Burkholderia pseudomallei BopE suppresses the Rab32-dependent defense pathway to promote its intracellular replication and virulence.

Melioidosis is a serious infectious disease caused by the Gram-negative bacterium Burkholderia pseudomallei. Recently, Rab32-dependent immune vesicles emerge as a critical defense pathway to restrict the intracellular B. pseudomallei. However, B. pseudomallei can evade host immune vesicles and survive in the cytoplasm, although this mechanism is not well understood. In this study, we found Rab32-dependent vesicles could effectively combat B. pseudomallei infection, but not all intracellular B. pseudomallei were encapsulated in Rab32-positive vesicles. To explore how B. pseudomallei counteracted the Rab32-dependent defense pathway, transcriptomic profiling of B. pseudomallei was performed to characterize the response dynamics during infection. We found that the type III secretion system of B. pseudomallei was activated, and a variety of effector proteins were highly upregulated. Among them, BopE, BprD, and BipC were shown to interact with Rab32. Interestingly, BopE directly interacts with host Rab32, potentially suppressing Rab32 function by interfering with nucleotide exchange, which in turn restricts the recruitment of Rab32 to bacterial-containing vesicles. Knocking out of BopE can increase the proportion of Rab32-positive vesicles, suppressing the intracellular replication and virulence of B. pseudomallei. Collectively, our findings have demonstrated that BopE may be an important effector for B. pseudomallei to evade from the Rab32-dependent killing vesicles into the cytosol for survival and replication. Therefore, a deeper understanding of the interaction between BopE and the host Rab32-dependent restriction pathway may provide an effective therapeutic strategy for the elimination of intracellular B. pseudomallei.IMPORTANCEB. pseudomallei is facultative intracellular bacterium that has evolved numerous strategies to evade host immune vesicles and survive in the cytoplasm. Rab32-dependent vesicles are one of these immune vesicles, but the mechanism by which B. pseudomallei escape Rab32-dependent vesicles remains elusive. Here, we find B. pseudomallei infection leading the activation of the type III secretion system (T3SS-3) and increasing the expression of various effectors. Specifically, we identify that BopE, an effector secreted by T3SS-3, triggers vesicle escape to promote B. pseudomallei pathogenicity and survival. Mechanistically, BopE suppresses the activation of Rab32 by interfering with nucleotide exchange, ultimately triggering vesicle escape and intracellular survival. We also find knocking out the bopE gene can increase the proportion of Rab32-positive vesicles that trap B. pseudomallei, dampening the survival of B. pseudomallei both in vitro and in vivo. Taken together, our findings provide insights into the molecular mechanisms of pathogen effector-induced vesicle escape, indicating a potential melioidosis treatment via blocking B. pseudomallei BopE-host Rab32 interaction.

Burkholderia pseudomallei BipD initiates mitophagy to evade killing by hijacking host KLHL9-KLHL13-CUL3 E3 ligase to ubiquitinate IMMT.

Burkholderia pseudomallei (B. pseudomallei) is a facultative intracellular parasitic pathogen with multiple immune escape mechanisms. Mitophagy is critical for mitochondrial quality control and function in various biological processes. We reported that B. pseudomallei infection induces mitophagy to promote its intracellular survival by decreasing mitochondrial reactive oxygen species (mtROS). Mechanically, B. pseudomallei infection leads to the rupture of host outer mitochondrial membrane (OMM) by DNM1L/DRP1 (dynamin 1-like). Furthermore, BipD, the type III secretion system (T3SS) needle tip protein of B. pseudomallei, hijacks the host KLHL9 (kelch-like 9)-KLHL13 (kelch-like 13)-CUL3 (cullin 3) E3 ubiquitin ligase complex to promote the K63-linked ubiquitination of IMMT/mitofilin (inner membrane protein, mitochondrial) at the K211 site. Then BipD-initiated mitophagy, via the conventional macroautophagy/autophagy pathway with the receptor SQSTM1 (sequestosome 1) involvement, decreases the mtROS production, which in turn facilitates the intracellular survival of B. pseudomallei. Here, our findings reveal an unexpected function of BipD and the KLHL9-KLHL13-CUL3 E3 ligase complex and suggest a novel mechanism used by bacterial pathogens that hijack host mitophagy for their survival.

