Compromised autophagy by MIR30B benefits the intracellular survival of Helicobacter pylori

Helicobacter pylori evade immune responses and achieve persistent colonization in the stomach. However, the mechanism by which H. pylori infections persist is not clear. In this study, we showed that MIR30B is upregulated during H. pylori infection of an AGS cell line and human gastric tissues. Upregulation of MIR30B benefited bacterial replication by compromising the process of autophagy during the H. pylori infection. As a potential mechanistic explanation for this observation, we demonstrate that MIR30B directly targets ATG12 and BECN1, which are important proteins involved in autophagy. These results suggest that compromise of autophagy by MIR30B allows intracellular H. pylori to evade autophagic clearance, thereby contributing to the persistence of H. pylori infections.     

Methods to monitor autophagy in H. pylori vacuolating cytotoxin A (VacA)-treated cells

Helicobacter pylori is a gram negative pathogen that infects at least half of the world’s population and is associated not only with gastric cancer but also with other diseases such as gastritis and peptic ulcers. Indeed, H. pylori is considered the single most important risk factor for the development of gastric cancer. The vacuolating cytotoxin, VacA, secreted by H. pylori promotes intracellular survival of the bacterium and modulates host immune responses. In a recent study, we reported that VacA induces autophagy. Multilamellar autophagosomes are detected in gastric epithelial cells that are distinct from the large vacuoles formed by VacA. Furthermore, inhibition of autophagy stabilizes VacA and reduces vacuolation in the cells indicating that the toxin is being degraded by autophagy, thus limiting toxin-induced host cell damage. Many of the methods that were used for this study are commonly employed techniques that were adapted for H. pylori infection and VacA intoxication. In this paper, we describe the various methods and specific protocols used for the assessment and monitoring of autophagy during H. pylori infection.     

Crohn disease ATG16L1 polymorphism increases susceptibility to infection with Helicobacter pylori in humans

Autophagy plays key roles both in host defense against bacterial infection and in tumor biology. Helicobacter pylori (H. pylori) infection causes chronic gastritis and is the single most important risk factor for the development of gastric cancer in humans. Its vacuolating cytotoxin (VacA) promotes gastric colonization and is associated with more severe disease. Acute exposure to VacA initially triggers host autophagy to mitigate the effects of the toxin in epithelial cells. Recently, we demonstrated that chronic exposure to VacA leads to the formation of defective autophagosomes that lack CTSD/cathepsin D and have reduced catalytic activity. Disrupted autophagy results in accumulation of reactive oxygen species and SQSTM1/p62 both in vitro and in vivo in biopsy samples from patients infected with VacA⁺ but not VacA^- strains. We also determined that the Crohn disease susceptibility polymorphism in the essential autophagy gene ATG16L1 increases susceptibility to H. pylori infection. Furthermore, peripheral blood monocytes from individuals with the ATG16L1 risk variant show impaired autophagic responses to VacA exposure. This is the first study to identify both a host autophagy susceptibility gene for H. pylori infection and to define the mechanism by which the autophagy pathway is affected following H. pylori infection. Collectively, these findings highlight the synergistic effects of host and bacterial autophagy factors on H. pylori pathogenesis and the potential for subsequent cancer susceptibility.     

Effect of Helicobacter pylori’s vacuolating cytotoxin on the autophagy pathway in gastric epithelial cells

Host cell responses to Helicobacter pylori infection are complex and incompletely understood. Here, we report that autophagy is induced within human-derived gastric epithelial cells (AGS) cells in response to H. pylori infection. These autophagosomes were distinct and different from the large vacuoles induced during H. pylori infection. Autophagosomes were detected by transmission electron microscopy, conversion of LC3-I to LC3-II, GFP-LC3 recruitment to autophagosomes, and depended on Atg5 and Atg12. The induction of autophagy depended on the vacuolating cytotoxin (VacA) and, moreover, VacA was sufficient to induce autophagosome formation. The channel forming activity of VacA was necessary for inducing autophagy. Intracellular VacA partially co-localized with GFP-LC3, indicating that the toxin associates with autophagosomes. The inhibition of autophagy increased the stability of intracellular VacA, which in turn resulted in enhanced toxin-mediated cellular vacuolation. These findings suggest that the induction of autophagy by VacA may represent a host mechanism to limit toxin-induced cellular damage.     

