Proteomic Characterization of <i>Helicobacter pylori</i> CagA Antigen Recognized by Child Serum Antibodies and Its Epitope Mapping by Peptide Array

<div><p>Serum antibodies against pathogenic bacteria play immunologically protective roles, and can be utilized as diagnostic markers of infection. This study focused on Japanese child serum antibodies against <i>Helicobacter pylori</i>, a chronically-infected gastric bacterium which causes gastric cancer in adults. Serological diagnosis for <i>H. pylori</i> infection is well established for adults, but it needs to be improved for children. Serum samples from 24 children, 22 <i>H. pylori</i> (<i>Hp</i>)-positive and 2 <i>Hp</i>-negative children, were used to catalogue antigenic proteins of a Japanese strain CPY2052 by two-dimensional electrophoresis followed by immunoblot and LC-MS/MS analysis. In total, 24 proteins were identified as candidate antigen proteins. Among these, the major virulence factor, cytotoxin-associated gene A protein (CagA) was the most reactive antigen recognized by all the <i>Hp</i>-positive sera even from children under the age of 3 years. The major antigenic part of CagA was identified in the middle region, and two peptides containing CagA epitopes were identified using a newly developed peptide/protein-combined array chip method, modified from our previous protein chip method. Each of the epitopes was found to contain amino acid residue(s) unique to East Asian CagA. Epitope analysis of CagA indicated importance of the regional CagA antigens for serodiagnosis of <i>H. pylori</i> infection in children.</p></div

A CBB stained 2DE pattern of whole-cell lysates of <i>H. pylori</i> strain CPY2052 and representative images of 2D-immunoblots probed with sera from children.

<p><b>A.</b> See Pico CBB-stained 2-DE gel containing proteins from <i>H. pylori</i> CPY2052. Spots of antigenic proteins recognized by at least one serum sample are indicated by arrows with numbers in red. Abbreviated names of proteins identified by MS analysis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104611#pone-0104611-t001" target="_blank">Table 1</a>) are indicated in black. The red number of each spot corresponds to a W number in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104611#pone-0104611-t001" target="_blank">Table 1</a>. The p<i>I</i> and molecular size markers are indicated at the bottom and to the right, respectively, and cup-loading site in IEF is indicated as a black bar at the top, left end of the gel. <b>B</b>. Representative images of immunoblots probed with sera from asymptomatic children. <b>C</b>. Representative images of immunoblots probed with sera from symptomatic children. Serum No. 15, juvenile rheumatoid arthritis, No. 4, epilepsia, and No. 22, iron-deficiency anemia, (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104611#pone.0104611.s001" target="_blank">Table S1</a>). D. A image of immunoblot probed with serum from <i>Hp</i>-negative child. Serum No. and age of children are indicated at the top of each panel.</p

Alignment of epitope sequences in the CagA middle region and consensus sequences of epitopes in East Asian and western CagA.

<p>Amino acid sequences of seven East Asian and five western CagA corresponding to peptides m8 and m24–25 were taken from the NCBI database. Each sequence is denoted as EA-CagA (East Asian) or W-CagA (western) and written by single letters. Amino acids indicated with black boxes, tryptophan (W) in peptide m8 and glutamic acid (E) and- threonine (T) in peptide m24–25 are unique to EA-CagA. *: Location in tertiary structure of CagA²⁶⁶⁹⁵<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104611#pone.0104611-Hayashi1" target="_blank">[36]</a>. Consensus sequence among 7 EA-CagA or 5 W-CagA are shown by capital letters for all conserved amino acids, small letters for mainly conserved amino acids, or dot for no conserved amino acids.</p

Antigenic region of CagA^CPY2052 protein recognized by IgG antibodies in <i>H. pylori (Hp)</i>-positive child sera.

<p>Antigenic region of CagA^CPY2052 protein recognized by IgG antibodies in <i>H. pylori (Hp)</i>-positive child sera.</p

2D-immunoblot analysis of CagA and CagA fragments with serum from <i>H. pylori-</i>positive child and CagA-specific antibodies.