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.

Effect of O antigen glycosyl isomerase gene mutation on biological property and pathogenicity of Burkholderia pseudomallei strain BPC006.

Burkholderia pseudomallei, an intracellular pathogen, is responsible for melioidosis, a zoonotic disease. Its pathogenesis involves several virulence factors, among which lipopolysaccharide (LPS) plays a crucial role. Our research reveals that the O antigen present within the LPS significantly regulates the host immune response. In a previous study, we obtained a B. pseudomallei mutant strain ΔwbiI. Here, the purification of LPS from ΔwbiI and a gas chromatography-mass spectrometry (GC-MS) analysis were conducted. The results confirmed the absence of specific sugar 6-deoxy-Talp, which is a typical component of the O antigen in the wild type B. pseudomallei. Our findings underscore the potent impact the O antigen exerts on the virulence of B. pseudomallei. The ΔwbiI strain displayed significantly increased invasiveness and cytotoxicity in vitro. This enhanced cytotoxicity seems to be related to the exposure of lipid A and an increased cell membrane hydrophobicity resulting from the deletion of the O antigen. Additionally, in mouse models, the ΔwbiI strain resulted in a heightened host lethality and an excessive inflammatory response in mice. These findings indicate that the O-antigenic polysaccharide moiety of B. pseudomallei plays a role in its pathogenicity in vitro and in vivo.

Shiga toxin 2 A-subunit induces mitochondrial damage, mitophagy and apoptosis via the interaction of Tom20 in Caco-2 cells.

Shiga toxin type 2 (Stx2) is the primary virulence factor produced by Shiga toxin-producing enterohemorrhagic Escherichia coli (STEC), which causes epidemic outbreaks of gastrointestinal sickness and potentially fatal sequela hemolytic uremic syndrome (HUS). Most studies on Stx2-induced apoptosis have been performed with holotoxins, but the mechanism of how the A and B subunits of Stx2 cause apoptosis in cells is not clear. Here, we found that Stx2 A-subunit (Stx2A) induced mitochondrial damage, PINK1/Parkin-dependent mitophagy and apoptosis in Caco-2 cells. PINK1/Parkin-dependent mitophagy caused by Stx2A reduced apoptosis by decreasing the accumulation of reactive oxidative species (ROS). Mechanistically, Stx2A interacts with Tom20 on mitochondria to initiate the translocation of Bax to mitochondria, leading to mitochondrial damage and apoptosis. Overall, these data suggested that Stx2A induces mitochondrial damage, mitophagy and apoptosis via the interaction of Tom20 in Caco-2 cells and that mitophagy caused by Stx2A ameliorates apoptosis by eliminating damaged mitochondria. These findings provide evidence for the potential use of Tom20 inhibition as an anti-Shiga toxin therapy.

Serodiagnosis of Abdominal Abscess Caused by Burkholderia pseudomallei: Case Report and Literature Review.

Burkholderia pseudomallei, the causative agent of melioidosis can be responsible for a wide spectrum of clinical manifestations and heterogeneous prognoses, with a high mortality in the acute onset. We report a case of a deep abdominal abscess with sepsis secondary to melioidosis in a young farmer from a non-high-risk population. Emergency medical treatment was administered according to the detection of serum antibodies against Hcp1, the results of which provided etiological evidence of B. pseudomallei infection for the timely and properly antimicrobial therapy in the absence of direct evidence of melioidosis. To our knowledge, this is the first reported case of serodiagnosis of acute exacerbation of melioidosis in China.

MicroRNA-31 induced by Fusobacterium nucleatum infection promotes colorectal cancer tumorigenesis.