The association of intestinal parasitosis and H. pylori infection in children and adults from a Mexican community with high prevalence of parasitosis

Background. ABSTExperimental evidences have suggested that a Th1 response is unable to eliminate H. pylori colonization; whereas a Th2 response, like the one induced by vaccination, reduces H. pylori infection in animal models. Some parasitic infections induce a polarized Th2 response, which theoretically would favor a reduced H. pylori prevalence. The aim of this work was to study the possible association between parasitic infections and H. pylori prevalence. Materials and Methods. The study population included 120 children and 188 adults from a low socioeconomic level village. H. pylori prevalence was determined in serum by ELISA; parasitic infections were identified in feces by microscopic examination; and total serum IgE levels, as an indirect indicator of some parasitic infections, were determined by ELISA. Results. In children, H. pylori prevalence was no different between those with and without intestinal parasitic infection. By contrast, adults with intestinal parasitic infection had a significantly lower H. pylori prevalence than adults without parasites (62.6% compared with 80.4%; p = 0.006, OR 2.45). Also in adults, but not in children, total IgE levels were significantly higher in those without H. pylori infection than in those with H. pylori infection (p < 0.001). Conclusions. Intestinal parasitic infections and serum IgE levels showed an age‐dependent association with H. pylori prevalence. In adults, but not in children, intestinal parasitic infections and increased IgE levels where associated with a reduced H. pylori prevalence.     

MicroRNA targets autophagy in pancreatic cancer cells during cancer therapy

The therapeutic outcome of pancreatic cancer is generally poor due to the inherent or acquired resistance of cancer cells to treatment. Pancreatic cancer cells have higher basal autophagy levels than other cancer cell types, which may correlate with their nonresponsiveness to the available cancer therapy. Therefore, understanding the mechanisms behind autophagy activation in pancreatic cancer cells may ultimately improve therapeutic outcomes. Here we demonstrated that MIR23B is a potent inhibitor of autophagy. MIR23B targets the 3′UTR of the autophagy-related gene ATG12, thereby decreasing autophagic activity and ultimately promoting radiation-induced pancreatic cancer cell death. Thus, our study clarified some of the underlying molecular mechanisms of activated autophagy in response to cancer therapy in pancreatic cancer.     

MicroRNA expression profiling of Leishmania donovani-infected host cells uncovers the regulatory role of MIR30A-3p in host autophagy

Leishmania is an obligate intracellular parasite that replicates inside phagolysosomes or parasitophorous vacuoles (PV) of the monocyte/macrophage lineage. It reprograms macrophages and produces a metabolic state conducive to successful infection and multiplication. MicroRNAs (miRNAs), a class of small 22 to 24 nucleotide noncoding regulatory RNAs alter the gene expression and consequently proteome output by targeting mRNAs, may play a regulatory role in modulating host cell functions. In the present study, we demonstrate the novel regulatory role of host microRNA, MIR30A-3p in modulation of host cell macroautophagy/autophagy after infection with L. donovani. Our in vitro studies showed that MIR30A-3p expression was significantly enhanced after L. donovani infection in a time-dependent manner. Transient transfection with a MIR30A-3p inhibitor followed by L. donovani infection promoted the autophagic response and decreased the intracellular parasite burden in both THP-1 cells and human monocyte-derived macrophages (HsMDM). BECN1/Beclin 1, the mammalian ortholog of yeast Vps30/Atg6, is a key autophagy-promoting protein that plays a key role in the regulation of cell death and survival. We report BECN1-dependent modulation of host cell autophagy in response to L. donovani infection. Pretreatment of L. donovani-infected macrophages with the MIR30A-3p mimic decreased and with antagomir increased the expression of BECN1 protein. We demonstrate that BECN1 is a potential target of MIR30A-3p and this miRNA negatively regulates BECN1 expression. Our present study reveals for the first time a novel role of MIR30A-3p in regulating autophagy-mediated L. donovani elimination by targeting BECN1. The present study has significant impact for the treatment of visceral leishmaniasis.     

