Rab32 GTPase, as a direct target of miR-30b/c, controls the intracellular survival of Burkholderia pseudomallei by regulating phagosome maturation.

Burkholderia pseudomallei is a gram-negative, facultative intracellular bacterium, which causes a disease known as melioidosis. Professional phagocytes represent a crucial first line of innate defense against invading pathogens. Uptake of pathogens by these cells involves the formation of a phagosome that matures by fusing with early and late endocytic vesicles, resulting in killing of ingested microbes. Host Rab GTPases are central regulators of vesicular trafficking following pathogen phagocytosis. However, it is unclear how Rab GTPases interact with B. pseudomallei to regulate the transport and maturation of bacterial-containing phagosomes. Here, we showed that the host Rab32 plays an important role in mediating antimicrobial activity by promoting phagosome maturation at an early phase of infection with B. pseudomallei. And we demonstrated that the expression level of Rab32 is increased through the downregulation of the synthesis of miR-30b/30c in B. pseudomallei infected macrophages. Subsequently, we showed that B. pseudomallei resides temporarily in Rab32-positive compartments with late endocytic features. And Rab32 enhances phagosome acidification and promotes the fusion of B. pseudomallei-containing phagosomes with lysosomes to activate cathepsin D, resulting in restricted intracellular growth of B. pseudomallei. Additionally, Rab32 mediates phagosome maturation depending on its guanosine triphosphate/guanosine diphosphate (GTP/GDP) binding state. Finally, we report the previously unrecognized role of miR-30b/30c in regulating B. pseudomallei-containing phagosome maturation by targeting Rab32 in macrophages. Altogether, we provide a novel insight into the host immune-regulated cellular pathway against B. pseudomallei infection is partially dependent on Rab32 trafficking pathway, which regulates phagosome maturation and enhances the killing of this bacterium in macrophages.

MicroRNA-146a inhibits autophagy to maintain the intracellular survival of Burkholderia pseudomallei by targeting LIPA.

Burkholderia pseudomallei is the etiological agent of melioidosis, which is an emerging infectious disease endemic to many tropical regions. Autophagy is an intrinsic cellular process that degrades cytoplasmic components and plays an important role in protecting the host against pathogens. Like many intracellular pathogens, B. pseudomallei can evade the autophagy-dependent cellular clearance. However, the underlying mechanism remains unclear. In this study, we applied a combination of multiple assays to monitor autophagy processes and found that B. pseudomallei induced an incomplete autophagic flux and eliminate autophagy clearance in macrophages by blocking autophagosome-lysosome fusion. Based on a high-throughput microarray screening, we found that LIPA (lysosomal acid LIPAse A) was downregulated during B. pseudomallei infection. MiR-146a was then identified to be specifically upregulated upon infection with B. pseudomallei and further regulated LIPA expression by interacting with 3'UTR of LIPA. Furthermore, overexpression of miR-146a contributed to the defect of autophagic flux caused by B. pseudomallei and was beneficial for the survival of B. pseudomallei in macrophages. Therefore, our findings suggest that miR-146a inhibits autophagy via posttranscriptional suppression of LIPA expression to maintain B. pseudomallei survival in macrophages.

Efficacy, safety, and immunogenicity of an oral recombinant Helicobacter pylori vaccine in children in China: a randomised, double-blind, placebo-controlled, phase 3 trial.