Persistent Fusobacterium nucleatum infection is associated with the development of human colorectal cancer (CRC) and promotes tumorigenicity, but the underlying mechanisms remain unclear. Here, we reported that F. nucleatum promoted the tumorigenicity of CRC, which was associated with F. nucleatum-induced microRNA-31 (miR-31) expression in CRC tissues and cells. F. nucleatum infection inhibited autophagic flux by miR-31 through inhibiting syntaxin-12 (STX12) and was associated with the increased intracellular survival of F. nucleatum. Overexpression of miR-31 in CRC cells promoted their tumorigenicity by targeting eukaryotic initiation factor 4F-binding protein 1/2 (eIF4EBP1/2), whereas miR-31 knockout mice were resistant to the formation of colorectal tumors. In conclusion, F. nucleatum, miR-31, and STX12 form a closed loop in the autophagy pathway, and continuous F. nucleatum-induced miR-31 expression promotes the tumorigenicity of CRC cells by targeting eIF4EBP1/2. These findings reveal miR-31 as a potential diagnostic biomarker and therapeutic target in CRC patients with F. nucleatum infection.

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.

Improvements in skills and knowledge after a comprehensive ELISA teaching course for biotechnology undergraduates.

As a universal and extensively adopted technique, enzyme-linked immunosorbent assay (ELISA) can be used to detect and quantify small molecules in many applications both clinical and analytical. However, generally, students experiment mechanically using commercial ELISA kits according to the instructions and eventually produce a standard curve to calculate the concentration of the sample to be measured, cannot understand the critical factors and process of method establishment. This study systematically introduced undergraduates to using the pathogen-specific antigen and establishing an indirect ELISA method to detect the diagnostic target pathogen Burkholderia pseudomallei. This course aimed to develop the experimental skills of the students and improve their scientific research knowledge, which fully embody the organic combination of scientific research and teaching. Students independently selected the diagnostic antigen target of interest, obtained the antigen proteins using genetic engineering techniques, and established an ELISA method through a series of conditional optimization experiments. In addition, typical student-generated data, experimental methods, and a student feedback interpretation are presented in this study. Overall, the students were able to combine abstract knowledge with practice and understand the principles and applications of antigen-antibody interactions, thus enabling them to gain practical experience in molecular biology techniques, and learn how to use this principle to establish an ELISA method for detecting infectious diseases.

One-pot RPA-Cas12a assay for instant and visual detection of Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of melioidosis, a potentially life-threatening infectious disease, and poses public health risks in endemic areas. Due to the high mortality, intrinsic antibiotic resistance, and atypical manifestations, establishing a rapid, accurate, and sensitive identification of B. pseudomallei enables earlier diagnosis, proper treatments, and better outcomes of melioidosis. Herein, we present a One-Pot CRISPR-integrated assay for Instant and Visual Detection (termed OPC-IVD) of B. pseudomallei. The integration of recombinase polymerase amplification and CRISPR-Cas12a recognition-activated trans-cleavage, achieved a true all-in-one single-tube reaction system, initiating the amplification and cleavage simultaneously, which realized a facile sample-to-answer assay. This approach could be performed with simplified DNA extraction and completed around 30 min by holding the reaction tube in the hand. The detection limit of our OPC-IVD was determined to be 2.19 copy/uL of plasmid DNA, 12.5 CFU/mL of B. pseudomallei, and 61.5 CFU/mL of bacteria in spiked blood samples, respectively. Furthermore, the introduction of internal amplification control effectively reduced the occurrence of false negatives, which was incorporated in the reaction system, and amplified simultaneously with the target and read by naked eyes. The assay exhibited 100% accuracy when evaluated in clinical isolates and samples. The streamlined workflow of our OPC-IVD of B. pseudomallei enables a field-deployable, instrument-free, and ultra-fast approach that can be utilized by non-expert personnel in the field of molecular diagnosis of melioidosis especially in under-resourced setting.

