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.

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.

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.

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.

An immunopotentiator, ophiopogonin D, encapsulated in a nanoemulsion as a robust adjuvant to improve vaccine efficacy.

As an ingredient of vaccines, adjuvants are indispensable for enhancing and directly inducing robust and extensive adaptive immune responses associated with vaccine antigens. In this study, we initially determined that a new molecular immunopotentiator, ophiopogonin D (OP-D), enhanced the antibody response to antigen. Because OP-D has certain disadvantages, including poor solubility, we next encapsulated OP-D in a nanoemulsion adjuvant (nanoemulsion-encapsulated OP-D, NOD) using low-energy emulsification methods. The NOD thus produced was small, with an average size of 76.45 nm, and exhibited good distribution (PdI value 0.16), significantly increasing the solubility of OP-D. Furthermore, NOD exhibited reduced cellular toxicity and acute toxicity. Our results showed that a fusion antigen of MRSA (HlaH35LIsdB348-465) formulated with NOD significantly improved humoral and cellular immune responses compared to those observed in the antigen/OP-D and antigen/AlPO4 groups. Compared with antigen/OP-D, the antigen formulated with NOD more effectively promoted antigen uptake by dendritic cells (DCs) and the activation of antigen-presenting cells (APCs). Moreover, the NOD-formulated antigen had ideal protective efficacy in a MRSA sepsis model by inducing more potent antibody responses and a Th1/Th17-biased CD4+ T cell immune response. Therefore, these results suggest that NOD is a promising and robust adjuvant platform for a MRSA vaccine. STATEMENT OF SIGNIFICANCE: We first identified a new powerful immunopotentiator, Ophiopogonin D, among dozens of natural products and then used nanotechnology to construct a highly efficient and low toxic adjuvant system (NOD). Our approach intersects natural medicinal chemistry, nanomaterials and immunology, revealing that a strong adjuvant activity of this adjuvant system was verified in vitro and in vivo, and the application of MRSA subunit vaccine model for survival experiments achieved a 100% protection rate. This research illustrate that NOD is a promising and robust adjuvant platform for subunit vaccines.

A case report: Splenic abscess caused by Burkholderia pseudomallei.

RATIONALE: Melioidosis is an emerging infectious disease caused by Burkholderia pseudomallei. To our knowledge, there have been very few cases of splenic abscesses due to melioidosis in Hainan, China. PATIENT CONCERNS: The patient was a 55-year-old male farmer, who was admitted in our hospital with persistent left epigastric dull pain accompanied by chills and febrile. One month before, the patient presented with persistent abdominal pain. After received anti-infection therapy, the subjective symptoms eased slightly, but recently he suffered from intermittent abdominal pain again. DIAGNOSES: Bacteria isolated from splenic pus were identified as B. pseudomallei by the Phoenix-100 system and indirect immunofluorescence. INTERVENTIONS: The patient was treated by surgical excision and anti-infection therapy. OUTCOMES: The patient was then treated with intravenous ceftazidime and oral trimethoprim-sulfamethoxazole for 2 weeks and his clinical symptoms improved. LESSONS: In endemic areas, B. pseudomallei should be considered as a causative organism of splenic abscess in patients with established risk factors. The isolation of B. pseudomallei from abscess sites is crucial to improve clinical outcomes by appropriate antimicrobial therapy coupled with surgical drainage.

Helicobacter pylori VacA induces autophagic cell death in gastric epithelial cells via the endoplasmic reticulum stress pathway.

The Helicobacter pylori vacuolating cytotoxin (VacA) can promote progressive vacuolation and gastric injury and may be associated with human gastric cancer. Increasing evidence indicates that autophagy is involved in the cell death induced by VacA, but the specific mechanisms need to be further elucidated. We show here that VacA could induce autophagy and increase cell death in human gastric cancer cell lines. Further investigations revealed that inhibition of autophagy could decrease the VacA-induced cell death in AGS cells. Furthermore, numerous dilated endoplasmic reticula (ER) were observed, and the phosphorylation of a subunit of eukaryotic translation initiation factor 2 subunit 1 also increased in the VacA-treated AGS cells, while repression of ER stress could reduce autophagy and cell death through knockdown of activating transcription factor 4 and DNA-damage-inducible transcript 3. In addition, the expression of pseudokinase tribbles homolog 3 (TRIB3) upon ER stress was triggered by VacA, and knockdown of TRIB3 could also decrease VacA-induced cell death. Finally, inhibition of autophagy could decrease VacA s1m1 -induced cell death and apoptosis, and apoptosis inhibitor Z-VAD had no significant effect on autophagy induced by VacA s1m1 . Thus, these results suggested that VacA causes autophagic cell death via ER stress in gastric epithelial cells.

Helicobacter pylori CagA Protein Negatively Regulates Autophagy and Promotes Inflammatory Response via c-Met-PI3K/Akt-mTOR Signaling Pathway.

