Mechanism of H. pylori Intracellular Entry: An in vitro Study

The majority of H. pylori reside on gastric epithelial cell surfaces and in the overlying mucus, but a small fraction of H. pylori enter host epithelial and immune cells. To explore the role of the nudA invasin in host cell entry, a ΔnudA deletion derivative of strain J99 was constructed and transformants were verified by PCR and by fluorescence in situ hybridization. AGS cells were inoculated with either wild type (WT) strain J99 or its ΔnudA mutant to determine the fraction of bacteria that were bound to the cells and inside these cells using the gentamicin protection assay. We observed no significant difference between either the density of H. pylori bound to AGS cell membranes or the density of intracellular H. pylori. To further explore this finding, separate chambers of each culture were fixed in glutaraldehyde for transmission electron microscopy (TEM) and immunogold TEM. This addition to the classical gentamicin assay demonstrated that there were significantly more intracellular, and fewer membrane-bound, H. pylori in WT-infected AGS cells than in ΔnudA allele infected cells. Thus, the sum of intracellular and membrane-bound H. pylori was similar in the two groups. Since no other similar TEM study has been performed, it is at present unknown whether our observations can be reproduced by others Taken together however, our observations suggest that the classical gentamicin protection assay is not sufficiently sensitive to analyze H. pylori cell entry and that the addition of TEM to the test demonstrate that nudA plays a role in H. pylori entry into AGS cells in vitro. In addition, deletion of the invasin gene appears to limit H. pylori to the AGS cell surface, where it may be partly protected against gentamicin. In contrast, this specific environment may render H. pylori more vulnerable to host defense and therapeutic intervention, and less prone to trigger normal immune, carcinogenic, and other developmental response pathways

Role of ABO Secretor Status in Mucosal Innate Immunity and H. pylori Infection

The fucosylated ABH antigens, which constitute the molecular basis for the ABO blood group system, are also expressed in salivary secretions and gastrointestinal epithelia in individuals of positive secretor status; however, the biological function of the ABO blood group system is unknown. Gastric mucosa biopsies of 41 Rhesus monkeys originating from Southern Asia were analyzed by immunohistochemistry. A majority of these animals were found to be of blood group B and weak-secretor phenotype (i.e., expressing both Lewis a and Lewis b antigens), which are also common in South Asian human populations. A selected group of ten monkeys was inoculated with Helicobacter pylori and studied for changes in gastric mucosal glycosylation during a 10-month period. We observed a loss in mucosal fucosylation and concurrent induction and time-dependent dynamics in gastric mucosal sialylation (carbohydrate marker of inflammation), which affect H. pylori adhesion targets and thus modulate host–bacterial interactions. Of particular relevance, gastric mucosal density of H. pylori, gastritis, and sialylation were all higher in secretor individuals compared to weak-secretors, the latter being apparently “protected.” These results demonstrate that the secretor status plays an intrinsic role in resistance to H. pylori infection and suggest that the fucosylated secretor ABH antigens constitute interactive members of the human and primate mucosal innate immune system

Microarray analysis of Shigella flexneri-infected epithelial cells identifies host factors important for apoptosis inhibition

<p>Abstract</p> <p>Background</p> <p><it>Shigella flexneri </it>inhibits apoptosis in infected epithelial cells. In order to understand the pro-survival effects induced by the bacteria, we utilized apoptosis-specific microarrays to analyze the changes in eukaryotic gene expression in both infected and uninfected cells in the presence and absence of staurosporine, a chemical inducer of the intrinsic pathway of apoptosis. The goal of this research was to identify host factors that contribute to apoptosis inhibition in infected cells.</p> <p>Results</p> <p>The microarray analysis revealed distinct expression profiles in uninfected and infected cells, and these changes were altered in the presence of staurosporine. These profiles allowed us to make comparisons between the treatment groups. Compared to uninfected cells, <it>Shigella-</it>infected epithelial cells, both in the presence and absence of staurosporine, showed significant induced expression of <it>JUN</it>, several members of the inhibitor of apoptosis gene family, nuclear factor κB and related genes, genes involving tumor protein 53 and the retinoblastoma protein, and surprisingly, genes important for the inhibition of the extrinsic pathway of apoptosis. We confirmed the microarray results for a selection of genes using <it>in situ </it>hybridization analysis.</p> <p>Conclusion</p> <p>Infection of epithelial cells with <it>S. flexneri </it>induces a pro-survival state in the cell that results in apoptosis inhibition in the presence and absence of staurosporine. The bacteria may target these host factors directly while some induced genes may represent downstream effects due to the presence of the bacteria. Our results indicate that the bacteria block apoptosis at multiple checkpoints along both pathways so that even if a cell fails to prevent apoptosis at an early step, <it>Shigella </it>will block apoptosis at the level of caspase-3. Apoptosis inhibition is most likely vital to the survival of the bacteria <it>in vivo</it>. Future characterization of these host factors is required to fully understand how <it>S. flexneri </it>inhibits apoptosis in epithelial cells.</p

