coli</italic> LPS standard (<xref ref-type="fig" rid="f6">
<bold>Figures&#xa0;6B, C</bold>
</xref>). The &#x394;579 LPS fingerprint exhibited bands at higher molecular weights (e.g., &gt;29 kDa) than wildtype <italic>H. pylori</italic>, implying this mutant synthesized more elaborated O-antigen. This strain also induced higher CXCL-8 levels (<xref ref-type="fig" rid="f3">
<bold>Figure&#xa0;3</bold>
</xref>), possibly implicating elaborated O-antigen presentation in higher CXCL-8 levels. We noted that bands corresponding to high molecular weight O-antigen in &#x394;579 were less prominent than lower molecular weight O-antigen produced by wildtype <italic>H. pylori</italic>, suggesting there may be a heterogeneous population of &#x394;579 with different phase types. In contrast to elaborated LPS produced by &#x394;579, LPS produced by &#x394;waaL and &#x394;wzk was limited to a prominent band at&lt;14 kDa corresponding to the lipid A core, thus confirming truncated LPS biosynthesis (<xref ref-type="fig" rid="f6">
<bold>Figures&#xa0;6B, C</bold>
</xref>). However, the glycoprotein mutant &#x394;580 had variable LPS elaboration, with differences in relative production of high molecular weight O-antigen (<xref ref-type="fig" rid="f6">
<bold>Figures&#xa0;6B, C</bold>
</xref>).</p>
<p>Within the context of immune response data, we noticed apparent correlations with extent of LPS elaboration and extent of cytokine secretion elicited. For example, &#x394;579 displayed relatively high molecular weight LPS structures (<xref ref-type="fig" rid="f6">
<bold>Figures&#xa0;6B, C</bold>
</xref>) and elicited heightened pro- and anti-inflammatory cytokine secretion from challenged host cells (<xref ref-type="fig" rid="f3">
<bold>Figures&#xa0;3B</bold>
</xref>, <xref ref-type="fig" rid="f5">
<bold>5B, C, E</bold>
</xref>). By contrast, &#x394;wzk and &#x394;waaL displayed truncated LPS (<xref ref-type="fig" rid="f6">
<bold>Figures&#xa0;6B, C</bold>
</xref>) and elicited somewhat mitigated IL-6 secretion from challenged host cells (<xref ref-type="fig" rid="f5">
<bold>Figure&#xa0;5</bold>
</xref>). Taken together, these data indicate that relative elaboration of LPS may play a role in extent of immune recognition and response by dendritic cells, but more detailed methods need to be developed for LPS analysis to fully support this claim. The role of <italic>H. pylori&#x2019;</italic>s general protein glycosylation system in modulating immune response is less clear.</p>
</sec>
</sec>
<sec id="s4" sec-type="discussion">
<label>4</label>
<title>Discussion</title>
<p>
<italic>H. pylori</italic> are a compelling pathogen to study in the context of the immune system. Previous work showed that <italic>H. pylori</italic> incorporate Lewis antigen epitopes within the terminus of their LPS structures, suggesting that their glycans feign self in the host environment (<xref ref-type="bibr" rid="B90">Wang et&#xa0;al., 2000</xref>; <xref ref-type="bibr" rid="B78">Simoons-Smit et&#xa0;al., 1996</xref>; <xref ref-type="bibr" rid="B44">Lee et&#xa0;al., 2006</xref>). Given the established role that LPS plays in immune stimulation by similar pathogens, and the characterized role of <italic>H. pylori</italic>&#x2019;s glycans in bacterial fitness and binding to host cells, our study aimed to investigate the role of <italic>H. pylori</italic> cell surface glycosylation on host immune cell signaling and response. Here, we surveyed a small panel of glycosylation mutants bearing insertional inactivation of genes implicated in glycan biosynthesis (<xref ref-type="bibr" rid="B61">Moulton et&#xa0;al., 2020</xref>) for their ability to invoke an immune response (<xref ref-type="fig" rid="f7">
<bold>Figure&#xa0;7</bold>
</xref>). Our data implicate extent of <italic>H. pylori</italic> LPS elaboration with extent of host immune response, with glycoprotein elaboration playing a less clear role. This work offers a novel approach to assess the role of <italic>H. pylori</italic> glycan biosynthesis in modulating host immune recognition and response.</p>
<fig id="f7" position="float">
<label>Figure&#xa0;7</label>
<caption>
