Abstract
<p style="margin-bottom:11px"><span style="font-size:11pt"><span style="line-height:normal"><span style="font-family:Calibri,sans-serif"><b><span lang="EN-IE" style="font-family:"Arial",sans-serif">Aim:</span></b><span lang="EN-IE" style="font-family:"Arial",sans-serif"> <i>Helicobacter pylori</i></span><i> </i><span lang="EN-IE" style="font-family:"Arial",sans-serif">can</span><span lang="EN-IE" style="font-family:"Arial",sans-serif"> release outer membrane vesicles (OMVs) </span><span lang="EN-IE" style="font-family:"Arial",sans-serif"><span style="color:black">from the cell surface</span></span><span lang="EN-IE" style="font-family:"Arial",sans-serif">.</span><span lang="EN-IE" style="font-family:"Arial",sans-serif"> Lipopolysaccharide (</span><span lang="EN-IE" style="font-family:"Arial",sans-serif">LPS</span><span lang="EN-IE" style="font-family:"Arial",sans-serif">)</span> <span lang="EN-IE" style="font-family:"Arial",sans-serif">rich in the outer leaflet of bacterial </span><span lang="EN-IE" style="font-family:"Arial",sans-serif">outer membrane </span><span lang="EN-IE" style="font-family:"Arial",sans-serif">(OM) is the major constituent of OMVs. Here, we tried to explore the involvement of </span><i><span lang="EN-IE" style="font-family:"Arial",sans-serif">H. pylori</span></i> <span lang="EN-IE" style="font-family:"Arial",sans-serif">LPS in OMV biogenesis, protein cargo packaging and virulence. </span></span></span></span></p><p style="margin-bottom:11px"><span style="font-size:11pt"><span style="line-height:normal"><span style="font-family:Calibri,sans-serif"><b><span lang="EN-IE" style="font-family:"Arial",sans-serif">Method:</span></b><b> </b><span lang="EN-IE" style="font-family:"Arial",sans-serif">V</span><span lang="EN-IE" style="font-family:"Arial",sans-serif">arious <i>H. pylori </i>LPS</span><span lang="EN-IE" style="font-family:"Arial",sans-serif">-</span><span lang="EN-IE" style="font-family:"Arial",sans-serif">truncated mutants, including those </span><span lang="EN-IE" style="font-family:"Arial",sans-serif">with</span><span lang="EN-IE" style="font-family:"Arial",sans-serif"> defects in the inner-core</span><span lang="EN-IE" style="font-family:"Arial",sans-serif"> (</span><span lang="EN-IE" style="font-family:"Arial",sans-serif">heptose biosynthetic pathway</span><span lang="EN-IE" style="font-family:"Arial",sans-serif"> in particular)</span><span lang="EN-IE" style="font-family:"Arial",sans-serif">, outer-core</span><span lang="EN-IE" style="font-family:"Arial",sans-serif"> and </span><span lang="EN-IE" style="font-family:"Arial",sans-serif">O-antigen</span><span lang="EN-IE" style="font-family:"Arial",sans-serif">, were constructed.</span> <span lang="EN-IE" style="font-family:"Arial",sans-serif"><span style="color:black">OMVs </span></span><span lang="EN-IE" style="font-family:"Arial",sans-serif"><span style="color:black">isolated </span></span><span lang="EN-IE" style="font-family:"Arial",sans-serif"><span style="color:black">from </span></span><span lang="EN-IE" style="font-family:"Arial",sans-serif"><span style="color:black">these</span></span><span lang="EN-IE" style="font-family:"Arial",sans-serif"><span style="color:black"> mutants</span></span> <span lang="EN-IE" style="font-family:"Arial",sans-serif">were applied for </span><span lang="EN-IE" style="font-family:"Arial",sans-serif">biochemical and biophysical</span><span lang="EN-IE" style="font-family:"Arial",sans-serif"> characterization, and for virulence analysis by co-culture with gastric AGS cells.