Current information on the association of Helicobacter pylori with autophagy and gastric cancer

Helicobacter pylori (H. pylori) is a Gram-negative bacterium and causative agent of gastric cancer. H. pylori induce defective autophagy or inhibit it by means of CagA and vacuolating cytotoxin A (VacA) toxins leading to the gastric cancer induction. Impaired or defective autophagy leads to the accumulation of cytotoxic materials, such as ROS and P62 that lead to increased mutations in the DNA, genome instability, and risk of cancer formation. H. pylori CagA may inhibit autophagy through the c-Met-PI3k/Akt-mTOR signaling pathway. However, VacA induces autophagy by some signaling pathways. In the gastric epithelial cells, VacA is a necessary and sufficient factor for the creation of autophagy. While CagA is a negative regulator of this phenomenon, the elimination of this gene from H. pylori has increased autophagy and the production of inflammatory cytokines is reduced. In gastrointestinal cancers, some of the microRNAs (miRNAs) act as tumor suppressors and some other are oncogenes by regulating various genes expression. H. pylori can also modify autophagy through a mechanism that includes the function of miRNAs. In autophagy, oncogenic miRNAs inhibit activation of some tumor suppressor signaling pathways (e.g., ULK1 complex, Beclin-1 function, and Atg4 messaging), whereas tumor suppressor miRNAs can block the activation of oncogenic signaling pathways. For instance, Beclin-1 is negatively regulated by miRNA-376b (oncogenic miRNA) and miRNA-30a (tumor suppressor miRNA). Similarly, Atg4 by miRNA-376b (oncogenic miRNA) and miRNA-101 (tumor suppressor miRNA). So, this apparent paradox can be explained as that both Beclin-1 and Atg4 play different roles in a particular cell or tissue.     

Reactive oxygen species and gastric carcinogenesis: The complex interaction between Helicobacter pylori and host

Helicobacter pylori (H. pylori) is a highly successful human pathogen that colonizes stomach in around 50% of the global population. The colonization of bacterium induces an inflammatory response and a substantial rise in the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS), mostly derived from host neutrophils and gastric epithelial cells, which play a crucial role in combating bacterial infections. However, H. pylori has developed various strategies to quench the deleterious effects of ROS, including the production of antioxidant enzymes, antioxidant proteins as well as blocking the generation of oxidants. The host's inability to eliminate H. pylori infection results in persistent ROS production. Notably, excessive ROS can disrupt the intracellular signal transduction and biological processes of the host, incurring chronic inflammation and cellular damage, such as DNA damage, lipid peroxidation, and protein oxidation. Markedly, the sustained inflammatory response and oxidative stress during H. pylori infection are major risk factor for gastric carcinogenesis. In this context, we summarize the literature on H. pylori infection-induced ROS production, the strategies used by H. pylori to counteract the host response, and subsequent host damage and gastric carcinogenesis.     

Burkholderia cenocepacia J2315 escapes to the cytosol and actively subverts autophagy in human macrophages

Selective autophagy functions to specifically degrade cellular cargo tagged by ubiquitination, including bacteria. Strains of the Burkholderia cepacia complex (Bcc) are opportunistic pathogens that cause life‐threatening infections in patients with cystic fibrosis (CF) and chronic granulomatous disease (CGD). While there is evidence that defective macrophage autophagy in a mouse model of CF can influence B. cenocepacia susceptibility, there have been no comprehensive studies on how this bacterium is sensed and targeted by the host autophagy response in human macrophages. Here, we describe the intracellular life cycle of B. cenocepacia J2315 and its interaction with the autophagy pathway in human cells. Electron and confocal microscopy analyses demonstrate that the invading bacteria interact transiently with the endocytic pathway before escaping to the cytosol. This escape triggers theselective autophagy pathway, and the recruitment of ubiquitin, the ubiquitin‐binding adaptors p62 and NDP52 and the autophagosome membrane‐associated protein LC3B, to the bacterial vicinity. However, despite recruitment of these key autophagy pathway effectors, B. cenocepacia blocks autophagosome completion and replicates in the host cytosol. We find that a pre‐infection increase in cellular autophagy flux can significantly inhibit B. cenocepacia replication and that lower autophagy flux in macrophages from immunocompromised CGD patients could contribute to increased B. cenocepacia susceptibility, identifying autophagy manipulation as a potential therapeutic approach to reduce bacterial burden in B. cenocepacia infections.     

