Antimicrobial Effect of a Proteolytic Enzyme From the Fruits of Solanum granuloso-leprosum (Dunal) Against Helicobacter pylori

Helicobacter pylori is a gram-negative, helix-shaped, and microaerophilic bacteria that colonizes the human gastric mucosa, causing chronic infections, gastritis, peptic ulcer, lymphomas associated with lymphoid mucosa tissue, and gastric cancer. H. pylori is considered a Type 1 human carcinogen by WHO. The prevalence of the infection is estimated in more than half of the world population. Treatment of H. pylori infection includes antibiotics and proton pump inhibitors, but the increasing antibiotic resistance promotes the research of novel, more effective, and natural antibacterial compounds. The aim of this work was to study the effect of the partially purified proteolytic extract (RAP) of the fruits from Solanum granuloso-leprosum (Dunal), a South American native plant, and a purified fraction named granulosain I, against H. pylori, to obtain natural food additives for the production of anti-H. pylori functional foods. Furthermore, granulosain I and RAP could be used as natural adjuncts to conventional therapies. Granulosain I and RAP antibacterial activity was evaluated as minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against H. pylori NCTC 11638 (reference strain) and twelve H. pylori wild strains, using a microdilution plating technique (Clinical and Laboratory Standards Institute). All the strains tested were susceptible to granulosain I with MIC from 156.25 to 312.5 μg/mL and MBC from 312.5 to 625 μg/mL, respectively. Besides, all the strains tested were susceptible to the RAP with MIC from 312.5 to 625 μg/mL and MBC from 625 to 1,250 μg/mL, respectively. The effect of granulosain I and RAP on the transcription of H. pylori genes encoding pathogenic factors, omp18, ureA, and flaA, with respect to a housekeeping gene (16S rRNA), was evaluated by RT-PCR technique. The band intensity between pathogenic factors and control gene was correlated under treated or untreated conditions, using the ImageJ program. Granulosain I and RAP significantly decreased the expression of pathogenic factors: omp18, ureA, and flaA. The combined inhibitory effect of granulosain I or RAP and an antibiotic such as, amoxicillin (AML, 10 μg), clarithromycin (CLA, 15 μg), levofloxacin (LEV, 5 μg), and metronidazole (MTZ, 5 μg) was evaluated, using the agar diffusion technique. Granulosain I and RAP showed significant synergistic effect on AML, CLA, and LEV, but no significant effect on MTZ was observed. Besides, granulosain I and RAP did not show toxicological effects at the concentrations studied. Finally, granulosain I and RAP could be used as safe natural food additives and as adjuvants for conventional therapies against H. pylori.

MNPmApp: An image analysis tool to quantify mononuclear phagocyte distribution in mucosal tissuesa, b

Mononuclear phagocytes (MNPs) such as dendritic cells and macrophages perform key sentinel functions in mucosal tissues and are responsible for inducing and maintaining adaptive immune responses to mucosal pathogens. Positioning of MNPs at the mucosal epithelial interface facilitates their access to luminally-derived antigens and may regulate MNP function through soluble mediators or surface receptor interactions. Therefore, accurately quantifying the distribution of MNPs within mucosal tissues as well as their spatial relationship with other cells is important to infer functional cellular interactions in health and disease. In this study, we developed and validated a MATLAB-based tissue cytometry platform, termed “MNP mapping application” (MNPmApp), that performs high throughput analyses of MNP density and distribution in the gastrointestinal mucosa based on digital multicolor fluorescence microscopy images and that integrates a Monte Carlo modeling feature to assess randomness of MNP distribution. MNPmApp identified MNPs in tissue sections of the human gastric mucosa with a specificity of 98.3 ± 1.6% and a sensitivity of 76.4 ± 15.1%. Monte Carlo modeling revealed that mean MNP-MNP distances were significantly lower than anticipated based on random cell placement, whereas MNP-epithelial distances did not significantly differ from those of randomly placed cells. Interestingly, H. pylori infection had no significant impact on MNP density or distribution with regards to MNP-epithelial distances or MNP-MNP distances in gastric tissue. Overall, our analysis demonstrates that MNPmApp is a useful tool for unbiased quantitation of MNPs and their distribution at mucosal sites.

Adhesion of Helicobacter Species to the Human Gastric Mucosa: A Deep Look Into Glycans Role

