Burkholderia pseudomallei BipD modulates host mitophagy to evade killing.

Mitophagy is critical for mitochondrial quality control and function to clear damaged mitochondria. Here, we found that Burkholderia pseudomallei maneuvered host mitophagy for its intracellular survival through the type III secretion system needle tip protein BipD. We identified BipD, interacting with BTB-containing proteins KLHL9 and KLHL13 by binding to the Back and Kelch domains, recruited NEDD8 family RING E3 ligase CUL3 in response to B. pseudomallei infection. Although evidently not involved in regulation of infectious diseases, KLHL9/KLHL13/CUL3 E3 ligase complex was essential for BipD-dependent ubiquitination of mitochondria in mouse macrophages. Mechanistically, we discovered the inner mitochondrial membrane IMMT via host ubiquitome profiling as a substrate of KLHL9/KLHL13/CUL3 complex. Notably, K63-linked ubiquitination of IMMT K211 was required for initiating host mitophagy, thereby reducing mitochondrial ROS production. Here, we show a unique mechanism used by bacterial pathogens that hijacks host mitophagy for their survival.

Lopinavir enhances anoikis by remodeling autophagy in a circRNA-dependent manner.

Macroautophagy/autophagy-mediated anoikis resistance is crucial for tumor metastasis. As a key autophagy-related protein, ATG4B has been demonstrated to be a prospective anti-tumor target. However, the existing ATG4B inhibitors are still far from clinical application, especially for tumor metastasis. In this study, we identified a novel circRNA, circSPECC1, that interacted with ATG4B. CircSPECC1 facilitated liquid-liquid phase separation of ATG4B, which boosted the ubiquitination and degradation of ATG4B in gastric cancer (GC) cells. Thus, pharmacological addition of circSPECC1 may serve as an innovative approach to suppress autophagy by targeting ATG4B. Specifically, the circSPECC1 underwent significant m6A modification in GC cells and was subsequently recognized and suppressed by the m6A reader protein ELAVL1/HuR. The activation of the ELAVL1-circSPECC1-ATG4B pathway was demonstrated to mediate anoikis resistance in GC cells. Moreover, we also verified that the above pathway was closely related to metastasis in tissues from GC patients. Furthermore, we determined that the FDA-approved compound lopinavir efficiently enhanced anoikis and prevented metastasis by eliminating repression of ELAVL1 on circSPECC1. In summary, this study provides novel insights into ATG4B-mediated autophagy and introduces a viable clinical inhibitor of autophagy, which may be beneficial for the treatment of GC with metastasis.

Recombinant Pseudomonas aeruginosa flagellin delivered using ferritin nanoparticles provides enhanced cross-protection against lung infection in mice.

Increasing prevalence of multidrug- and pan-drug-resistant Pseudomonas aeruginosa (PA) strains has created an urgent need for an effective vaccine. Flagellin is an essential vaccine target because of its contribution to bacterial motility and other pathogenic processes. However, flagellin-based vaccines have not been successful thus far, probably due to a lack of efficient adjuvants or delivery systems. In this study, we genetically fused an A-type flagellin (FliC) to the self-assembled nanocarrier ferritin to construct the nanoparticle vaccine, reFliC-ferritin (reFliC-FN). reFliC-FN formed homogenous nanoparticles and induced a quick T helper 1 (Th1)-predominant immune response, which was quite different from that induced by recombinant FliC alone. In addition, reFliC-FN provided enhanced protection against PA strains carrying the A-type and heterogeneous B-type flagellins. Preliminary safety assays revealed the good biocompatibility and biosafety of reFliC-FN. Therefore, our data highlight the potential of ferritin as an ideal delivery system and suggest reFliC-FN as a promising PA vaccine candidate.

A novel structurally identified epitope delivered by macrophage membrane-coated PLGA nanoparticles elicits protection against Pseudomonas aeruginosa.