BACKGROUND: Helicobacter pylori is one of the most common gastric pathogens, affecting at least half the world's population, and is strongly associated with gastritis, peptic ulcer, gastric adenocarcinoma, and lymphoma. We aimed to assess the efficacy, safety, and immunogenicity of a three-dose oral recombinant H pylori vaccine in children in China. METHODS: We did this randomised, double-blind, placebo-controlled, phase 3 trial at one centre in Ganyu County, Jiangsu Province, China. Healthy children aged 6-15 years without past or present H pylori infection were randomly assigned (1:1), via computer-generated randomisation codes in blocks of ten, to receive the H pylori vaccine or placebo. Participants, their guardians, and study investigators were masked to treatment allocation. The primary efficacy endpoint was the occurrence of H pylori infection within 1 year after vaccination. We did analysis in the per-protocol population. This trial is registered with ClinicalTrials.gov, number NCT02302170. FINDINGS: Between Dec 2, 2004, and March 19, 2005, we randomly assigned 4464 participants to either the vaccine group (n=2232) or the placebo group (n=2232), of whom 4403 (99%) participants completed the three-dose vaccination schedule and were included in the per-protocol efficacy analysis. We extended follow-up to 3 years. We recorded 64 events of H pylori infection within the first year (14 events in 2074·3 person-years at risk in the vaccine group vs 50 events in 2089·6 person-years at risk in the placebo group), resulting in a vaccine efficacy of 71·8% (95% CI 48·2-85·6). 157 (7%) participants in the vaccine group and 161 (7%) participants in the placebo group reported at least one adverse reaction. Serious adverse events were reported in five (<1%) participants in the vaccine group and seven (<1%) participants in the placebo group, but none was considered to be vaccination related. INTERPRETATION: The oral recombinant H pylori vaccine was effective, safe, and immunogenic in H pylori-naive children. This vaccine could substantially reduce the incidence of H pylori infection; however, follow up over a longer period is needed to confirm the protection of the vaccine against H pylori-associated diseases. FUNDING: Chongqing Kangwei Biological Technology.

A pro-inflammatory role for Th22 cells in Helicobacter pylori-associated gastritis.

OBJECTIVE: Helper T (Th) cell responses are critical for the pathogenesis of Helicobacter pylori-induced gastritis. Th22 cells represent a newly discovered Th cell subset, but their relevance to H. pylori-induced gastritis is unknown. DESIGN: Flow cytometry, real-time PCR and ELISA analyses were performed to examine cell, protein and transcript levels in gastric samples from patients and mice infected with H. pylori. Gastric tissues from interleukin (IL)-22-deficient and wild-type (control) mice were also examined. Tissue inflammation was determined for pro-inflammatory cell infiltration and pro-inflammatory protein production. Gastric epithelial cells and myeloid-derived suppressor cells (MDSC) were isolated, stimulated and/or cultured for Th22 cell function assays. RESULTS: Th22 cells accumulated in gastric mucosa of both patients and mice infected with H. pylori. Th22 cell polarisation was promoted via the production of IL-23 by dendritic cells (DC) during H. pylori infection, and resulted in increased inflammation within the gastric mucosa. This inflammation was characterised by the CXCR2-dependent influx of MDSCs, whose migration was induced via the IL-22-dependent production of CXCL2 by gastric epithelial cells. Under the influence of IL-22, MDSCs, in turn, produced pro-inflammatory proteins, such as S100A8 and S100A9, and suppressed Th1 cell responses, thereby contributing to the development of H. pylori-associated gastritis. CONCLUSIONS: This study, therefore, identifies a novel regulatory network involving H. pylori, DCs, Th22 cells, gastric epithelial cells and MDSCs, which collectively exert a pro-inflammatory effect within the gastric microenvironment. Efforts to inhibit this Th22-dependent pathway may therefore prove a valuable strategy in the therapy of H. pylori-associated gastritis.

A dominant CD4(+) T-cell response to Helicobacter pylori reduces risk for gastric disease in humans.