Helicobacter species infections may be associated with the development of gastric disorders, such as gastritis, peptic ulcers, intestinal metaplasia, dysplasia and gastric carcinoma. Binding of these bacteria to the gastric mucosa occurs through the recognition of specific glycan receptors expressed by the host epithelial cells. This review addresses the state of the art knowledge on these host glycan structures and the bacterial adhesins involved in Helicobacter spp. adhesion to gastric mucosa colonization. Glycans are expressed on every cell surface and they are crucial for several biological processes, including protein folding, cell signaling and recognition, and host-pathogen interactions. Helicobacter pylori is the most predominant gastric Helicobacter species in humans. The adhesion of this bacterium to glycan epitopes present on the gastric epithelial surface is a crucial step for a successful colonization. Major adhesins essential for colonization and infection are the blood-group antigen-binding adhesin (BabA) which mediates the interaction with fucosylated H-type 1 and Lewis B glycans, and the sialic acid-binding adhesin (SabA) which recognizes the sialyl-Lewis A and X glycan antigens. Since not every H. pylori strain expresses functional BabA or SabA adhesins, other bacterial proteins are most probably also involved in this adhesion process, including LabA (LacdiNAc-binding adhesin), which binds to the LacdiNAc motif on MUC5AC mucin. Besides H. pylori, several other gastric non-Helicobacter pylori Helicobacters (NHPH), mainly associated with pigs (H. suis) and pets (H. felis, H. bizzozeronii, H. salomonis, and H. heilmannii), may also colonize the human stomach and cause gastric disease, including gastritis, peptic ulcers and mucosa-associated lymphoid tissue (MALT) lymphoma. These NHPH lack homologous to the major known adhesins involved in colonization of the human stomach. In humans, NHPH infection rate is much lower than in the natural hosts. Differences in the glycosylation profile between gastric human and animal mucins acting as glycan receptors for NHPH-associated adhesins, may be involved. The identification and characterization of the key molecules involved in the adhesion of gastric Helicobacter species to the gastric mucosa is important to understand the colonization and infection strategies displayed by different members of this genus.

Hsp60 Quantification in Human Gastric Mucosa Shows Differences between Pathologies with Various Degrees of Proliferation and Malignancy Grade

Background: Stomach diseases are an important sector of gastroenterology, including proliferative benign; premalignant; and malignant pathologies of the gastric mucosa, such as gastritis, hyperplastic polyps, metaplasia, dysplasia, and adenocarcinoma. There are data showing quantitative changes in chaperone system (CS) components in inflammatory pathologies and tumorigenesis, but their roles are poorly understood, and information pertaining to the stomach is scarce. Here, we report our findings on one CS component, the chaperone Hsp60, which we studied first considering its essential functions inside and outside mitochondria. Methods: We performed immunohistochemical experiments for Hsp60 in different samples of gastric mucosa. Results: The data obtained by quantitative analysis showed that the average percentages of Hsp60 were of 32.8 in normal mucosa; 33.5 in mild-to-moderate gastritis; 51.8 in severe gastritis; 58.5 in hyperplastic polyps; 67.0 in intestinal metaplasia; 89.4 in gastric dysplasia; and 92.5 in adenocarcinomas. Noteworthy were: (i) the difference between dysplasia and adenocarcinoma with the other pathologies; (ii) the progressive increase in Hsp60 from gastritis to hyperplastic polyp, gastric dysplasia, and gastric carcinoma; and (iii) the correlation of Hsp60 levels with histological patterns of cell proliferation and, especially, with tissue malignancy grades. Conclusions: This trend likely reflects the mounting need for cells for Hsp60 as they progress toward malignancy and is a useful indicator in differential diagnosis, as well as the call for research on the mechanisms underpinning the increase in Hsp60 and its possible roles in carcinogenesis.

Helicobacter pylori infection rates in dyspeptic Serbian HIV-infected patients compared to HIV-negative controls

Helicobacter pylori infection does not belong to the spectrum of opportunistic infections in people living with HIV (PLHIV). To evaluate the Helicobacter pylori infection prevalence rate trends in HIV co-infected individuals in comparison to the HIV-negative population, we compared histopathological findings of H. pylori positive gastritis (gastritis topography and histopathology) between 303 PLHIV and 2642 HIV-negative patients who underwent esophagogastroduodenoscopy (EGD) between 1993 and 2014 due to dyspeptic symptoms. The prevalence of H. pylori infection was significantly higher in HIV-negative controls than in PLHIV (50.2% vs. 28.1%). A significantly positive linear trend of H. pylori co-infection in PLHIV was revealed in the observed period (b = 0.030, SE = 0.011, p = 0.013), while this trend was significantly negative in HIV-negative patients (b = - 0.027, SE = 0.003, p < 0.001). Patients with HIV/H. pylori co-infection had significantly higher CD4+ T cell counts and more often had undetectable HIV viremia, due to successful anti-retroviral therapy (ART). Stomach histopathological findings differed between HIV co-infected and H. pylori mono-infected patients. Our findings confirm that the ART has changed the progression of HIV infection, leading to a significant increase in the prevalence of H. pylori infection in dyspeptic PLHIV over time. Our data also suggests that a functional immune system may be needed for H. pylori-induced human gastric mucosa inflammation.

Disruption of gastric mucous granule exocytosis by Helicobacter pylori virulence factor CagA

Helicobacter pylori infection is the strongest known risk factor of stomach cancer. Strains harboring the virulence factor CagA (cytotoxin-associated gene A) significantly stimulate host inflammatory response, which increases the risk of ulceration and cancer. However, the mechanisms by which CagA triggers prolonged inflammation with mucosal damage remain elusive. Based on a large-scale genetic screen using Drosophila, we identified a novel CagA target Synaptotagmin-like protein 2-a, Slp2-a, an effector of small GTPase Rab27. Using gastric organoid-derived monolayers of polarized mucous cells, we demonstrated that CagA inhibited Slp2-a-mediated docking of mucous granules to the plasma membrane by direct binding to Slp2-a. We further observed aberrant cytoplasmic retention of mucus in human gastric mucosa infected with CagA-expressing strains. These results suggest that CagA could be disrupting the protective mucous barrier by inhibiting Slp2-a-mediated mucous granule exocytosis, which may lead to mucosal damage from luminal acid and pepsin to promote inflammation leading to cancer.