Specific and Sensitive Detection of H. pylori in Biological Specimens by Real-Time RT-PCR and In Situ Hybridization

PCR detection of H. pylori in biological specimens is rendered difficult by the extensive polymorphism of H. pylori genes and the suppressed expression of some genes in many strains. The goal of the present study was to (1) define a domain of the 16S rRNA sequence that is both highly conserved among H. pylori strains and also specific to the species, and (2) to develop and validate specific and sensitive molecular methods for the detection of H. pylori. We used a combination of in silico and molecular approaches to achieve sensitive and specific detection of H. pylori in biologic media. We sequenced two isolates from patients living in different continents and demonstrated that a 546-bp domain of the H. pylori 16S rRNA sequence was conserved in those strains and in published sequences. Within this conserved sequence, we defined a 229-bp domain that is 100% homologous in most H. pylori strains available in GenBank and also is specific for H. pylori. This sub-domain was then used to design (1) a set of high quality RT-PCR primers and probe that encompassed a 76-bp sequence and included at least two mismatches with other Helicobacter sp. 16S rRNA; and (2) in situ hybridization antisense probes. The sensitivity and specificity of the approaches were then demonstrated by using gastric biopsy specimens from patients and rhesus monkeys. This H. pylori-specific region of the 16S rRNA sequence is highly conserved among most H. pylori strains and allows specific detection, identification, and quantification of this bacterium in biological specimens

Generation of ρ0 cells utilizing a mitochondrially targeted restriction endonuclease and comparative analyses

Eukaryotic cells devoid of mitochondrial DNA (ρ0 cells) were originally generated under artificial growth conditions utilizing ethidium bromide. The chemical is known to intercalate preferentially with the mitochondrial double-stranded DNA thereby interfering with enzymes of the replication machinery. ρ0 cell lines are highly valuable tools to study human mitochondrial disorders because they can be utilized in cytoplasmic transfer experiments. However, mutagenic effects of ethidium bromide onto the nuclear DNA cannot be excluded. To foreclose this mutagenic character during the development of ρ0 cell lines, we developed an extremely mild, reliable and timesaving method to generate ρ0 cell lines within 3–5 days based on an enzymatic approach. Utilizing the genes for the restriction endonuclease EcoRI and the fluorescent protein EGFP that were fused to a mitochondrial targeting sequence, we developed a CMV-driven expression vector that allowed the temporal expression of the resulting fusion enzyme in eukaryotic cells. Applied on the human cell line 143B.TK− the active protein localized to mitochondria and induced the complete destruction of endogenous mtDNA. Mouse and rat ρ0 cell lines were also successfully created with this approach. Furthermore, the newly established 143B.TK− ρ0 cell line was characterized in great detail thereby releasing interesting insights into the morphology and ultra structure of human ρ0 mitochondria

High Diversity of vacA and cagA Helicobacter pylori Genotypes in Patients with and without Gastric Cancer

BACKGROUND: Helicobacter pylori is associated with chronic gastritis, peptic ulcers, and gastric cancer. The aim of this study was to assess the topographical distribution of H. pylori in the stomach as well as the vacA and cagA genotypes in patients with and without gastric cancer. METHODOLOGY/PRINCIPAL FINDINGS: Three gastric biopsies, from predetermined regions, were evaluated in 16 patients with gastric cancer and 14 patients with dyspeptic symptoms. From cancer patients, additional biopsy specimens were obtained from tumor centers and margins; among these samples, the presence of H. pylori vacA and cagA genotypes was evaluated. Positive H. pylori was 38% and 26% in biopsies obtained from the gastric cancer and non-cancer groups, respectively (p = 0.008), and 36% in tumor sites. In cancer patients, we found a preferential distribution of H. pylori in the fundus and corpus, whereas, in the non-cancer group, the distribution was uniform (p = 0.003). A majority of the biopsies were simultaneously cagA gene-positive and -negative. The fundus and corpus demonstrated a higher positivity rate for the cagA gene in the non-cancer group (p = 0.036). A mixture of cagA gene sizes was also significantly more frequent in this group (p = 0.003). Ninety-two percent of all the subjects showed more than one vacA gene genotype; s1b and m1 vacA genotypes were predominantly found in the gastric cancer group. The highest vacA-genotype signal-sequence diversity was found in the corpus and 5 cm from tumor margins. CONCLUSION/SIGNIFICANCE: High H. pylori colonization diversity, along with the cagA gene, was found predominantly in the fundus and corpus of patients with gastric cancer. The genotype diversity observed across systematic whole-organ and tumor sampling was remarkable. We find that there is insufficient evidence to support the association of one isolate with a specific disease, due to the multistrain nature of H. pylori infection shown in this work