Cytotoxin-associated-gene A (CagA) of Helicobacter pylori (H. pylori) is a virulence factor that plays critical roles in H. pylori-induced gastric inflammation. In the present study, gastric biopsies were used for genotyping cagA and vacA genes, determining the autophagic activity, and the severity of gastric inflammation response. It was revealed that autophagy in gastric mucosal tissues infected with cagA+H. pylori strains was lower than the levels produced by cagA-H. pylori strains, accompanied with accumulation of SQSTM1 and decreased LAMP1 expression. In vitro, deletion mutant of cagA gene resulted in increased autophagic activity, and decreased expression of SQSTM1 and cytokines, whereas over-expression of CagA down-regulated the starvation-induced autophagy, and induced more production of the cytokines. Moreover, the production of the cytokines was increased by inhibition of autophagy, but decreased by enhancement of autophagy. Deletion of CagA decreased the ability to activate Akt kinase at Ser-473 site and increased autophagy. c-Met siRNA significantly affected CagA-mediated autophagy, and decreased the level of p-Akt, p-mTOR, and p-S6. Both c-Met siRNA and MK-2206 could reverse inflammatory response. H. pylori CagA protein negatively regulates autophagy and promotes the inflammation in H. pylori infection, which is regulated by c-Met-PI3K/Akt-mTOR signaling pathway activation.

TLR2/TLR4 activation induces Tregs and suppresses intestinal inflammation caused by Fusobacterium nucleatum in vivo.

Toll-like receptors (TLRs) 2 and 4 play critical roles in intestinal inflammation caused by Fusobacterium nucleatum (F. nucleatum) infection, but the role of TLR2/TLR4 in regulation of proinflammatory cytokines remains unknown. In this study, through microarray analysis and qRT-PCR, we showed that TLR2/TLR4 are involved in the F. nucleatum-induced inflammatory signaling pathway in Caco-2 cells, C57BL/6 mice and human clinical specimens. In TLR2-/- and TLR4-/- mice, F. nucleatum infection resulted in increased colonization of the bacteria and production of the proinflammatory cytokines IL-8, IL-1ß and TNF-α. In addition, the ratio of Foxp3+ CD4+ T cells in the total CD4+ T cells in TLR2-/- and TLR4-/- mice was less than that in wild-type mice, and the ratio in hybrid mice was more than that in knockout mice, which suggested that TLR2/TLR4 mediated the number of Tregs. Furthermore, it was observed that inflammatory cytokine levels were reduced in TLR2-/- mice after Treg transfer. Thus, these data indicate that TLR2/TLR4 regulate F. nucleatum-induced inflammatory cytokines through Tregs in vivo.

Fusobacterium nucleatum-Induced Impairment of Autophagic Flux Enhances the Expression of Proinflammatory Cytokines via ROS in Caco-2 Cells.

Fusobacterium nucleatum (F. nucleatum) plays a critical role in gastrointestinal inflammation. However, the exact mechanism by which F. nucleatum contributes to inflammation is unclear. In the present study, it was revealed that F. nucleatum could induce the production of proinflammatory cytokines (IL-8, IL-1ß and TNF-α) and reactive oxygen species (ROS) in Caco-2 colorectal) adenocarcinoma cells. Furthermore, ROS scavengers (NAC or Tiron) could decrease the production of proinflammatory cytokines during F. nucleatum infection. In addition, we observed that autophagy is impaired in Caco-2 cells after F. nucleatum infection. The production of proinflammatory cytokines and ROS induced by F. nucleatum was enhanced with either autophagy pharmacologic inhibitors (3-methyladenine, bafilomycin A1) or RNA interference in essential autophagy genes (ATG5 or ATG12) in Caco-2 cells. Taken together, these results indicate that F. nucleatum-induced impairment of autophagic flux enhances the expression of proinflammatory cytokines via ROS in Caco-2 Cells.

Melioidosis in Hainan, China: a restrospective study.

BACKGROUND: Melioidosis is a tropical disease caused by infection with Burkholderia pseudomallei. Here, we report an 11 year (2002-2013) retrospective study of melioidosis cases in China. METHODS: A total of 170 culture-confirmed melioidosis cases were included in our analysis, with culture-positive confirmation, biochemical identification and 16S DNA sequencing. A retrospective study design was employed and a correlational analysis of potential risk factors for mortality was carried out with logistic regression. RESULTS: We observed a year-over-year increasing trend in the incidence of melioidosis in Hainan, particularly after 2007 (annual peak of 64 cases in 2012). Farmers and fishers were the main group susceptible to melioidosis (75/170; 44.1%). Forty-six (27.1%) of the cases were fatal. Pneumonia (58/170; 34.1%) and septicaemia (44/170; 25.9%) were common presentations. Meanwhile, pre-existing diabetes (74/170; 43.5%) and being employed in a job that involves outdoor labour (148/170; 87.1%) emerged as common factors among affected patients. We did not observe a significant effect of seasonal variation on melioidosis mortality, but the greatest number of cases did occur in the rainiest season. CONCLUSIONS: This was the first clinical retrospective study of melioidosis in Hainan, China. The present data will be a useful resource to melioidosis researchers worldwide.

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.

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.

Shiga toxins induce autophagic cell death in intestinal epithelial cells via the endoplasmic reticulum stress pathway.