BACKGROUND & AIMS: Immunodominance is an important feature of antiviral, antitumor, and antibacterial cellular immune responses, but it is not well demonstrated in the immune responses against Helicobacter pylori. Antigen-specific CD4(+) T cells protect mice against infection with H pylori. We investigated the immunodominant CD4(+) T-cell response to neuraminyllactose-binding hemagglutinin (HpaA), which is a conserved, H pylori-specific colonization factor that is being investigated as an antigen for vaccination strategies. METHODS: HpaA-specific CD4(+) T cells were expanded with autologous peripheral blood mononuclear cells that had been incubated with recombinant HpaA and characterized using overlapping synthetic peptides. We compared the percentage of CD4(+) T cells with specificity for HpaA(88-100), restricted to HLA-DRB1*1501, among 59 H pylori-infected subjects with different gastric diseases. RESULTS: We identified and characterized several immunodominant CD4(+) T-cell epitopes derived from HpaA. The immunodominant CD4(+) T-cell responses specific to HpaA(88-100) were observed in most H pylori-infected individuals who expressed HLA-DRB1*1501 and were significantly more abundant in patients with less severe diseases (P < .05). CONCLUSIONS: The HLA-DRB1*1501-restricted immunodominant CD4(+) T-cell response to HpaA(88-100) is associated with reduced risk of severe gastric diseases. Further study of these and other immunodominant CD4(+) T-cell responses to H pylori will provide insight into mechanisms of protective immunity and aid in vaccine design.

CD8(+) T cells that produce interleukin-17 regulate myeloid-derived suppressor cells and are associated with survival time of patients with gastric cancer.

BACKGROUND & AIMS: CD8(+) T cells that produce interleukin (IL)-17 (Tc17 cells) promote inflammation and have been identified in tumors. We investigated their role in the pathogenesis of gastric cancer. METHODS: We used flow cytometry analyses to determine levels and phenotype of Tc17 cells in blood and tumor samples from 103 patients with gastric cancer. We performed multivariate analysis to identify factors associated with overall survival using the Cox proportional hazards model. CD8(+) T cells and monocytes were isolated and cocultured in an assay for induction of Tc17 cells. Tumor cells and myeloid-derived suppressor cells (MDSCs) were isolated and used in assays of Tc17 cell function. RESULTS: Tc17 cells with distinct cytokine and functional profiles were found in gastric tumor samples from patients. The percentage of Tc17 cells increased with tumor progression and was associated with overall survival time. Tumor-activated monocytes secreted IL-6, IL-1ß, and IL-23, which promoted development of Tc17 cell populations. Supernatants from cultured Tc17 cells induced production of the chemokine CXCL12 by tumor cells; this promoted CXCR4-dependent migration of MDSCs and impaired functions of anti-tumor CD8(+) cytotoxic T cells via a cell contact-dependent mechanism. CONCLUSIONS: Percentages of Tc17 cells in gastric tumors are associated with survival times of patients. These cells promote chemotaxis of MDSCs, which might promote tumor progression.

Detection of Burkholderia pseudomallei with CRISPR-Cas12a based on specific sequence tags.

Melioidosis is a bacterial infection caused by Burkholderia pseudomallei (B. pseudomallei), posing a significant threat to public health. Rapid and accurate detection of B. pseudomallei is crucial for preventing and controlling melioidosis. However, identifying B. pseudomallei is challenging due to its high similarity to other species in the same genus. To address this issue, this study proposed a dual-target method that can specifically identify B. pseudomallei in less than 40 min. We analyzed 1722 B. pseudomallei genomes to construct large-scale pan-genomes and selected specific sequence tags in their core genomes that effectively distinguish B. pseudomallei from its closely related species. Specifically, we selected two specific tags, LC1 and LC2, which we combined with the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated proteins (Cas12a) system and recombinase polymerase amplification (RPA) pre-amplification. Our analysis showed that the dual-target RPA-CRISPR/Cas12a assay has a sensitivity of approximately 0.2 copies/reaction and 10 fg genomic DNA for LC1, and 2 copies/reaction and 20 fg genomic DNA for LC2. Additionally, our method can accurately and rapidly detect B. pseudomallei in human blood and moist soil samples using the specific sequence tags mentioned above. In conclusion, the dual-target RPA-CRISPR/Cas12a method is a valuable tool for the rapid and accurate identification of B. pseudomallei in clinical and environmental samples, aiding in the prevention and control of melioidosis.

First genome sequence of a Burkholderia pseudomallei Isolate in China, strain BPC006, obtained from a melioidosis patient in Hainan.

