Pylori Membrane Research Articles

Helicobacter pylori-targeting multiligand photosensitizer for effective antibacterial endoscopic photodynamic therapy

Helicobacter pylori (H. pylori) infection is closely associated with the development of gastric inflammatory diseases and cancer. However, the continued abuse and misuse of antibiotics has accelerated the spread of antibiotic-resistant strains, which poses a tremendous challenge for antibiotic-based H. pylori treatment. In this study, a H. pylori-targeting photodynamic therapy (PDT) system is proposed that multiple 3′-sialyllactose (3SL)-conjugated, poly-l-lysine-based photosensitizer (p3SLP). p3SLP facilitates H. pylori-targeting PDT via the specific interaction between 3SL and sialic acid-binding adhesin (SabA) in the H. pylori membrane. p3SLP can be orally administered to H. pylori infected mice and irradiated using an endoscopic laser system. The gastrointestinal pathological analysis of the H. pylori-infected mice demonstrated significant H. pylori specific antibacterial effects of PDT without side effects to normal tissue. In addition, an anti-inflammatory response was observed at the site of infection after p3SLP treatment. Consequently, this study demonstrates the superior efficacy of anti-H. pylori PDT with p3SLP in H. pylori-infected mice, and this approach shows great potential for replacing antibiotic-based therapy.

In vitro antimicrobial activity of immobilised essential oil components against Helicobacter pylori.

Available disinfection methods and therapies against Helicobacter pylori have multiple disadvantages, such as increased prevalence of antibiotic-resistant strains, which requires the search for novel effective antimicrobial agents against H. pylori. Among them, naturally-occurring antimicrobial compounds, like essential oil components (EOCs), have been reported as substances with anti-H. pylori potential. To avoid the disadvantages associated with using EOCs in their free form, including volatility, low water solubility and intense sensory properties, their immobilisation in inert supports has recently been developed. This study sought to evaluate the inhibitory properties of EOCs immobilised on silica microparticles against H. pylori and to elucidate the mechanism of action of the immobilised antimicrobials. After the preparation and characterisation of the antimicrobial supports, the susceptibility of H. pylori in the presence of the immobilised compounds was assessed by plate count, fluorescent viability staining and direct viable count-fluorescent in situ hybridisation analyses. The antimicrobial supports were found to inhibit H. pylori growth, and to induce morphological and metabolic alterations to the H. pylori membrane, with a minimum bactericidal concentration value between 25 and 50μg/ml according to the tested EOC. These findings indicate that immobilised EOCs can be used as potential antimicrobial agents for H. pylori clearance and treatment.

Docosahexaenoic acid loaded lipid nanoparticles with bactericidal activity against Helicobacter pylori

Docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid present in fish oil, has been described as a promising molecule to the treatment of Helicobacter pylori gastric infection. However, due to its highly unsaturated structure, DHA can be easily oxidized loosing part of its bioactivity. This work aims the nanoencapsulation of DHA to improve its bactericidal efficacy against H. pylori.DHA was loaded into nanostructured lipid carriers (NLC) produced by hot homogenization and ultrasonication using a blend of lipids (Precirol ATO5®, Miglyol-812®) and a surfactant (Tween 60®). Homogeneous NLC with 302±14nm diameter, −28±3mV surface charge (dynamic and electrophoretic light scattering) and containing 66±7% DHA (UV/VIS spectroscopy) were successfully produced.Bacterial growth curves, performed over 24h in the presence of different DHA concentrations (free or loaded into NLC), demonstrated that nanoencapsulation enhanced DHA bactericidal effect, since DHA-loaded NLC were able to inhibit H. pylori growth in a much lower concentrations (25μM) than free DHA (>100μM).Bioimaging studies, using scanning and transmission electron microscopy and also imaging flow cytometry, demonstrated that DHA-loaded NLC interact with H. pylori membrane, increasing their periplasmic space and disrupting membrane and allowing the leakage of cytoplasmic content. Furthermore, the developed nanoparticles are not cytotoxic to human gastric adenocarcinoma cells at bactericidal concentrations. DHA-loaded NLC should, therefore, be envisaged as an alternative to the current treatments for H. pylori infection.

Critical Role of Antimicrobial Peptide Cathelicidin for Controlling Helicobacter pylori Survival and Infection.