<p>Summary of immune responses elicited by challenge with glycosylation mutants of <italic>H. pylori</italic> bearing defects in glycoprotein and/or LPS biosynthesis, as indicated. Effects are relative to immune response elicited by challenge with wildtype <italic>H. pylori</italic> in the same experiment. Data are based on biological replicates (n &gt; 3). &#x201c;Truncated&#x201d; glycoprotein indicates reduction in glycoprotein biosynthesis relative to wildtype; &#x201c;Truncated&#x201d; LPS indicates lower molecular weight LPS biosynthesized than wildtype; &#x201c;Higher MW&#x201d; LPS indicates higher molecular weight LPS biosynthesized than wildtype; &#x201c;Intact&#x201d; LPS indicates LPS with similar molecular weight pattern as wildtype; &#x201c;Increase&#x201d; indicates a significant increase relative to wildtype; &#x201c;Decrease&#x201d; indicates a significant decrease relative to wildtype; &#x201c;Same&#x201d; reflects equivalent levels relative to wildtype; &#x201c;Variable&#x201d; indicates the effect depended on the replicate, with significant increases and decreases observed.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fcimb-14-1377077-g007.tif"/>
</fig>
<p>Gastric epithelial cells produced heightened levels of the pro-inflammatory cytokine CXCL-8 upon challenge with &#x394;579, a glycoprotein mutant that synthesizes elaborated LPS (<xref ref-type="fig" rid="f3">
<bold>Figure&#xa0;3B</bold>
</xref>). In contrast, &#x394;wzk, which bears truncated LPS and glycoproteins, induced decreased CXCL-8 expression relative to wildtype bacteria. These data support previous findings that attribute epithelial cell cytokine production to bacterial LPS stimulation (<xref ref-type="bibr" rid="B55">Lotz et&#xa0;al., 2007</xref>). It is well established that CXCL-8 is produced by gastric cells during <italic>H. pylori</italic> infection (<xref ref-type="bibr" rid="B28">Eftang et&#xa0;al., 2012</xref>). In addition, CXCL-8 is linked to gastric cancer proliferation; it promotes chronic inflammation and propagates infection (<xref ref-type="bibr" rid="B91">Waugh and Wilson, 2008</xref>; <xref ref-type="bibr" rid="B54">Liu et&#xa0;al., 2016</xref>). In fact, CXCL-8 levels positively correlate to poor clinical outcomes for patients who have gastric cancer, decreasing CD8+ T cell infiltration and increasing immunosuppressor programmed death ligand 1 (PD-L1) expression on macrophages (<xref ref-type="bibr" rid="B50">Lin et&#xa0;al., 2019</xref>). Simultaneously, LPS has been found to stimulate significant upregulation of the cannabinoid receptor 1 in the gastrointestinal tract, which promotes cancer cell proliferation (<xref ref-type="bibr" rid="B76">Sedighzadeh et&#xa0;al., 2020</xref>). Other studies implicate LPS in cancer immune suppression, demonstrating that LPS contributes to T cell exhaustion and upregulates PD-L1 in lung cancer, establishing a need to explore this link in gastric cancer (<xref ref-type="bibr" rid="B53">Liu et&#xa0;al., 2021</xref>; <xref ref-type="bibr" rid="B77">Shi et&#xa0;al., 2022</xref>). Our data add nuance to these reports by implicating <italic>H. pylori</italic> LPS with CXCL-8 upregulation, suggesting possible roles for glycan structures in pathogenesis, chronic infection, and cancer development upon infection.</p>
<p>Our study demonstrates that alteration and truncation of glycan structures on <italic>H. pylori</italic>&#x2019;s cell surface do not hinder the activation of immature dendritic cells and their concomitant expression of CD80 and CD86 (<xref ref-type="fig" rid="f4">
<bold>Figure&#xa0;4</bold>
</xref>), consistent with previous reports measuring iDC activation via biomarker upregulation (<xref ref-type="bibr" rid="B42">Kranzer et&#xa0;al., 2004</xref>). We found that glycan alteration and truncation yielded differences in cytokine expression profiles from iDCs (<xref ref-type="fig" rid="f5">
<bold>Figure&#xa0;5</bold>
</xref>). These results indicate that the downstream activation and polarization of CD4 T cells might be different between the different glycosylation mutants. As such, there may be value in assessing glycan structures, the type of adaptive immune response triggered, and the extent to which downstream responses are useful against extracellular pathogens or harmful to the host. Follow up studies could reveal glycan targets that modulate inflammatory cytokine signaling and downstream immune response while maintaining upregulated CD80/CD86 expression in ways that benefit the host.</p>
<p>A confounding observation in our studies was the variable cytokine signaling responses across multiple freeze lots of the same glycosylation mutant or of the same mutant in biological replicates (e.g., &#x394;580, &#x394;1179 and &#x394;waaL; <xref ref-type="fig" rid="f3">
<bold>Figures&#xa0;3</bold>
</xref>, <xref ref-type="fig" rid="f5">
<bold>5</bold>