Burkholderia pseudomallei survival in lung epithelial cells benefits from miRNA-mediated suppression of ATG10

Burkholderia pseudomallei is the causative agent of melioidosis, a disease with high mortality, which is prevalent in tropical regions of the world. A recent study shows that B. pseudomallei can survive inside mammalian cells because of its ability to actively evade cell autophagy. However, the underlying mechanisms remain unclear. In the present study, based on microarray screening, we found that ATG10 was downregulated following B. pseudomallei infection in A549 human lung epithelial cells. Forced expression of ATG10 accelerated the elimination of intracellular B. pseudomallei by enhancing the process of autophagy. Moreover, MIR4458, MIR4667-5p, and MIR4668-5p were found, by microarray screening, to be upregulated in response to B. pseudomallei infection. These 3 novel miRNAs, MIR4458, MIR4667-5p, and MIR4668-5p, targeted to the 3′-untranslated region of ATG10 in different time-course and spatial manners. Upregulation of these miRNAs reduced the level of ATG10 and inhibited autophagy, leading to increasing survival rate of intracellular B. pseudomallei. Furthermore, the increase of these miRNAs was correlated with the reduced promoter methylation status in A549 cells in response to B. pseudomallei infection. Our results reveal that 3 novel miRNAs regulate autophagy-mediated elimination of B. pseudomallei by targeting ATG10, and provide potential targets for clinical treatment.     

Autophagy Limits Listeria monocytogenes Intracellular Growth in the Early Phase of Primary Infection

Autophagy has been recently proposed to be a component of the innate cellular immune response against several types of intracellular microorganisms. However, other intracellular bacteria including Listeria monocytogenes have been thought to evade the autophagic cellular surveillance. Here, we show that cellular infection by L. monocytogenes induces an autophagic response, which inhibits the growth of both the wild-type and a delta actA mutant strain, the latter being impaired in cell-to-cell spreading. The onset of early intracellular growth is accelerated in autophagy-deficient cells, but the growth rate once bacteria begin to multiply in the cytosol does not change. Moreover, a significant fraction of the intracellular bacteria co-localize with autophagosomes at the early time-points after infection. Thus, autophagy targets L. monocytogenes during primary infection by limiting the onset of early bacterial growth. The bacterial expression of listeriolysin O but not phospholipases is necessary for the induction of autophagy, suggesting a possible role for permeabilization of the vacuole in the induction of autophagy. Interestingly, the growth of a delta plcA/B L. monocytogenes strain deficient for bacterial phospholipases is impaired in wild-type cells, but restored in the absence of autophagy, suggesting that bacterial phospholipases may facilitate the escape of bacteria from autophagic degradation. We conclude that L. monocytogenes are targeted for degradation by autophagy during the primary infection, in the early phase of the intracellular cycle, following listeriolysin O-dependent vacuole perforation but preceding active multiplication in the cytosol, and that expression of bacterial phospholipases is necessary for the evasion of autophagy.     

Impact of Helicobacter pylori on the development of vitamin B12 deficiency in the absence of gastric atrophy

Background. Cobalamin (vitamin B₁₂) deficiency is associated with Helicobacter pylori infection. This study examined how serum vitamin B₁₂ levels relate to gastric mucosa H. pylori density and histology, and to hematological findings in patients with minimal or no gastric atrophy. A second aim was to confirm that H. pylori eradication therapy increases serum B₁₂. Materials and Methods. Biopsies of the gastric mucosa from a population of dyspeptic patients were graded for level of chronic inflammation, neutrophil activity, atrophy, and H. pylori density. A total of 145 H. pylori‐infected patients with minimal or no atrophy were included in the study. Serum cobalamin level, hemoglobin level, and mean corpuscular volume were measured in the 145 patients before eradication therapy, and in 65 of the subjects after treatment. The hematologic findings before and after eradication therapy and correlations between serum vitamin B₁₂ level and histologic parameters, hematologic findings, and patient age were statistically analyzed. Results. There was no significant correlation between serum cobalamin level and patient age. Before treatment all the histopathological scores were inversely correlated with serum vitamin B₁₂ level (p < .01) on univariate analysis. Only H. pylori density was significantly associated with B₁₂ level on multivariate analysis. Serum hemoglobin and cobalamin levels were significantly increased after treatment, regardless of H. pylori eradication status (p < .001). Conclusion. The findings provide strong evidence that H. pylori infection is associated with cobalamin deficiency, and show that this is true even in patients with nonulcer dyspepsia and minimal or no gastric atrophy.     