The antimicrobial peptide cathelicidin is critical for protection against different kinds of microbial infection. This study sought to elucidate the protective action of cathelicidin against Helicobacter pylori infection and its associated gastritis. Exogenous cathelicidin was found to inhibit H. pylori growth, destroy the bacteria biofilm, and induce morphological alterations in H. pylori membrane. Additionally, knockdown of endogenous cathelicidin in human gastric epithelial HFE-145 cells markedly increased the intracellular survival of H. pylori. Consistently, cathelicidin knockout mice exhibited stronger H. pylori colonization, higher expression of proinflammatory cytokines IL-6, IL-1β, and ICAM1, and lower expression of the anti-inflammatory cytokine IL-10 in the gastric mucosa upon H. pylori infection. In wild-type mice, H. pylori infection also stimulated gastric epithelium-derived cathelicidin production. Importantly, pretreatment with bioengineered Lactococcus lactis that actively secretes cathelicidin significantly increased mucosal cathelicidin levels and reduced H. pylori infection and the associated inflammation. Moreover, cathelicidin strengthened the barrier function of gastric mucosa by stimulating mucus synthesis. Collectively, these findings indicate that cathelicidin plays a significant role as a potential natural antibiotic for H. pylori clearance and a therapeutic agent for chronic gastritis.

Cathelicidin is a host defense peptide in controlling Helicobacter pylori survival and infection

Cathelicidin, a host defense antibacterial peptide in humans can eradicate different kinds of microbial infection. This study sought to elucidate the actions of cathelicidin in protection against Helicobacter pylori (H. pylori) infection both in vitro and in vivo. To examine the direct antimicrobial action of cathelicidin, H. pylori survival, biofilm formation and morphology change were determined after exposure to different doses of cathelicidin in vitro. Intracellular H. pylori in gastric epithelial cells and in cathelicidin wild‐type (Cnlp+/+) and knockout (Cnlp−/−) mice stomachs were also investigated. Results showed that exogenous cathelicidin could affect H. pylori growth, destroy bacteria biofilm and cause pore formation in H. pylori membranes. Endogenous cathelicidin in gastric epithelial cells could be induced by active form of vitamin D (1,25D3). We also showed that 1,25D3 eliminated the intracellular H. pylori in vitro. Furthermore, Cnlp−/−mice exhibited stronger H. pylori colonization in both acute and chronic H. pylori infection when compared with Cnlp+/+ mice. To further confirm this antibacterial action was due to endogenous cathelicidin, a high level of mouse gastric cathelicidin (CRAMP) production was found in Cnlp+/+ mice but not in Cnlp−/− animals. This finding could partially explain why there was less bacterial infection in wild type mice. Taken together, these results indicate that human cathelicidin plays a significant role as a host defense peptide for H. pylori clearance and infection.

IgG2 response and low IgG titre specific to Helicobacter pylori CagA as serological markers for gastric cancer

Infection with Helicobacter pylori cytotoxin-associated gene A (CagA)-positive strains is associated with the development of gastric cancer (GC). However, some reports have failed to demonstrate an increased frequency of CagA antibodies in GC patients. This study evaluated the response of IgG antibody and subclasses IgG1 and IgG2 against both CagA and H. pylori membrane antigens in patients with pre-cancerous lesions and cases with GC. A total of 137 patients with a positive serum IgG response to H. pylori were selected: 46 with intestinal metaplasia, 41 with gastric adenocarcinoma and 50 with non-atrophic gastritis (NAG) considered as controls. The response of total IgG, IgG1 and IgG2 was investigated by immunoblot and ELISA using an in-house recombinant CagA and membrane antigens from a local strain, and possible associations were estimated using a logistic regression model. Compared with NAG patients, GC patients showed a higher frequency of IgG2 CagA antibodies (55.2 vs 15.4 %, P = 0.001), but a lower frequency (80.5 vs 96.0 %, P = 0.021) and diminished levels of IgG2 H. pylori antibodies [12.5 vs 21.9 ELISA units (EU), P = 0.007]. GC patients also presented lower levels of CagA (32.6 vs 42.4 EU, P = 0.004) and H. pylori total IgG (33.7 vs 38.7 EU, P = 0.029). GC was associated with a positive IgG2 CagA response [odds ratio (OR) = 3.74, 95 % confidence interval (CI) 1.81-5.37; P = 0.002] and with a low titre of total IgG CagA antibodies (OR = 2.18, 95 % CI 1.35-2.69; P = 0.006). These results suggest that the IgG2 response to CagA could be used as a novel serological marker to identify patients with H. pylori-associated GC.