</xref>). These results prompted us to probe phase variable Lewis Y presentation in our <italic>H. pylori</italic> mutants to assess whether there is a correlation between Lewis Y expression and relative immune response. Previous reports implicate the tetrasaccharide blood-group antigen Lewis Y in <italic>H. pylori</italic> immune modulation, evasion, and pathogenicity (<xref ref-type="bibr" rid="B60">Mandrell and Apicella, 1993</xref>; <xref ref-type="bibr" rid="B3">Appelmelk et&#xa0;al., 2000</xref>; <xref ref-type="bibr" rid="B9">Bergman et&#xa0;al., 2004</xref>; <xref ref-type="bibr" rid="B88">van der Woude and B&#xe4;umler, 2004</xref>). Some attribute <italic>H. pylori</italic> colonization persistence and homeostasis between host and pathogen to the phase variable expression of Lewis blood-group antigens, suggesting a commensal origin commandeered for opportunistic pathogenicity (<xref ref-type="bibr" rid="B8">Bergman et&#xa0;al., 2006</xref>). To query the effect of Lewis Y variation on our mutant immune response data, we probed Lewis Y expression in bacterial lysates used in iDC challenge experiments. We observed no clear correlation with Lewis Y expression and cytokine secretion (<xref ref-type="fig" rid="f5">
<bold>Figures&#xa0;5</bold>
</xref>, <xref ref-type="fig" rid="f6">
<bold>6</bold>
</xref>). This result was surprising due to the reported role of Lewis Y in engaging DC-SIGN on dendritic cells to suppress immune response (<xref ref-type="bibr" rid="B9">Bergman et&#xa0;al., 2004</xref>). Our data suggest that Lewis Y does not appear to be the sole proprietor for regulating <italic>H. pylori-</italic>induced pro-inflammatory responses and suggest that other genes are regulated by the phase variation phenomena.</p>
<p>There appears to be a more direct connection between LPS elaboration and immunogenicity. Although previous studies have demonstrated that <italic>H. pylori</italic> LPS is less endotoxic compared to other bacterial counterparts, the pathogen&#x2019;s modified lipid A and O-antigen are believed to permit selectivity of binding to certain host receptor molecules, enabling the bacterium&#x2019;s persistence (<xref ref-type="bibr" rid="B20">Cullen et&#xa0;al., 2011</xref>; <xref ref-type="bibr" rid="B16">Chmiela et&#xa0;al., 2014</xref>; <xref ref-type="bibr" rid="B51">Lina et&#xa0;al., 2014</xref>). Our results provide novel insight into the role of <italic>H. pylori</italic> LPS in the context of whole cells. In particular, elaborated LPS present on &#x394;579 appears to be correlated with increased pro-inflammatory cytokines including CXCL-8 (<xref ref-type="fig" rid="f3">
<bold>Figure&#xa0;3</bold>
</xref>), TNF-&#x3b1;, and IL-6 (<xref ref-type="fig" rid="f5">
<bold>Figure&#xa0;5</bold>
</xref>). Furthermore, truncation of the LPS moiety correlates to somewhat mitigated cytokine secretion (e.g., IL-6) from challenged host cells (<xref ref-type="fig" rid="f3">
<bold>Figures&#xa0;3</bold>
</xref>, <xref ref-type="fig" rid="f5">
<bold>5</bold>
</xref>).</p>
<p>The precise mechanisms by which <italic>H. pylori</italic> bearing elaborated LPS and glycoproteins engage immune receptors was not explored in this work. Expanding our understanding of the structures of <italic>H. pylori</italic>&#x2019;s glycans and their influence on immune recognition and response will allow us to pinpoint structures, and their corresponding biosynthesis enzymes, that contribute to the pathogen&#x2019;s engagement of immune receptors. Further, determining the relative heterogeneity of glycans in a single <italic>H. pylori</italic> population, assessing the phenotypes of complemented strains, and probing immune response elicited by independent mutants are important future directions. Moreover, exploring the total number of protein antigens modified by glycans in wildtype bacteria versus glycosylation mutants, and probing how glycan modification events impact antigenicity of proteins, relative protein stability, and protein abundance, will be important steps to tease out the molecular mechanisms by which <italic>H. pylori</italic>&#x2019;s general protein glycosylation system influences host immune responses. Our results suggest the potential to manipulate host immune tolerance and activation through targeting select glycans on <italic>H. pylori</italic>&#x2019;s cell surface to quell pro-inflammatory responses characteristic of gastritis and gastric cancer while maintaining dendritic cell activation necessary for adaptive immune system upregulation. Finally, our results indicate that <italic>H. pylori</italic> LPS plays an important role in stimulating the immune response.</p>
</sec>
<sec id="s5" sec-type="conclusions">
<label>5</label>
<title>Conclusion</title>
<p>