Melioidosis, caused by Burkholderia pseudomallei, is considered to be endemic to Northern Australia and Southeast Asia, with high mortality and relapse rates, regardless of powerful antibiotic therapy. Here we report the first genome sequence of Burkholderia pseudomallei strain BPC006, obtained from a melioidosis patient in Hainan, China. The genome sizes of the 2 chromosomes were determined to be 4,001,777 bp and 3,153,284 bp.

Role of gamma-delta T cells in host response against Staphylococcus aureus-induced pneumonia.

BACKGROUND: Staphylococcus aureus is the major cause of hospital-acquired and community-acquired pneumonia. Host defense to S.aureus infection is largely mediated by the innate immune system. γδ T cells play an important role in innate immunity to many infectious diseases. However, less is known about the role of these cells during S.aureus-induced pneumonia. In this study, we examined the response and the role of γδ T cells to pulmonary S.aureus infection. RESULTS: Mice infected with S. aureus intranasally showed rapid γδ T cells accumulation in the lung. Deficiency of γδ T cells led to attenuated bacterial clearance and less tissue damage in lung compared with WT mice. Moreover, TCR-δ-/- mice exhibited impaired neutrophil recruitment and reduced cytokine production at the site of infection. The γδ T cells in response to pulmonary S. aureus infection mainly secreted IL-17 and γδ T cells deficiency reduced IL-17 production, which might regulate the production of neutrophil-inducing cytokine/chemokine in the S. aureus-infected lungs. CONCLUSIONS: Accumulation of γδ T cells in the lungs to S. aureus infection is beneficial for bacteria clearance and also contributes to the tissue damage. These cells were the primary source of IL-17, which might influence the recruitment of neutrophils at the early stage of infection.

Increased intratumoral IL-22-producing CD4(+) T cells and Th22 cells correlate with gastric cancer progression and predict poor patient survival.

IL-22-producing CD4(+) T cells (IL-22(+)CD4(+) T cells) and Th22 cells (IL-22(+)IL-17(-)IFN-γ(-)CD4(+) T cells) represent newly discovered T-cell subsets, but their nature, regulation, and clinical relevance in gastric cancer (GC) are presently unknown. In our study, the frequency of IL-22(+)CD4(+) T cells in tumor tissues from 76 GC patients was significantly higher than that in tumor-draining lymph nodes, non-tumor, and peritumoral tissues. Most intratumoral IL-22(+)CD4(+) T cells co-expressed IL-17 and IFN-γ and showed a memory phenotype. Locally enriched IL-22(+)CD4(+) T cells positively correlated with increased CD14(+) monocytes and IL-6 and IL-23 detection ex vivo, and in vitro IL-6 and IL-23 induced the polarization of IL-22(+)CD4(+) T cells in a dose-dependent manner and the polarized IL-22(+)CD4(+) T cells co-expressed of IL-17 and IFN-γ. Moreover, IL-22(+)CD4(+) T-cell subsets (IL-22(+)IL-17(+)CD4(+), IL-22(+)IL-17(-)CD4(+), IL-22(+)IFN-γ(+)CD4(+), IL-22(+)IFN-γ(-)CD4(+), and IL-22(+)IL-17(+)IFN-γ(+)CD4(+) T cells), and Th22 cells were also increased in tumors. Furthermore, higher intratumoral IL-22(+)CD4(+) T-cell percentage and Th22-cell percentage were found in patients with tumor-node-metastasis stage advanced and predicted reduced overall survival. In conclusion, our data indicate that IL-22(+)CD4(+) T cells and Th22 cells are likely important in establishing the tumor microenvironment for GC; increased intratumoral IL-22(+)CD4(+) T cells and Th22 cells are associated with tumor progression and predict poorer patient survival, suggesting that tumor-infiltrating IL-22(+)CD4(+) T cells and Th22 cells may be suitable therapeutic targets in patients with GC.

Increased tumor-infiltrating CD8(+)Foxp3(+) T lymphocytes are associated with tumor progression in human gastric cancer.