Role of the HefC Efflux Pump inHelicobacter pyloriCholesterol-Dependent Resistance to Ceragenins and Bile Salts

The human gastric pathogen Helicobacter pylori modifies host cholesterol via glycosylation and incorporates the glycosylated cholesterol into its membrane; however, the benefits of cholesterol to H. pylori are largely unknown. We speculated that cholesterol in the H. pylori membrane might alter the susceptibility of these organisms to membrane-disrupting antibacterial compounds. To test this hypothesis, H. pylori strains were cultured in Ham's F-12 chemically defined medium in the presence or absence of cholesterol. The two cultures were subjected to overnight incubations with serial 2-fold dilutions of 10 bile salts and four ceragenins, which are novel bile salt derivatives that mimic membrane-disrupting activity of antimicrobial peptides. H. pylori cultured with cholesterol was substantially more resistant to seven of the bile salts and three ceragenins than H. pylori cultured without cholesterol. In most cases, these cholesterol-dependent differences ranged from 2 to 7 orders of magnitude; this magnitude depended on concentration of the agent. Cholesterol is modified by glycosylation using Cgt, a cholesteryl glycosyltransferase. Surprisingly, a cgt knockout strain still maintained cholesterol-dependent resistance to bile salts and ceragenins, indicating that cholesterol modification was not involved in resistance. We then tested whether three putative, paralogous inner membrane efflux pumps, HefC, HefF, or HefI, played a role. While HefF and HefI appeared unimportant, HefC was shown to play a critical role in the resistance to bile salts and ceragenins by multiple methods in multiple strain backgrounds. Thus, both cholesterol and the putative bile salt efflux pump HefC play important roles in H. pylori resistance to bile salts and ceragenins.

Interleukin‐6 Induction by Helicobacter pylori in Human Macrophages is Dependent on Phagocytosis

The colonization of the gastric mucosa with Helicobacter pylori is accompanied by elevated levels of proinflammatory cytokines, such as interleukin-1 (IL-1), IL-6, and IL-8. The aim of our study was to determine the mechanisms of IL-6 stimulation in phagocytes upon H. pylori infection. We investigated the secretion of IL-6 by different professional phagocytes from murine and human origin, including granulocyte- and monocyte-like cells and macrophages derived from human peripheral blood monocytes (PBMCs). The influence of viability, phagocytosis, and the impact of different subcellular fractions of H. pylori bacteria were evaluated. IL-6 levels induced by H. pylori were low in cell lines derived from murine and human monocytes and in human granulocyte-like cells. By contrast, macrophages derived from human PBMCs were highly responsive to both H. pylori and Escherichia coli. IL-6 induction was blocked by inhibition of actin-dependent processes prior to infection with H. pylori, but not with E. coli or E. coli lipopolysaccharide (LPS). Using cell fractionation, the most activity was found in the H. pylori membrane. H. pylori LPS exhibited a 10(3)- to 10(4)-fold lower biologic activity than E. coli LPS, suggesting a minor role for toll-like receptor 4 (TLR4)-mediated signalling from the exterior. From these data, we conclude that macrophages may be a major source of IL-6 in the gastric mucosa upon H. pylori infection. The IL-6 induction by H. pylori in these cells is a multifactorial process, which requires the uptake and presumably degradation of H. pylori bacteria.

Mechanism of strong resistance of Helicobacter pylori respiration to nitric oxide

The aim of the present work is to elucidate the mechanism by which the respiration of Helicobacter pylori but not of Escherichia coli shows a strong resistance to nitric oxide (NO). Nitric oxide strongly but reversibly inhibited the oxygen consumption by sonicated membranes from H. pylori and Triton X-100-treated cells. Although the sensitivity of the H. pylori respiration to cyanide was low, it also increased after the treatment with Triton X-100. Kinetic analyses revealed that NO was rapidly degraded by E. coli and the Triton X-100-treated H. pylori, but not by the intact H. pylori. Thus, the low sensitivity to NO might reflect the low affinity of the cytochrome c oxidase for this radical within the membrane/lipid bilayers of H. pylori. Such properties of the oxidase in H. pylori membranes may, at least in part, underlie the mechanism by which this bacterium thrives in NO-enriched gastric juice.