When Helicobacter pylori invades and replicates in the cells

Autophagosome formation is induced by Helicobacter pylori infection and these autophagic vesicles are adopted for replication of H. pylori and subsequent eradication of the invading H. pylori in macrophages. Some Taiwanese clinical isolates of H. pylori can replicate in certain macrophage cell lines. After entry, there was a 5-10 fold increment of re-cultivable H. pylori from the infected permissible cells at 12 h post infection. The dividing H. pylori are observed to reside in double-layered autophagosomes. Therefore, H. pylori can be considered as a kind of intracellular microorganism. The autophagy induction by H. pylori is not only found in macrophages, but also in dendritic cells and gastric epithelial cells. This new finding has several implications for the life cycle of H. pylori in the host. The bacterium’s residence inside infected cells will increase its resistance to antimicrobial treatment, avoid neutralization by anti-H. pylori antibodies, impair antigen presentation, and alter the cellular immune response. The replication of H. pylori in autophagic vesicles, and the consequences of this provide an important hint as to why this microorganism causes so such a broad spectrum of diseases.     

Helicobacter pylori: immunoproteomics related to different pathologies

Helicobacter pylori is a Gram-negative bacterium that causes ulcer, atrophic gastritis, adenocarcinoma and mucosa-associated lymphoid tissue lymphoma. Moreover, an ongoing controversial role of this bacterium infection has been suggested in the etiopathogenesis of some extradigestive diseases. The humoral response to H. pylori during a natural infection can be used for diagnostic purposes and as a basis for vaccine development. Host–pathogen interactions may be investigated by means of immunoproteomics, which provides global information about relevant specific and nonspecific antigens, and thus might be suitable to identify novel vaccine candidates or serological markers of H. pylori infection as well as of different related diseases. In this review, we describe how several research groups used H. pylori proteomics combined with western blotting analysis, using sera from patients affected with different H. pylori-related pathologies, to investigate potential associations between host immune response and clinical outcomes of H. pylori infection, resulting in the rapid identification of novel, highly immunoreactive antigens.     

Presence of Helicobacter pylori in a Sibling is Associated with a Long‐Term Increased Risk of H. pylori Infection in Israeli Arab Children

Background and Objectives: We examined the dynamics of Helicobacter pylori infection between pre‐school and school ages and compared the determinants of late acquisition of H. pylori infection with determinants of early and persistent H. pylori infection. Methods: ELISA was used to detect H. pylori antigens in stool specimens collected from children at preschool age (3–5 years) and from their mothers and siblings in 2004. The children were tested again for H. pylori at school age (6–9 years) in 2007–2009. Household and socioeconomic characteristics were obtained by interviews. Results: The prevalence of H. pylori infection increased from 49.7% (95% CI 42.8, 56.7) in 2004 to 58.9% (95% CI 51.8, 65.6) in 2007–2009. Among children tested in both examinations, 69 (49.3%) had persistent infection, 14 (10.0%) were new cases, 56 (40.0%) remained uninfected, and one (0.7%) had lost H. pylori infection. The approximate annual incidence of infection during 2004–2009 was 5%. Sibling’s H. pylori positivity at baseline increased the risk for late acquisition of H. pylori infection; adjusted prevalence ratio (PR) 4.62 (95% CI 0.76, 28.23) (p = .09), while maternal education lowered the risk; adjusted PR 0.84 (95% CI 0.69, 1.01) (p = .06). Sibling’s H. pylori positivity was the only significant variable associated with early and persistent H. pylori infection in multivariate analysis. Conclusions: Most H. pylori infections are acquired at preschool age and transient infection beyond this age is uncommon in this population. Helicobacter pylori‐infected siblings are the major reservoir of H. pylori in early and late childhood demonstrating sustained intra‐familial transmission of H. pylori.