The increasing prevalence of antibiotic resistance by Pseudomonas aeruginosa (PA) raises an urgent need for an effective vaccine. The outer membrane proteins of PA, especially those that are upregulated during infection, are ideal vaccine targets. However, the strong hydrophobicity of these proteins hinders their application for this purpose. In this study, we selected eight outer membrane proteins from PA with the most significantly upregulated expression. Their extracellular loops were analyzed and screened by using sera from patients who had recovered from PA infection. As a result, a novel immunogenic epitope (Ep167-193) from PilY1 (PA4554) was found. Moreover, we constructed a macrophage membrane-coated PLGA (poly lactic-co-glycolic acid) nanoparticle vaccine carrying PilY1 Ep167-193 (PNPs@M-Ep167-193) that elicits a Th2 immune response and confers adequate protection in mice. Our data furnished the promising vaccine candidate PNPs@M-Ep167-193 while providing additional evidence for structure-based epitope identification and vaccine design.

Helicobacter pylori-induced REDD1 modulates Th17 cell responses that contribute to gastritis.

OBJECTIVE: Regulated in development and DNA damage responses-1 (REDD1) is a conserved and ubiquitous protein, which is induced in response to multiple stimuli. However, the regulation, function and clinical relevance of REDD1 in Helicobacter pylori-associated gastritis are presently unknown. APPROACH: Immunohistochemistry, real-time PCR and Western blot analyses were performed to examine the levels of REDD1 in gastric samples from H. pylori-infected patients and mice. Gastric tissues from Redd1-/- and wildtype (WT, control) mice were examined for inflammation. Gastric epithelial cells (GECs), monocytes and T cells were isolated, stimulated and/or cultured for REDD1 regulation and functional assays. RESULTS: REDD1 was increased in gastric mucosa of H. pylori-infected patients and mice. H. pylori induced GECs to express REDD1 via the phosphorylated cytotoxin associated gene A (cagA) that activated MAPKp38 pathway to mediate NF-κB directly binding to REDD1 promoter. Human gastric REDD1 increased with the severity of gastritis, and mouse REDD1 from non-marrow chimera-derived cells promoted gastric inflammation that was characterized by the influx of MHCII+ monocytes. Importantly, gastric inflammation, MHCII+ monocyte infiltration, IL-23 and IL-17A were attenuated in Redd1-/- mice. Mechanistically, REDD1 in GECs regulated CXCL1 production, which attracted MHCII+ monocytes migration by CXCL1-CXCR2 axis. Then H. pylori induced MHCII+ monocytes to secrete IL-23, which favored IL-17A-producing CD4+ cell (Th17 cell) polarization, thereby contributing to the development of H. pylori-associated gastritis. CONCLUSIONS: The present study identifies a novel regulatory network involving REDD1, which collectively exert a pro-inflammatory effect within gastric microenvironment. Efforts to inhibit this REDD1-dependent pathway may prove valuable strategies in treating of H. pylori-associated gastritis.

COVID-19 patients with hypertension are at potential risk of worsened organ injury.

In less than 6 months, COVID-19 spread rapidly around the world and became a global health concern. Hypertension is the most common chronic disease in COVID-19 patients, but its impact on these patients has not been well described. In this retrospective study, 82 patients diagnosed with COVID-19 were enrolled, and epidemiological, demographic, clinical, laboratory, radiological and therapy-related data were analyzed and compared between COVID-19 patients with (29 cases) or without (53 cases) hypertension. The median age of the included patients was 60.5 years, and the cohort included 49 women (59.8%) and 33 (40.2%) men. Hypertension (31 [28.2%]) was the most common chronic illness, followed by diabetes (16 [19.5%]) and cardiovascular disease (15 [18.3%]). The most common symptoms were fatigue (55 [67.1%]), dry cough (46 [56.1%]) and fever ≥ 37.3 °C (46 [56.1%]). The median time from illness onset to positive RT-PCR test was 13.0 days (range 3-25 days). There were 6 deaths (20.7%) in the hypertension group and 5 deaths (9.4%) in the nonhypertension group, and more hypertensive patients with COVID-19 (8 [27.6%]) than nonhypertensive patients (2 [3.8%]) (P = 0.002) had at least one comorbid disease. Compared with nonhypertensive patients, hypertensive patients exhibited higher neutrophil counts, serum amyloid A, C-reactive protein, and NT-proBNP and lower lymphocyte counts and eGFR. Dynamic observations indicated more severe disease and poorer outcomes after hospital admission in the hypertension group. COVID-19 patients with hypertension have increased risks of severe inflammatory reactions, serious internal organ injury, and disease progression and deterioration.