Helicobacter pylori infection: pathogenesis

This review covers progress in identifying Helicobacter pylori-derived factors that are involved in survival and virulence of the organism and in elucidating host response pathways that can limit the infection but are also susceptible to dysregulation. Recent work has identified genes of the cytotoxin-associated gene (cag) pathogenicity island (PAI) involved in regulating signaling, interleukin-8 secretion, and phenotypic events in epithelial cells. New roles in pathogenesis have been recognized for vacuolating toxin A (VacA) and urease, H. pylori membrane and secreted factors, and host epithelial surface molecules. Molecular pathways involved in H. pylori-induced apoptosis in epithelial cells, T cells, and macrophages are being dissected. Activation of toll-like receptors and bacterial factors involved in nitric oxide (NO) and reactive oxygen species induction were also described. The ability of H. pylori to limit NO production by several mechanisms may be an important part of its ability to evade the host immune response.

Interactions of plaunotol with bacterial membranes.

Plaunotol, a cytoprotective antiulcer agent, has a bactericidal effect against Helicobacter pylori, which may result from interaction of this compound with the bacterial cell membrane. The purpose of the present study was to confirm that plaunotol interacts with the H. pylori membrane. Membrane fluidities were measured using two stearic acid spin labels, namely 5-doxyl-stearic acid (in which the nitroxide group is located in the upper portion of the bacterial cell membrane) and 16-doxyl-stearic acid methyl ester (in which the nitroxide group is located deeper in the bacterial cell membrane), by means of electron spin resonance. The membrane fluidities of plaunotol-treated cells were significantly increased in the measurements made using the two spin labels. We also attempted to isolate plaunotol-resistant H. pylori in vitro by two different methods. To assess the level of resistance that could be reached, H. pylori was passaged five times on an agar plate containing subinhibitory concentrations of plaunotol or metronidazole. To measure the rate of development of resistance, H. pylori was grown with subinhibitory concentrations (0.25 x MIC) of plaunotol or metronidazole, and quantitatively plated on to medium containing 4 x MIC of the compounds. This treatment was repeated once more. No plaunotol-resistant colonies were selected by the two methods. H. pylori developed resistance to metronidazole easily and at a relatively high rate. The mechanism by which plaunotol directly fluidizes and destroys the H. pylori membrane might make it difficult for this organism to develop resistance to plaunotol. It was confirmed that the bactericidal effects of plaunotol were also shown against Staphylococcus aureus, Streptococcus pneumoniae, Neisseria gonorrhoeae, Moraxella catarrhalis and Haemophilus influenzae. No such effect was seen against Escherichia coli and Pseudomonas aeruginosa.

Fumarate reductase of Helicobacter pylori--an immunogenic protein.

An immunogenic protein with an apparent mol. wt of 80 kDa that was recognised by 55% of sera from patients infected with Helicobacter pylori in Western blots was found in butanol extracts of H. pylori membranes. The N-terminal amino-acid sequence of the 80-kDa protein showed 80% identity with the N-terminal sequence of subunit A of the fumarate reductase of Wolinella succinogenes, suggesting the existence of a fumarate reductase in H. pylori. The membrane fraction of H. pylori catalysed succinate oxidation with methylene blue at a specific enzyme activity of 0.06 U/mg of protein. The enzyme was purified by Triton X100 extraction followed by ion-exchange chromatography. The purified enzyme contained an 80-kDa protein which was recognised by rabbit serum raised against subunit A of fumarate reductase of W. succinogenes. A second protein band with a mol. wt of 31 kDa was recognised by rabbit serum raised against subunit B of fumarate reductase of W. succinogenes. Two-dimensional gel electrophoresis demonstrated that the 80- and 31-kDa proteins were subunits of one protein complex. These results indicate that H. pylori contains an enzyme that is very similar to W. succinogenes fumarate reductase. The 80-kDa subunit was recognised in sonicates of all 32 H. pylori strains tested by rabbit antibodies raised against subunit A of fumarate reductase of W. succinogenes, indicating that fumarate reductase is a common protein in H. pylori. The fumarate reductase of H. pylori might enable the bacterium to perform anaerobic respiration in a similar fashion to other anaerobic or facultative bacteria.