Shiga toxins (Stxs) are a family of cytotoxic proteins that lead to the development of bloody diarrhea, hemolytic-uremic syndrome, and central nervous system complications caused by bacteria such as S. dysenteriae, E. coli O157:H7 and E. coli O104:H4. Increasing evidence indicates that macroautophagy (autophagy) is a key factor in the cell death induced by Stxs. However, the associated mechanisms are not yet clear. This study showed that Stx2 induces autophagic cell death in Caco-2 cells, a cultured line model of human enterocytes. Inhibition of autophagy using pharmacological inhibitors, such as 3-methyladenine and bafilomycin A1, or silencing of the autophagy genes ATG12 or BECN1 decreased the Stx2-induced death in Caco-2 cells. Furthermore, there were numerous instances of dilated endoplasmic reticulum (ER) in the Stx2-treated Caco-2 cells, and repression of ER stress due to the depletion of viable candidates of DDIT3 and NUPR1. These processes led to Stx2-induced autophagy and cell death. Finally, the data showed that the pseudokinase TRIB3-mediated DDIT3 expression and AKT1 dephosphorylation upon ER stress were triggered by Stx2. Thus, the data indicate that Stx2 causes autophagic cell death via the ER stress pathway in intestinal epithelial cells.

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.

miR-30b, down-regulated in gastric cancer, promotes apoptosis and suppresses tumor growth by targeting plasminogen activator inhibitor-1.

BACKGROUND: Gastric cancer is one of the most common malignant diseases worldwide. Emerging evidence has shown that microRNAs (miRNAs) are associated with tumor development and progression. Our previous studies have revealed that H. pylori infection was able to induce the altered expression of miR-30b in gastric epithelial cells. However, little is known about the potential role of miR-30b in gastric cancer. METHODS: We analyzed the expression of miR-30b in gastric cancer cell lines and human gastric cancer tissues. We examined the effect of miR-30b mimics on the apoptosis of gastric cancer cells in vitro by flow cytometry (FCM) and caspase-3/7 activity assays. Nude mouse xenograft model was used to determine whether miR-30b is involved in tumorigenesis of gastric cancer. The target of miR-30b was identified by bioinformatics analysis, luciferase assay and Western blot. Finally, we performed the correlation analysis between miR-30b and its target expression in gastric cancer. RESULTS: miR-30b was significantly down-regulated in gastric cancer cells and human gastric cancer tissues. Enforced expression of miR-30b promoted the apoptosis of gastric cancer cells in vitro, and miR-30b could significantly inhibit tumorigenicity of gastric cancer by increasing the apoptosis proportion of cancer cells in vivo. Moreover, plasminogen activator inhibitor-1 (PAI-1) was identified as the potential target of miR-30b, and miR-30b level was inversely correlated with PAI-1 expression in gastric cancer. In addition, silencing of PAI-1 was able to phenocopy the effect of miR-30b overexpression on apoptosis regulation of cancer cells, and overexpression of PAI-1 could suppressed the effect of promoting cell apoptosis by miR-30b, indicating PAI-1 is potentially involved in miR-30b-induced apoptosis on cancer cells. CONCLUSION: miR-30b may function as a novel tumor suppressor gene in gastric cancer by targeting PAI-1 and regulating the apoptosis of cancer cells. miR-30b could serve as a potential biomarker and therapeutic target against gastric cancer.

Proteomic analysis of the Ehrlichia chaffeensis phagosome in cultured DH82 cells.

Ehrlichia chaffeensis is an obligately intracellular bacterium that resides and multiplies within cytoplasmic vacuoles of phagocytes. The Ehrlichia-containing vacuole (ECV) does not fuse with lysosomes, an essential condition for Ehrlichia to survive inside phagocytes, but the mechanism of inhibiting the fusion of the phagosome with lysosomes is not clear. Understanding the ECV molecular composition may decipher the mechanism by which Ehrlichia inhibits phagosome-lysosome fusion. In this study, we obtained highly purified ECVs from E. chaffeensis-infected DH82 cells by sucrose density gradient centrifugation and analyzed their composition by mass spectrometry-based proteomics. The ECV composition was compared with that of phagolysosomes containing latex beads. Lysosomal proteins such as cathepsin D, cathepsin S, and lysosomal acid phosphatase were not detected in E. chaffeensis phagosome preparations. Some small GTPases, involved in membrane dynamics and phagocytic trafficking, were detected in ECVs. A notable finding was that Rab7, a late endosomal marker, was consistently detected in E. chaffeensis phagosomes by mass spectrometry. Confocal microscopy confirmed that E. chaffeensis phagosomes contained Rab7 and were acidified at approximately pH 5.2, suggesting that the E. chaffeensis vacuole was an acidified late endosomal compartment. Our results also demonstrated by mass spectrometry and immunofluorescence analysis that Ehrlichia morulae were not associated with the autophagic pathway. Ehrlichia chaffeensis did not inhibit phagosomes containing latex beads from fusing with lysosomes in infected cells. We concluded that the E. chaffeensis vacuole was a late endosome and E. chaffeensis might inhibit phagosome-lysosome fusion by modifying its vacuolar membrane composition, rather than by regulating the expression of host genes involved in trafficking.

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.

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.