<p><b>A</b>. See Pico CBB-stained 2-DE gels and immunoblots of CPY2052, HPK5 and NCTC11637 and their <i>cagA</i> deletion strains probed with <i>Hp</i>-positive serum No. 15 serum. <b>B</b>. Immuoblots probed with antibodies for EPIYA motif-containing fragment of western-CagA (anti-western-CagA-C) and East Asian CagA (anti-EAS). Regions of CagA to produce two antibodies are diagrammed schematically. Spot numbers are corresponded to those in Fig. 2. 1(C) or C is CagA, and 2(V) or V is VacA. Spots of a-f or g-k are CagA-fragments in 2-D immunoblot of HPK5 and NCTC11637 lysate, respectively.</p

Antigenic region of CagA^CPY2052 determined by immunoblotting analysis of <i>E. coli</i> lysates containing recombinant proteins.

<p><b>A</b>. A series of eight distinct CagA fragments fused with GFP at N-terminus expressed in <i>E. coli</i>. <b>B</b>. CBB-stained SDS-PAGE gel of <i>E. coli</i> lysates, immunoblots probed with anti-GFP, and immunoblots probed with anti-CagA C-terminus specific antibody (anti-western-CagA-C). Asterisks in the images indicate each full-length recombinant protein. Four-times more volumes of lysates expressing CagA C-terminal fragments, MC’, C3, C2, and C1, were loaded to compensate the small amount of antigens. <b>C</b>. Immunoblots probed with sera from <i>Hp-</i>positive, asymptomatic children. <b>D</b>. Immunoblots probed with sera from <i>Hp-</i>positive symptomatic children. Three highly reactive proteins possibly from <i>E. coli</i> are indicated by blue arrowheads. <b>E</b>. Immunoblots probed with sera from <i>Hp-</i>negative children showing <i>E. coli</i>-derived bands similar to those in D.</p

Epitope peptides in CagA^CPY2052 middle region recognized by IgG antibodies in <i>H. pylori (Hp)</i>-positive child sera.

<p><b>*</b> Peptides reacted with one of Hp (-) serum samples.</p

Immunoblot analysis of whole-cell lysate of East Asian and western <i>Helicobacter pylori</i> strains using serum from Japanese child.

<p><b>A</b>. A Coomarssie Brilliant Blue R-250 (CBB)-stained SDS-PAGE gels containing whole-cell lysate of three East Asian <i>Helicobacter pylori</i> strains (HPK5, CPY2052, and CPY3401) and two western <i>H. pylori</i> strains (26695 and NCTC11637). The 130–140 kDa band (black arrowhead) is CagA by LC-MS/MS. <b>B</b>. Representative immunoblot. A SDS-PAGE gel, was blotted onto PVDF membrane, and probed with diluted serum No. 15 from a child (4-year-old) with juvenile rheumatoid arthritis. The serum reacted with CagA (black arrowhead) and several other proteins with low molecular weights.</p

Candidates of <i>Helicobacter pylori</i> antigenic proteins recognized by IgG antibodies in sera of Japanese <i>H. pylori</i>-positive children as determined by LC-MS/MS.

<p>1) Protein names with * are newly identified <i>H. pylori</i> antigenic protein candidates in this study. Abbreviated names are indidcated in parentheses.</p><p>Alternative name is indicated after/. W38, W57 and W69 spots included two antigen candidate proteins with high MS scores.</p><p>2) Proteins which were identified from more than two distinctive peptides by Spectrum Mill MS Proteomics Workbench, except W2 and W15.</p><p>3) Proteins which were identified as more than 20 MS/MS Search Score by Spectrum Mill MS Proteomics Workbench, except W2 and W15.</p><p>4) SP: Swiss Prot, EA: East-Asia 7 strains, othrers are all by NCBI.</p><p>5) Identification by immunoblot analysis using wild type and knockout strains of the coressponding genes.</p><p>6) FlaA was also reacted with two of <i>H. pylori</i>-negative child serum samples, not included in this number**.</p