<italic>H. pylori</italic> is a gut pathogen that evades clearance by the immune system. <italic>H. pylori&#x2019;s</italic> glycans play an essential role in the bacteria&#x2019;s ability to colonize and thrive in the gastrointestinal tract (<xref ref-type="bibr" rid="B72">Rad et&#xa0;al., 2002</xref>; <xref ref-type="bibr" rid="B75">Schirm et&#xa0;al., 2003</xref>; <xref ref-type="bibr" rid="B51">Lina et&#xa0;al., 2014</xref>; <xref ref-type="bibr" rid="B61">Moulton et&#xa0;al., 2020</xref>). This work investigates the role of cell surface glycans in modulating host immune response to <italic>H. pylori</italic> and demonstrates the potential for selective perturbation of glycan structures to diminish pro-inflammatory responses in severe infections that lead to gastritis and gastric cancer. These studies offer insight into immune response data in conjunction with <italic>H. pylori</italic> glycan architecture and antigen presentation. Broadly, this work suggests that glycan-specific targeting of <italic>H. pylori</italic> could pose a means to dampen chronic inflammatory responses while maintaining cell-mediated adaptive immune system upregulation.</p>
</sec>
<sec id="s6" sec-type="data-availability">
<title>Data availability statement</title>
<p>The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.</p>
</sec>
<sec id="s7" sec-type="ethics-statement">
<title>Ethics statement</title>
<p>Ethical approval was not required for the studies on humans in accordance with the local legislation and institutional requirements because only commercially available established cell lines were used.</p>
</sec>
<sec id="s8" sec-type="author-contributions">
<title>Author contributions</title>
<p>KB: Formal analysis, Investigation, Methodology, Validation, Visualization, Writing &#x2013; original draft, Writing &#x2013; review &amp; editing. FK: Conceptualization, Formal analysis, Investigation, Methodology, Validation, Writing &#x2013; review &amp; editing. WS: Formal analysis, Investigation, Methodology, Validation, Visualization, Writing &#x2013; review &amp; editing. AM: Investigation, Methodology, Writing &#x2013; review &amp; editing. KM: Investigation, Methodology, Project administration, Supervision, Writing &#x2013; review &amp; editing. DD: Conceptualization, Funding acquisition, Project administration, Supervision, Writing &#x2013; original draft, Writing &#x2013; review &amp; editing.</p>
</sec>
</body>
<back>
<sec id="s9" sec-type="funding-information">
<title>Funding</title>
<p>The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. National Institutes of Health grant numbers P20GM103423 and R15GM109397, Arnold and Mabel Beckman Foundation, Littlefield and Fall Research Awards from Bowdoin College. Research reported in this publication was supported by the National Institutes of Health (NIH) under grant number R15GM109397 to DD and by an Institutional Development Award (IDeA) under grant number P20GM10342, as well as a Beckman Scholar Award to KB from the Arnold and Mabel Beckman Foundation, and a Fall Research Award and Littlefield Summer Research Award from Bowdoin College to WS.</p>
</sec>
<ack>
<title>Acknowledgments</title>
<p>We gratefully acknowledge insightful conversations with C. Isabella, D. Calles, A. McBride, and members of our research laboratories for support and guidance.</p>
</ack>
<sec id="s10" sec-type="COI-statement">
<title>Conflict of interest</title>
<p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p>
</sec>
<sec id="s11" sec-type="disclaimer">
<title>Publisher&#x2019;s note</title>
<p>All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.</p>
</sec>
<sec id="s12" sec-type="supplementary-material">
<title>Supplementary material</title>
<p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type="uri" xlink:href="https://www.frontiersin.org/articles/10.3389/fcimb.2024.1377077/full#supplementary-material">https://www.frontiersin.org/articles/10.3389/fcimb.2024.1377077/full#supplementary-material</ext-link>
</p>
<supplementary-material xlink:href="DataSheet_1.pdf" id="SM1" mimetype="application/pdf"/>
</sec>
<fn-group>
<title>Abbreviations</title>
<fn fn-type="abbr">
<p>
<italic>H. pylori</italic>, <italic>Helicobacter pylori</italic>; LPS, lipopolysaccharide; AGS, adenocarcinoma-derived gastric epithelial cells; IL-8 or CXCL-8, interleukin 8; Cag-T4SS, type IV secretion system; iDCs, immature dendritic cells; WT, wildtype; ELISA, enzyme-linked immunosorbent assay; PD-L1, programmed death ligand 1; HBA, horse blood agar; Ac<sub>4</sub>GlcNAz, peracetylated <italic>N</italic>-azidoacetylglucosamine; Phos-FLAG, phosphine-FLAG conjugate; MOI, multiplicity of infection; ATCC, American Type Culture Collection; PBS, phosphate buffered saline; H<sub>2</sub>0, water; FITC, fluorescein isothiocyanate; PE, phycoerythrin; HRP, horse radish peroxidase; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoreses; TBS, tris-buffered saline.</p>
</fn>
</fn-group>
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