Transcriptome Analysis Reveals Unfolded Protein Response Was Induced During the Early Stage of Burkholderia pseudomallei Infection in A549 Cells.

Burkholderia pseudomallei is a zoonotic pathogen that usually affects patients' lungs and causes serious melioidosis. The interaction of B. pseudomallei with its hosts is complex, and cellular response to B. pseudomallei infection in humans still remains to be elucidated. In this study, transcriptomic profiling of B. pseudomallei-infected human lung epithelial A549 cells was performed to characterize the cellular response dynamics during the early infection (EI) stage. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed by using the online databases DAVID 6.8 and KOBAS 3.0. Real-time quantitative PCR and western blot were used for validation experiments. Compared with the negative control group (NC), a set of 36 common genes varied over time with a cut-off level of 1.5-fold change, and a P-value < 0.05 was identified. Bioinformatics analysis indicated that the PERK-mediated unfolded protein response (UPR) was enriched as the most noteworthy biological process category, which was enriched as a branch of UPR in the signaling pathway of protein processing in the endoplasmic reticulum. Other categories, such as inflammatory responses, cell migration, and apoptosis, were also focused. The molecular chaperone Bip (GRP78), PERK, and PERK sensor-dependent phosphorylation of eIF2α (p-eIF2α) and ATF4 were verified to be increasing over time during the EI stage, suggesting that B. pseudomallei infection activated the PERK-mediated UPR in A549 cells. Collectively, these results provide important initial insights into the intimate interaction between B. pseudomallei and lung epithelial cells, which can be further explored toward the elucidation of the cellular mechanisms of B. pseudomallei infections in humans.

Clinical characteristics of rehospitalized patients with COVID-19 in China.

This study aims to observe the clinical characteristics of recovered patients from Coronavirus Disease 2019 (COVID-19) with positive in reverse transcription-polymerase chain reaction (RT-PCR) or serum antibody. The profile, clinical symptoms, laboratory outcomes, and radiologic assessments were extracted on 11 patients, who tested positive for COVID-19 with RT-PCR or serum antibody after discharged and was admitted to Hubei No. 3 People's Hospital of Jianghan University for a second treatment in March 2020. The average interval time between the first discharge and the second admission measured 16.00 ± 7.14 days, ranging from 6 to 27 days. In the second hospitalization, one patient was positive for RT-PCR and serum antibody immunoglobulin M (IgM)-immunoglobulin G (IgG), five patients were positive for both IgM and IgG but negative for RT-PCR. Three patients were positive for both RT-PCR and IgG but negative for IgM. The main symptoms were cough (54.55%), fever (27.27%), and feeble (27.27%) in the second hospitalization. Compared with the first hospitalization, there were significant decreases in gastrointestinal symptoms (5 vs 0, P = .035), elevated levels of both white blood cell count (P = .036) and lymphocyte count (P = .002), remarkedly decreases in C-reactive protein and serum amyloid A (P < .05) in the second hospitalization. Additionally, six patients' chest computed tomography (CT) exhibited notable improvements in acute exudative lesions. There could be positive results for RT-PCR analysis or serum IgM-IgG in discharged patients, even with mild clinical symptoms, however, their laboratory outcomes and chest CT images would not indicate the on-going development in those patients.

Rational Design and Evaluation of an Artificial Escherichia coli K1 Protein Vaccine Candidate Based on the Structure of OmpA.