Characterization of Burkholderia pseudomallei O antigens in different clinical strains.

O antigen is the major component of lipopolysaccharide LPS. The chemical structure of the O antigen determines the LPS serospecificity of the bacteria, and the diversity of O antigen is the basis for serotyping Burkholderia pseudomallei. In this study, structural elucidation of type B O antigen obtained from a clinical B. pseudomallei strain was conducted, and the effects of different types of LPS on macrophage differentiation were investigated. The O antigen was found to be composed of repeating units of [→4)-α-L-Rhap(1 â†’ 4)-α-L-Rhap(1→2)-α-L-Rhap(1 â†’ 2)-α-L-Rhap(1 â†’ 3)-α-L-Rhap(1 â†’ 3)-α-L-Rhap(1 â†’ 4)-α-L-Rhap(1 â†’ 6)-α-D-Galp(1→]n, where some of the →4)-α-L-Rhap(1 â†’ units were substituted at O-3 by ß-D-Xylp(1 â†’ residues, and minor →3)-α-L-Rhap(1 â†’ units were substituted at O-2 by ß-D-Xylp(1 â†’ residues. Meahwhile, the →6)-α-D-Galp(1 â†’ units were substituted at O-3 by α-D-Galp(1 â†’ residues. Furthermore, both type A and type B O antigens of B. pseudomallei could polarize macrophages toward the M1 phenotype, but the core oligosaccharides had no such activity. Therefore, we deduced that this polarization relies on the O antigen of LPS and might be related to the ability of B. pseudomallei to survive and replicate within macrophages. Thus, the characterization of different types of O antigen structural motifs is essential for further clarifying the persistence/survival mechanisms and inflammatory potential of B. pseudomallei.

Anti-Hcp1 Monoclonal Antibody Is Protective against Burkholderia pseudomallei Infection via Recognizing Amino Acids at Asp95-Leu114.

Melioidosis, a severe tropical illness caused by Burkholderia pseudomallei, poses significant treatment challenges due to limited therapeutic options and the absence of effective vaccines. The pathogen's intrinsic resistance to numerous antibiotics and propensity to induce sepsis during acute infections further complicate management strategies. Thus, exploring alternative methods for prevention and treatment is crucial. Monoclonal antibodies (mAbs) have emerged as a promising strategy for the prevention and treatment of infectious diseases. This study focused on generating three mAbs (13F1, 14G11, and 15D9) targeting hemolysin-coregulated protein 1 (Hcp1), a protein involved in the type VI secretion system cluster 1 (T6SS1) of B. pseudomallei. Notably, pretreatment with 13F1 mAb significantly reduced the intracellular survival of B. pseudomallei and inhibited the formation of macrophage-derived multinucleated giant cells (MNGCs). This protective effect was also observed in vivo. We identified a sequence of amino acids (Asp95-Leu114) within Hcp1 as the likely binding site for 13F1 mAb. In summary, our findings reveal that 13F1 mAb counteracts infection by targeting Hcp1, offering potential new targets and insights for melioidosis prevention.

In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D.

As a principal ingredient of vaccines, adjuvants can directly induce or enhance the powerful, widespread, innate, and adaptive immune responses associated with antigens. Ophiopogonin D (OP-D), a purified component extracted from the plant Ophiopogon japonicus, has been found to be useful as a vaccine adjuvant. The problems of the low solubility and toxicity of OP-D can be effectively overcome by using a low-energy emulsification method to prepare nanoemulsion ophiopogonin D (NOD). In this article, a series of in vitro protocols for cellular activity evaluation are examined. The cytotoxic effects of L929 were determined using a cell counting kit-8 assay. Then, the secreted cytokine levels and corresponding immune cell numbers after the stimulation and culture of splenocytes from immunized mice were detected by ELISA and ELISpot methods. In addition, the antigen uptake ability in bone marrow-derived dendritic cells (BMDCs), which were isolated from C57BL/6 mice and matured after incubation with GM-CSF plus IL-4, was observed by laser scanning confocal microscopy (CLSM). Importantly, macrophage activation was confirmed by measuring the levels of IL-1ß, IL-6, and tumor necrosis factor alpha (TNF-α) cytokines by ELISA kits after coculturing peritoneal macrophages (PMs) from blank mice with the adjuvant for 24 h. It is hoped that this protocol will provide other researchers with direct and effective experimental approaches to evaluate the cellular response efficacies of novel vaccine adjuvants.

