Absence of Helicobacter pylori in the oral cavity of 10 non-dyspeptic subjects demonstrated by real-time polymerase chain reaction

Helicobacter pylori plays a significant role in gastric disease. However, the presence of this bacterium in the oral cavity remains controversial. The aim of the present study was to detect and quantify H. pylori in 29 different sites of the oral cavity in non-dyspeptic subjects by means of real-time polymerase chain reactions (PCR). Ten subjects without gastric symptoms were studied. Samples from unstimulated saliva, three sites of the tongue, oral mucosa, and 12 sites of both supragingival and subgingival plaque were collected from each subject. DNA was extracted from the oral samples and analysed for the presence of H. pylori by real-time PCR (LightCycler) using JW23/22 primers which targeted the 16S rRNA gene. DNA from H. pylori DSM 4867 was used as a positive control. Amplification efficiency for the LightCycler 2.0 runs ranged from 1.8 to 2.4. Melting curve analysis identified all the positive control capillaries, which contained H. pylori reference DNA, as a single and narrow peak at a melting temperature between 84.5 and 84.9 degrees C. All the negative control capillaries with no template control and the 29 oral samples from each subject showed either no melting peaks or broad melting peaks below 80 degrees C, which were considered as primer dimers. Therefore, H. pylori was not detected from any of the 290 oral samples. As a conclusion, H. pylori seems not to be permanently present in the oral cavity of a non-dyspeptic population

Absence of Helicobacter pylori in the oral cavity of 10 non-dyspeptic subjects demonstrated by real-time polymerase chain reaction

Helicobacter pylori plays a significant role in gastric disease. However, the presence of this bacterium in the oral cavity remains controversial. The aim of the present study was to detect and quantify H. pylori in 29 different sites of the oral cavity in non-dyspeptic subjects by means of real-time polymerase chain reactions (PCR). Ten subjects without gastric symptoms were studied. Samples from unstimulated saliva, three sites of the tongue, oral mucosa, and 12 sites of both supragingival and subgingival plaque were collected from each subject. DNA was extracted from the oral samples and analysed for the presence of H. pylori by real-time PCR (LightCycler) using JW23/22 primers which targeted the 16S rRNA gene. DNA from H. pylori DSM 4867 was used as a positive control. Amplification efficiency for the LightCycler 2.0 runs ranged from 1.8 to 2.4. Melting curve analysis identified all the positive control capillaries, which contained H. pylori reference DNA, as a single and narrow peak at a melting temperature between 84.5 and 84.9 degrees C. All the negative control capillaries with no template control and the 29 oral samples from each subject showed either no melting peaks or broad melting peaks below 80 degrees C, which were considered as primer dimers. Therefore, H. pylori was not detected from any of the 290 oral samples. As a conclusion, H. pylori seems not to be permanently present in the oral cavity of a non-dyspeptic population

A SNARE-Like Protein and Biotin Are Implicated in Soybean Cyst Nematode Virulence.

Phytoparasitic nematodes that are able to infect and reproduce on plants that are considered resistant are referred to as virulent. The mechanism(s) that virulent nematodes employ to evade or suppress host plant defenses are not well understood. Here we report the use of a genetic strategy (allelic imbalance analysis) to associate single nucleotide polymorphisms (SNPs) with nematode virulence genes in Heterodera glycines, the soybean cyst nematode (SCN). To accomplish this analysis, a custom SCN SNP array was developed and used to genotype SCN F3-derived populations grown on resistant and susceptible soybean plants. Three SNPs reproducibly showed allele imbalances between nematodes grown on resistant and susceptible plants. Two candidate SCN virulence genes that were tightly linked to the SNPs were identified. One SCN gene encoded biotin synthase (HgBioB), and the other encoded a bacterial-like protein containing a putative SNARE domain (HgSLP-1). The two genes mapped to two different linkage groups. HgBioB contained sequence polymorphisms between avirulent and virulent nematodes. However, the gene encoding HgSLP-1 had reduced copy number in virulent nematode populations and appears to produce multiple forms of the protein via intron retention and alternative splicing. We show that HgSLP-1 is an esophageal-gland protein that is secreted by the nematode during plant parasitism. Furthermore, in bacterial co-expression experiments, HgSLP-1 co-purified with the SCN resistance protein Rhg1 α-SNAP, suggesting that these two proteins physically interact. Collectively our data suggest that multiple SCN genes are involved in SCN virulence, and that HgSLP-1 may function as an avirulence protein and when absent it helps SCN evade host defenses

Multiple sequence alignment of the HgSLP-1 SNARE domain to related t-SNARE proteins.