Escherichia coli (E. coli) K1 causes meningitis and remains an unsolved problem in neonates, despite the application of antibiotics and supportive care. The cross-reactivity of bacterial capsular polysaccharides with human antigens hinders their application as vaccine candidates. Thus, protein antigens could be an alternative strategy for the development of an E. coli K1 vaccine. Outer membrane protein A (OmpA) of E. coli K1 is a potential vaccine candidate because of its predominant contribution to bacterial pathogenesis and sub-cellular localization. However, little progress has been made regarding the use of OmpA for this purpose due to difficulties in OmpA production. In the present study, we first investigated the immunogenicity of the four extracellular loops of OmpA. Using the structure of OmpA, we rationally designed and successfully generated the artificial protein OmpAVac, composed of connected loops from OmpA. Recombinant OmpAVac was successfully produced in E. coli BL21 and behaved as a soluble homogenous monomer in the aqueous phase. Vaccination with OmpAVac induced Th1, Th2, and Th17 immune responses and conferred effective protection in mice. In addition, OmpAVac-specific antibodies were able to mediate opsonophagocytosis and inhibit bacterial invasion, thereby conferring prophylactic protection in E. coli K1-challenged adult mice and neonatal mice. These results suggest that OmpAVac could be a good vaccine candidate for the control of E. coli K1 infection and provide an additional example of structure-based vaccine design.

Vaccination with a recombinant OprL fragment induces a Th17 response and confers serotype-independent protection against Pseudomonas aeruginosa infection in mice.

Pseudomonas aeruginosa (PA) is the major causative agent of nosocomial infection. Despite of adequate use of antibiotics, it still represents a major challenge in controlling PA infection. The local pulmonary Th17 response plays an important protective role against PA infection. And the Th17-mediated protection is antibody independent, so we hypothesized that it would be an optimal strategy of a vaccine for PA control to induce an effective Th17 response. Herein we report the successful production of a recombinant fragment of the OprL (reOprL) of PA. Purified reOprL forms homogeneous monomers in solution and vaccination with reOprL elicited a remarkable Th17 response. In addition, reOprL vaccination conferred effective serotype-independent protection against PA infection, which relied on the Th17 response. Our data suggest that reOprL is a good candidate for the future development of Th17 immunity based PA vaccines.

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.

Genome of Helicobacter pylori strain XZ274, an isolate from a tibetan patient with gastric cancer in China.

The infection rate of Helicobacter pylori is high all over the world, especially in the Chinese Tibetan Plateau. Here, we report the genome sequence of Helicobacter pylori strain XZ274 isolated from a Tibetan patient with gastric cancer. The strain contains 1,634,138 bp with 1,654 coding sequences and a pXZ274 plasmid of 22,406 bp with 26 coding sequences. This is the first complete genome sequence of Helicobacter pylori from the Tibetan Plateau in China.

Genotyping analysis of Helicobacter pylori using multiple-locus variable-number tandem-repeats analysis in five regions of China and Japan.

BACKGROUND: H. pylori (Helicobacter pylori) is the major causative agent of chronic active gastritis. The population of H. pylori shows a high genomic variability among isolates. And the polymorphism of repeat-units of genomics had participated the important process of evolution. Its long term colonization of the stomach caused different clinical outcomes, which may relate to the high degree of genetic variation of H. pylori. A variety of molecular typing tools have been developed to access genetic relatedness in H. pylori isolates. However, there is still no standard genotyping system of this bacterium. The MLVA (Multi-locus of variable number of tandem repeat analysis) method is useful for performing phylogenetic analysis and is widely used in bacteria genotyping; however, there's little application in H. pylori analysis. This article is the first application of the MLVA method to investigate H. pylori from different districts and ethnic groups of China. RESULTS: MLVA of 12 VNTR loci with high discrimination power based on 30 candidates were performed on a collection of 202 strains of H. pylori which originated from five regions of China and Japan. Phylogenetic tree was constructed using MLVA profiles. 12 VNTR loci presented with high various polymorphisms, and the results demonstrated very close relationships between genotypes and ethnic groups. CONCLUSIONS: This study used MLVA methodology providing a new perspective on the ethnic groups and distribution characteristics of H. pylori.