Fusobacterium nucleatum induces excess methyltransferase-like 3-mediated microRNA-4717-3p maturation to promote colorectal cancer cell proliferation.

Fusobacterium nucleatum infection plays vital roles in colorectal cancer (CRC) progression. Overexpression of microRNA-4717-3p (miR-4717) was reported to be upregulated in F. nucleatum positive CRC tissues, however, the underlying mechanism is unknown. In this study, we found that miR-4717 promoted CRC cell proliferation in vitro and growth of CRC in vivo following F. nucleatum infection. MicroRNA-4717 suppressed the expression of mitogen-activated protein kinase kinase 4 (MAP2K4), a tumor suppressor, by directly targeting its 3'-UTR. Furthermore, we confirmed that methyltransferase-like 3 (METTL3)-dependent m6 A methylation could methylate primary (pri)-miR-4717, which further promoted the maturation of pri-miR-4717, and METTL3 positively regulated CRC cell proliferation through miR-4717/MAP2K4 pathways. In conclusion, F. nucleatum-induced miR-4717 excessive maturation through METTL3-dependent m6 A modification promotes CRC cell proliferation, which provides a potential therapeutic target and diagnostic biomarker for CRC.

Long non-coding RNA EVADR induced by Fusobacterium nucleatum infection promotes colorectal cancer metastasis.

Both Fusobacterium nucleatum (F. nucleatum) and long non-coding RNA (lncRNA) EVADR are associated with colorectal cancer (CRC), but their relationship with CRC metastasis and the mechanisms by which EVADR promotes CRC metastasis are poorly understood. Here, we report that F. nucleatum promotes colorectal cancer cell metastasis to the liver and lung and that it can be detected in CRC-metastasis colonization in mouse models. Furthermore, F. nucleatum upregulates the expression of EVADR, which can increase the metastatic ability of CRC cells in vivo and in vitro. Mechanistically, elevated EVADR serves as a modular scaffold for the Y-box binding protein 1 (YBX1) to directly enhance the translation of epithelial-mesenchymal transition (EMT)-related factors, such as Snail, Slug, and Zeb1. These findings suggest that EVADR induced by F. nucleatum promotes colorectal cancer metastasis through YBX1-dependent translation. The EVADR-YBX1 axis may be useful for the prevention and treatment of patients with F. nucleatum-associated CRC metastasis.

Structural characterization of a novel pentasaccharide repeating unit from Burkholderia pseudomallei strain BPC006 and its role in diagnosis and immunogenicity.

Burkholderia pseudomallei causes melioidosis - an infectious disease with high mortality. Its varied clinical manifestations and resistance to many antibiotics make it a potential biothreat agent and calls for a robust diagnostic assay and effective vaccines. Bacterial cell surface polysaccharides are considered a valuable target for diagnostics and as protective antigen candidates. This study characterized the structure of polysaccharides of B. pseudomallei clinical strain from Hainan, China. A novel structural domain [→3-(α-D-Manp-1→3-α-D-Manp)2-2Me-α-L-6dTalp-1→] was identified by chemical analysis, gas chromatography-mass spectrometry (GC-MS), and 1D/2D nuclear magnetic resonance (NMR) spectroscopy. Immunofluorescence and enzyme-linked immunosorbent assay (ELISA) showed that the serum antibodies against the purified polysaccharide antigen could recognize and bind specifically to B. pseudomallei strains. Additionally, the assays revealed cross-reactivity with polysaccharides from different clinical strains. The polysaccharide antigen also exhibited a strong reaction with the sera from melioidosis patients. Thus, the pentasaccharide repeating unit residue could be a potential candidate antigen for the melioidosis serodiagnosis and vaccine development.