BACKGROUND: CD8(+)Foxp3(+) T lymphocytes have been detected in tumors. However, the distribution, phenotypic features, and regulation of these cells in gastric cancer remain unknown. METHODS: The levels of CD8(+)Foxp3(+) T lymphocytes in the peripheral blood, tumor-draining lymph nodes, non-tumor tissues, and tumor tissues of patients with gastric cancer were detected by flow cytometry. Foxp3 induction in CD8(+)Foxp3(-) T cells was investigated in vitro. The suppressive function of CD8(+)Foxp3(+) T lymphocytes was analyzed by their effect on CD4(+) T-cell proliferation and IFN-γ production. The percentages of CD8(+)Foxp3(+) T lymphocytes were evaluated for the association with tumor stage. RESULTS: The frequency of CD8(+)Foxp3(+) T lymphocytes in tumor tissues was significantly higher than that in non-tumor tissues, and similar results were also observed in tumor-draining lymph nodes compared with peripheral blood. Most intratumoral CD8(+)Foxp3(+) T lymphocytes were activated effector cells (CD45RA(-)CD27(-)). TGF-ß1 levels were positively correlated with the frequency of CD8(+)Foxp3(+) T lymphocytes in tumor tissues, and in vitro TGF-ß1 could induce the generation of CD8(+)Foxp3(+) T lymphocytes in a dose-dependent manner. Furthermore, intratumoral CD8(+)Foxp3(+) T lymphocytes suppressed the proliferation and IFN-γ production of CD4(+) T cells. Finally, intratumoral CD8(+)Foxp3(+) T lymphocytes were significantly increased with tumor progression in terms of tumor-node-metastasis (TNM) stage. CONCLUSIONS: Our data have shown that increased intratumoral CD8(+)Foxp3(+) T lymphocytes are associated with tumor stage and potentially influence CD4(+) T-cell functions, which may provide insights for developing novel immunotherapy protocols against gastric cancer.

Helicobacter pylori-induced Th17 responses modulate Th1 cell responses, benefit bacterial growth, and contribute to pathology in mice.

CD4(+) T cell responses are critical for the pathogenesis of Helicobacter pylori infection. The present study evaluated the role of the Th17 subset in H. pylori infection. H. pylori infection induced significant expression of IL-17 and IFN-gamma in mouse gastric tissue. IL-23 and IL-12 were increased in the gastric tissue and in H. pylori-stimulated macrophages. Cell responses were examined by intracellular staining for IFN-gamma, IL-4, and IL-17. Mice infected with H. pylori developed a mixed Th17/Th1 response; Th17 responses preceded Th1 responses. Treatment of mice with an anti-IL-17 Ab but not a control Ab significantly reduced the H. pylori burden and inflammation in the stomach. H. pylori colonization and gastric inflammation were also lower in IL-17(-/-) mice. Furthermore, administration of recombinant adenovirus encoding mouse IL-17 increased both H. pylori load and inflammation. Further analysis showed that the Th1 cell responses to H. pylori were downregulated when IL-17 is deficient. These results together suggest that H. pylori infection induces a mixed Th17/Th1 cell response and the Th17/IL-17 pathway modulates Th1 cell responses and contributes to pathology.

H. pylori related proinflammatory cytokines contribute to the induction of miR-146a in human gastric epithelial cells.

MicroRNAs have been implicated as a central regulator of the immune system. We have previously reported that Helicobacter pylori (H. pylori) was able to increase the expression of miR-146a, and miR-146a may negatively regulate H. pylori-induced inflammation, but the exact mechanism of how H. pylori contribute the induction of miR-146a is not clear. Here, we attempted to assess the role of H. pylori related proinflammatory cytokines including interleukin (IL)-8, tumor necrosis factor (TNF)-α, and interleukin (IL)-1ß, and cytotoxin-associated gene A (CagA) virulence factor on the induction of miR-146a. We found that IL-8, TNF-α, and IL-1ß could contribute to the induction of miR-146a in gastric epithelial cell HGC-27 in NF-κB-dependent manner, while the induction of miR-146a upon H. pylori stimulation was independent of above proinflammatory cytokines. Furthermore, overexpression of miR-146a reduced H. pylori-induced IL-8, TNF-α, and IL-1ß. However, CagA had no effect on the miR-146a induction. Taken together, our study suggest that proinflammatory cytokines IL-8, TNF-α, and IL-1ß could contribute to the induction of miR-146a during H. pylori infection, while CagA is not necessarily required for miR-146a induction. miR-146a may function as novel negative regulators to modulate the inflammation.