A core microbiome associated with the peritoneal tumors of pseudomyxoma peritonei

Pseudomyxoma peritonei (PMP) is a malignancy characterized by dissemination of mucus-secreting cells throughout the peritoneum. This disease is associated with significant morbidity and mortality and despite effective treatment options for early-stage disease, patients with PMP often relapse. Thus, there is a need for additional treatment options to reduce relapse rate and increase long-term survival. A previous study identified the presence of both typed and non-culturable bacteria associated with PMP tissue and determined that increased bacterial density was associated with more severe disease. These findings highlighted the possible role for bacteria in PMP disease. To more clearly define the bacterial communities associated with PMP disease, we employed a sequenced-based analysis to profile the bacterial populations found in PMP tumor and mucin tissue in 11 patients. Sequencing data were confirmed by in situ hybridization at multiple taxonomic depths and by culturing. A pilot clinical study was initiated to determine whether the addition of antibiotic therapy affected PMP patient outcome. We determined that the types of bacteria present are highly conserved in all PMP patients; the dominant phyla are the Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes. A core set of taxon-specific sequences were found in all 11 patients; many of these sequences were classified into taxonomic groups that also contain known human pathogens. In situ hybridization directly confirmed the presence of bacteria in PMP at multiple taxonomic depths and supported our sequence-based analysis. Furthermore, culturing of PMP tissue samples allowed us to isolate 11 different bacterial strains from eight independent patients, and in vitro analysis of subset of these isolates suggests that at least some of these strains may interact with the PMP-associated mucin MUC2. Finally, we provide evidence suggesting that targeting these bacteria with antibiotic treatment may increase the survival of PMP patients. Using 16S amplicon-based sequencing, direct in situ hybridization analysis and culturing methods, we have identified numerous bacterial taxa that are consistently present in all PMP patients tested. Combined with data from a pilot clinical study, these data support the hypothesis that adding antimicrobials to the standard PMP treatment could improve PMP patient survival.https://doi.org/10.1186/1750-1172-8-10

Archaeogenomic distinctiveness of the Isthmo-Colombian Area

The recently-enriched genomic history of Indigenous groups in the Americas is still meagre concerning continental Central America. Here, we report ten pre-Hispanic (plus two early colonial) genomes and 84 genome-wide profiles from seven groups presently living in Panama. Our analyses reveal that pre-Hispanic demographic events contributed to the extensive genetic structure currently seen in the area, which is also characterized by a distinctive Isthmo-Colombian Indigenous component. This component drives these populations on a specific variability axis and derives from the local admixture of different ancestries of northern North American origin(s). Two of these ancestries were differentially associated to Pleistocene Indigenous groups that also moved into South America leaving heterogenous genetic footprints. An additional Pleistocene ancestry was brought by UPopI, a still unsampled population that remained restricted to the Isthmian area, expanded locally during the early Holocene, and left genomic traces up to the present day

Archaeogenomic distinctiveness of the Isthmo-Colombian area

The recently enriched genomic history of Indigenous groups in the Americas is still meager concerning continental Central America. Here, we report ten pre-Hispanic (plus two early colonial) genomes and 84 genome-wide profiles from seven groups presently living in Panama. Our analyses reveal that pre-Hispanic demographic events contributed to the extensive genetic structure currently seen in the area, which is also characterized by a distinctive Isthmo-Colombian Indigenous component. This component drives these populations on a specific variability axis and derives from the local admixture of different ancestries of northern North American origin(s). Two of these ancestries were differentially associated to Pleistocene Indigenous groups that also moved into South America, leaving heterogenous genetic footprints. An additional Pleistocene ancestry was brought by a still unsampled population of the Isthmus (UPopI) that remained restricted to the Isthmian area, expanded locally during the early Holocene, and left genomic traces up to the present day