<p>The **marks the zero layer residue (red) critical for membrane fusion and * indicates conserved hydrophobic residues (green) in the flanking heptad repeat domains. The following sequences are in the alignment: 1). Locus: 2NPS_B; protein name: chain B; crystal structure of the early endosomal SNARE complex; accession: 2NPS_B; organism: <i>Rattus norvegicus</i> (Norway rat). 2). Locus: HgSLP; protein name: <i>Heterodera glycines</i> SNARE-like protein 1; accession: KM575849; organism: <i>Heterodera glycines</i> (soybean cyst nematode). 3). Locus: SYP24_ARATH, protein name: putative syntaxin-24; accession: Q9C615; organism: <i>Arabidopsis thaliana</i> (thale cress). 4. Locus: Q9SML5_CAPAN; protein name: syntaxin t-SNARE; accession: Q9SML5; organism: <i>Capsicum annuum</i> (peppers). 5). Locus: Q8S4W4_PORYE; protein name: Syntaxin PM. Accession: Q8S4W4; organism: <u><i>Pyropia yezoensis</i></u> (marine red alga). 6). Locus: SYP72_ARATH; protein name: Syntaxin-72; accession: Q94KK6; organism: <i>A</i>. <i>thaliana</i>. 7). Locus: BET1L_RAT; protein name: golgi SNARE 15 kDa; accession: O35152; organism: <i>R</i>. <i>norvegicus</i>. 8). Locus: SNP30_ARATH; putative SNAP25 homologous protein SNAP30; accession: Q9LMG8; organism: <i>A</i>. <i>thaliana</i>. 9). Locus: O44419_STRPU; Protein name: Synaptosomal-associated protein 25; accession: O44419; organism: <i>Strongylocentrotus purpuratus</i> (purple sea urchin). 10). Locus: O01389_HIRME; protein name: SNAP-25 homolog; accession: O01389; organism: <i>Hirudo medicinalis</i> (medicinal leech).</p

Alignment of paired SOLiD DNA sequencing reads from SCN inbred strain TN20 to the scaffold 385 reference sequence derived from TN10 genomic sequence.

<p>The Y-axis shows depth of coverage and the X-axis indicates the base position along the scaffold. The <i>P</i>. <i>dendritiformis</i>-like gene (<i>HgSLP-1</i>) spans bases 17816 to 21445.</p

SCN genetic linkage groups containing SCN SNPs linked to virulence.

<p>The left column has the map distance in centimorgans and the right column shows the SNP number. The SCN SNPs that show an allelic imbalance when grown on resistant and susceptible soybean plants are shown in red.</p

Large read mapping of TN10 cDNA sequences to the HgSLP-1 genomic sequence.

<p>The Y-axis shows depth of mapped cDNA coverage (99% identical with 99%overlap of each read) and the X-axis indicates the base position along the <i>HgSLP-1</i> gene. The numbers mark the exons of <i>HgSLP-1</i>.</p

Immunolocalization of HgSNARE-like protein-1 (HgSLP-1).

<p>Panels A—D are 40 x light field images matched with corresponding epiflorescent images of sections of SCN in soybean roots stained using HgSLP-1 antibodies. Arrows point to the basal cell of a subventral esophageal gland in A, the median bulb and esophageal lumen in B and the stylet in C. Panel D shows negative control sections lacking HgSLP-1 antibody staining in the nematode. Arrows in D point to the basal cell of an esophageal gland and the stylet. Panel D is a composite of two sequential sections from the same nematode. For all light field images, 20 micron scale bars are shown.</p

Quantitative PCR of <i>P</i>. <i>dendritiformis</i>-like gene (<i>HgSLP-1)</i> genomic copy number relative to <i>HgFAR-1</i> in inbred SCN strains, TN10, TN20 OP25, OP20 and OP50.

<p>Quantitative PCR of <i>P</i>. <i>dendritiformis</i>-like gene (<i>HgSLP-1)</i> genomic copy number relative to <i>HgFAR-1</i> in inbred SCN strains, TN10, TN20 OP25, OP20 and OP50.</p