Helicobacter pylori HP0876 is dispensable for heme-iron acquisition but attenuates bacterial adherence to gastric epithelial cells.

Helicobacter pylori can efficiently capture iron either from free heme or heme-containing compounds in the iron-limited gastric mucosa. However, the heme iron utilization systems of H. pylori have not been fully described to date. To investigate the contribution of genes involved in heme-iron utilization, a gene homologous to frpB, encode by hp0876 in H. pylori ATCC 26695, was inactivated by homologous recombination. Δhp0876 showed no demonstrable growth defects in the presence of the various concentrations of free iron. Moreover, when hemoglobin or heme was supplied as the sole iron sources, Δhp0876 had growth curves similar to the wild-type strain. The growth competition experiments in vitro also showed that Δhp0876 retained the ability for iron acquisition. Furthermore, IL-8 production in human gastric epithelial cells co-cultured with Δhp0876 and wild-type strain was compared, and our results indicated that lack of HP0876 affected the IL-8 release. And Δhp0876 exhibited significantly increased adherence to gastric epithelial cells. Together, our data suggests that HP0876 is dispensable for H. pylori heme-iron uptake, but it may attenuate H. pylori adherence to gastric epithelial cells, which induced decreased production of H. pylori-induced IL-8 production in gastric epithelial cells.

Identification of a novel linear epitope on EspA from enterohemorrhagic E. coli using a neutralizing and protective monoclonal antibody.

Enterohemorrhagic E. coli (EHEC) causes severe diseases in humans and animals via the production of Shiga toxins, and injection of effectors into epithelia using type III secretion system (TTSS). E. coli secreted protein A (EspA) forms the filamentous conduits of TTSS, which extends into the translocation pore embedded in host cell membranes and aids in the transportation of bacterial effectors. In addition, EspA is closely associated with initial bacterial adhesion and the formation of biofilms. EspA in its various forms elicits protective immune responses, although the epitope responsible has not to be identified. Here we report the presence of a linear, immunogenic, conserved and partially protective epitope E07 (100Lys-120Val) on EspA, which is recognized by the novel monoclonal antibody 1H10. This antibody blocks EHEC-induced actin polymerization and confers protection in mice. These findings provide a better understanding of EspA-induced immune responses and could lead to epitope-based vaccines and antibody-based therapies.

Efficient adenovirus-mediated gene transfer to gastric tissue by oral administration.

BACKGROUND: Recombinant adenoviruses (rAd) are well-characterized viral vectors and have been studied in many human diseases. However, there are no detailed methods for transferring genes to the stomach using rAd. METHODS: Gastric epithelial cells were infected with rAd encoding green fluorescence protein (AdGFP) for different times, or with AdGFP that had been incubated in artificial gastric juice at different pH values for 1 h. Gene expression was detected by fluorescence microscope and flow cytometry. Mice were infected via oral administration with rAd encoding red fluorescence protein and beta-galactosidase (AdRFP-lacZ) or rAd encoding mouse interleukin-17 (AdmIL-17), and tissues were collected at the indicated times after infection. LacZ expression in different tissues was detected by X-gal staining and IL-17 expression in the stomach was assessed by the real-time polymerase chain reaction and an enzyme-linked immunosorbent assay. Inflammation in the stomach was also assessed. RESULTS: rAd could infect the gastric epithelial cells and tolerate pH 5 for 1 h in vitro. Adenovirus-mediated genes were specifically expressed in the gastrointestinal tract and transgene expression persisted in gastric tissue for up to 7 days after oral administration of AdRFP-lacZ. Oral administration of AdmIL-17 induced mIL-17 expression in gastric tissue at the mRNA and protein levels and protein level peaked on day 5 post-infection. IL-6, a target protein of IL-17, and gastric inflammation also increased in AdmIL-17-infected mice. CONCLUSIONS: The present study has established a detailed method for transferring adenovirus-mediated gene to the stomach, which may provide a valuable approach for gene therapy or the study of the basic biology of gastric diseases.