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.

Tauroursodeoxycholic acid prevents Burkholderia pseudomallei-induced endoplasmic reticulum stress and is protective during melioidosis in mice.

BACKGROUND: Burkholderia pseudomallei, a facultative intracellular bacterium, is the aetiological agent of melioidosis that is responsible for up to 40% sepsis-related mortality in epidemic areas. However, no effective vaccine is available currently, and the drug resistance is also a major problem in the treatment of melioidosis. Therefore, finding new clinical treatment strategies in melioidosis is extremely urgent. RESULTS: We demonstrated that tauroursodeoxycholic acid (TUDCA), a clinically available endoplasmic reticulum (ER) stress inhibitor, can promote B. pseudomallei clearance both in vivo and in vitro. In this study, we investigated the effects of TUDCA on the survival of melioidosis mice, and found that treatment with TUDCA significantly decreased intracellular survival of B. pseudomallei. Mechanistically, we found that B. pseudomallei induced apoptosis and activated IRE1 and PERK signaling ways of ER stress in RAW264.7 macrophages. TUDCA treatment could reduce B. pseudomallei-induced ER stress in vitro, and TUDCA is protective in vivo. CONCLUSION: Taken together, our study has demonstrated that B. pseudomallei infection results in ER stress-induced apoptosis, and TUDCA enhances the clearance of B. pseudomallei by inhibiting ER stress-induced apoptosis both in vivo and in vitro, suggesting that TUDCA could be used as a potentially alternative treatment for melioidosis.

Burkholderia pseudomallei interferes with host lipid metabolism via NR1D2-mediated PNPLA2/ATGL suppression to block autophagy-dependent inhibition of infection.

Burkholderia pseudomallei: which causes melioidosis with high mortality in humans, has become a global public health concern. Recently, infection-driven lipid droplet accumulation has been related to the progression of host-pathogen interactions, and its contribution to the pathogenesis of infectious disease has been investigated. Here, we demonstrated that B. pseudomallei infection actively induced a time-dependent increase in the number and size of lipid droplets in human lung epithelial cells and macrophages. We also found that lipid droplet accumulation following B. pseudomallei infection was associated with downregulation of PNPLA2/ATGL (patatin like phospholipase domain containing 2) and lipophagy inhibition. Functionally, lipid droplet accumulation, facilitated via PNPLA2 downregulation, inhibited macroautophagic/autophagic flux and, thus, hindered autophagy-dependent inhibition of B. pseudomallei infection in lung epithelial cells. Mechanistically, we further revealed that nuclear receptor NR1D2 might be involved in the suppression of PNPLA2 after cell exposure to B. pseudomallei. Taken together, our findings unraveled an evolutionary strategy, by which B. pseudomallei interferes with the host lipid metabolism, to block autophagy-dependent suppression of infection. This study proposes potential targets for clinical therapy of melioidosis.Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; ATG7: autophagy related 7; B. pseudomallei: Burkholderia pseudomallei; CFU: colony-forming unit; DG: diglyceride; FASN: fatty acid synthase; GFP: green fluorescent protein; LAMP1: lysosomal associated membrane protein 1; LC-MS/MS: liquid chromatography-tandem mass spectrometry; LD: lipid droplet; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MG: monoglyceride; MOI: multiplicity of infection; mRFP: monomeric red fluorescent protein; NR1D2: nuclear receptor subfamily 1 group D member 2; p.i., post-infection; PLIN2/ADRP: perilipin 2; PNPLA2/ATGL: patatin like phospholipase domain containing 2; Rapa: rapamycin; SQSTM1/p62: sequestosome 1; shRNA: short hairpin RNA; TEM: transmission electron microscopy; TG: triglyceride.

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.