</span></span></span></span></p><p style="margin-bottom:11px"><span style="font-size:11pt"><span style="line-height:normal"><span style="font-family:Calibri,sans-serif"><b><span lang="EN-IE" style="font-family:"Arial",sans-serif">Results:</span></b> <span lang="EN-IE" style="font-family:"Arial",sans-serif">T</span><span lang="EN-IE" style="font-family:"Arial",sans-serif">he amount of OMV</span><span lang="EN-IE" style="font-family:"Arial",sans-serif"> production</span> <span lang="EN-IE" style="font-family:"Arial",sans-serif">was</span> <span lang="EN-IE" style="font-family:"Arial",sans-serif">significantly reduced and </span><span lang="EN-IE" style="font-family:"Arial",sans-serif">related to the structure of LPS</span><span lang="EN-IE" style="font-family:"Arial",sans-serif"> in these mutants</span><span lang="EN-IE" style="font-family:"Arial",sans-serif">, and the average sizes of OMVs from the</span><span lang="EN-IE" style="font-family:"Arial",sans-serif"> mutants with defects in the </span><span lang="EN-IE" style="font-family:"Arial",sans-serif">heptose biosynthetic pathway were</span> <span lang="EN-IE" style="font-family:"Arial",sans-serif">larger than others. </span><span lang="EN-IE" style="font-family:"Arial",sans-serif">In addition, the protein profile of OMVs from LPS-related mutants was also different from that of the wild-type <i>H. pylori</i>. </span><span lang="EN-IE" style="font-family:"Arial",sans-serif">Furthermore</span><span lang="EN-IE" style="font-family:"Arial",sans-serif"><span style="color:black">, </span></span><span lang="EN-IE" style="font-family:"Arial",sans-serif">the presence of CagA, VacA and </span><span lang="EN-IE" style="font-family:"Arial",sans-serif">several</span><span lang="EN-IE" style="font-family:"Arial",sans-serif"> adhesins on OMVs</span><span lang="EN-IE" style="font-family:"Arial",sans-serif"><span style="color:black"> was demonstrated in the wild-type <i>H. pylori</i>, but </span></span><span lang="EN-IE" style="font-family:"Arial",sans-serif"><span style="color:black">the </span></span><span lang="EN-IE" style="font-family:"Arial",sans-serif"><span style="color:black">levels of these virulence factors </span></span><span lang="EN-IE" style="font-family:"Arial",sans-serif">decreased variably among LPS mutants. Moreover</span><span lang="EN-IE" style="font-family:"Arial",sans-serif">,</span> <span lang="EN-IE" style="font-family:"Arial",sans-serif"><span style="color:black">AGS cell displayed </span></span><span lang="EN-IE" style="font-family:"Arial",sans-serif"><span style="color:black">a </span></span><span lang="EN-IE" style="font-family:"Arial",sans-serif">typical </span><span lang="EN-IE" style="font-family:"Arial",sans-serif">hummingbird/vacuolation phenotype</span><span lang="EN-IE" style="font-family:"Arial",sans-serif"> of <i>H. pylori</i> infection<span style="color:black"> after co-culture with </span></span><span lang="EN-IE" style="font-family:"Arial",sans-serif"><span style="color:black">wild-type </span></span><span lang="EN-IE" style="font-family:"Arial",sans-serif"><span style="color:black">OMVs</span></span><span lang="EN-IE" style="font-family:"Arial",sans-serif"><span style="color:black">, but the levels of these</span></span><span lang="EN-IE" style="font-family:"Arial",sans-serif"> morphological changes </span><span lang="EN-IE" style="font-family:"Arial",sans-serif">were reduced notably while AGS cells were treated with OMVs from LPS-disrupted mutants.</span></span></span></span></p><p style="margin-bottom:11px"><span style="font-size:11pt"><span style="line-height:normal"><span style="font-family:Calibri,sans-serif"><b><span lang="EN-IE" style="font-family:"Arial",sans-serif">Conclusion:</span></b><span lang="EN-IE" style="font-family:"Arial",sans-serif"> <span style="color:black">These findings suggest that LPS structure indeed plays essential roles in OMV formation, protein sorting, virulence factor delivery and thus contributes to the virulence of <i>H. pylori</i> infection.</span></span></span></span></span></p>