RGD-IL-24, a novel tumor-targeted fusion cytokine: expression, purification and functional evaluation.

Targeting drugs to tumor cells is a central challenge for improving existing cancer therapies. ACDCRGDCFCG peptide (RGD-4C) binds to alphavbeta3 integrin, which is selectively expressed in tumor blood vessels and on the surface of some tumor cells. Interleukin 24 (IL-24) is a novel cancer growth-suppressing and apoptosis-inducing cytokine. To enhance the antitumor effect, we coupled RGD-4C to the N-terminus of IL-24 and expressed RGD-IL-24 in Escherichia coli. Cell proliferation and adhesion experiments revealed that RGD-IL-24 specifically binds to MCF-7 cancer cells, and induces apoptosis of MCF-7 cancer cells. These studies support the use of the RGD-IL-24 protein in tumor-targeting therapy.

Screening and identification of natural antisense transcripts in Helicobacter pylori by a novel approach based on RNase I protection assay.

Natural antisense transcripts (NATs) are endogenous RNA molecules that exhibit partial or complete complementarity to other RNAs. Studies have shown that NATs may participate in a broad range of gene regulatory events. The identification of NATs in human, mouse and Escherichia coli has revealed their widespread occurrence in both eukaryotic and prokaryotic life. However, little is known about NATs in Helicobacter pylori (H. pylori), a human pathogen which is associated with gastric diseases. Here we systematically screened NATs in H. pylori by a novel experimental strategy based on RNase I protection assay. We successfully constructed a cDNA library of NATs and developed a novel poly(A)-tailed RT-PCR method to monitor the expression of NATs. After sequencing, bioinformatic analysis and expression detection, two novel NATs (NAT-39 and NAT-67) were confirmed. They were, respectively, complementary to the following genes: iron-regulated outer membrane protein (frpB) and periplasmic iron-binding protein (ceuE). Taken together, the results suggest that NAT-39 and NAT-67 may participate in the regulation of iron homeostasis in H. Pylori in a sequence complementary manner with target mRNAs.

Alteration of microRNA-4474/4717 expression and CREB-binding protein in human colorectal cancer tissues infected with Fusobacterium nucleatum.

Colorectal cancer (CRC) is a common and highly lethal form of cancer. Although the etiologic role of Fusobacterium nucleatum (F. nucleatum) in the development of CRC has been elucidated, the specific tumor molecules involved in the progression of CRC induced by F. nucleatum have not been identified. This study investigated several miRNAs and genes involved in the progression of F. nucleatum-induced CRC by Affymetrix miRNA microarray technology and GeneChip Human Transcriptome Array 2.0. The results suggest that miR-4474 and miR-4717 are up-regulated in CRC tissues in response to F. nucleatum infection, compared with the control group (paracancerous tissues), while other genes associated with signaling pathways in cancer, including CREB-binding protein (CREBBP), STAT1, PRKACB, CAMK2B, JUN, TP53 and EWSR1, were dysregulated. Bioinformatic analysis identified CREBBP as the primary aberrantly expressed gene in F. nucleatum-induced CRC. Consistent with the microarray analysis results, real-time RT-PCR analysis demonstrated that the expression of miR-4474/4717 was upregulated while that of CREBBP mRNA was downregulated in CRC patients infected with F. nucleatum. Additionally, CREBBP was identified as a novel target of miR-4474/4717. The results of this study suggest that miR-4474 and miR-4717 are involved in the progression of F. nucleatum-induced CRC by posttranscriptionally regulating the target gene CREBBP.