Burkholderia Pseudomallei Inhibits Autophagy To Benefit Its Intracellular Survival In Lung Epithelial Cells Through MiRNAs-mediated ATG10 Suppression

Burkholderia pseudomallei, a gram-negative pathogen, is the causative agent of melioidosis, which is classically characterized by pneumonia and multiple abscesses with a mortality rate of up to 40%. Melioidosis is endemic in southeast Asia, northern Australia and other tropical regions, and is the most common cause of pneumonia-derived sepsis in Thailand. Because melioidosis carries a high fatality rate, B. pseudomallei is classified as category B potential bioterrorism agent by the Center for Disease Control and NIAID. It has now been well accepted that a key component of the pathogenesis of B. pseudomallei is its ability to invade and survive intracellularly in both phagocytic and non-phagocytic cells, which explains numerous features of melioidosis including latency, recrudescence, and antibiotic resistance.Autophagy as one of the earliest defense responses encountered by intracellular pathogens, is a process that engulfs and delivers intracellular bacteria for lysosomal degradation. However, the battle between the human host and the infecting pathogens is continuously evolving. Preliminary evidence has indicated that several pathogens, such as Shigella, Salmonella, Mycobacteria and influenza A virus, have evolved mechanisms to evade the autophagic response. B. pseudomallei has the capacity to invade a number of cultured mammalian cell lines. After entry, B. pseudomallei utilizes numerous strategies that enable it to survive in such a specialized niche as the intracellular environment. Recently, Cullinane et al. have reported that B. pseudomallei can evade autophagy in an active behavior, although autophagy is induced in response to B. pseudomallei infection as early as 2 h infection period. Thus, the detailed mechanism by which B. pseudomallei-mediated subversion of autophagy should be further clarified.Micro RNAs(mi RNAs) are small, noncoding RNAs that regulate gene expression by sequence-specific binding to messenger RNA(m RNA). mi RNAs post-transcriptionally regulate gene expression by speci?cally targeting m RNAs for cleavage or inhibition of protein synthesis, regulating a wide spectrum of cellular processes such as cancers, immune response and autophagy. Growing evidence has indicated that dysregulated mi RNAs expression has been associated with several intracellular bacteria-mediated evasion of autophagy. Thus, understanding the molecular mechanisms of autophagy modulation in host response to B. pseudomallei infection is crucial to current and future therapeutic approaches.In this report, the relationships between mi RNA, autophagy and B. pseudomallei a re investigated in lung epithelial cell lines. We provide evidence showing that MIR4458, MIR4667-5p and MIR4668-5p suppress autophagy-mediated removal of B. pseudomallei in epithelial cells by targeting ATG10.【Objectives】In this study, we aim to clarify the detailed mechanisms involved in the impaired autophagic clearance of B. pseudomallei in human lung epithelium cells, and search for new approaches to the treatment of B. pseudomallei infection to provide theoretical guidance.【Methods】1. To determine the status of autophagy in response to B. pseudomallei infection.(1) Western blot and GFP-MAP1LC3 B puncta assay were used to determine whether B. pseudomallei modulates autophagy.(2) To estimate the number of live internalized B. pseudomallei in A549 cells, bacterial colony forming units were performed in a time course experiment.2. To elucidate the underlying mechanisms that B. pseudomallei inhibits the autophagy pathway to avoid clearance by the host cells.We used Human Affymetrix Gene Chips to determine the gene expression profile of A549 cells at 6 h p.i..3. To elucidate the underlying mechanisms for downregulated ATG10 expression.(1) The luciferase assay, Western blot and q RT-PCR analysis were used to identify whether ATG10 is targeted by MIR4458, MIR4667-5p and MIR4668-5p.(2) Transfection with the three mi RNAs mimic, we used Western blot, GFP-MAP1LC3 B puncta assay and bacterial colony forming assay to test the role of MIR4458, MIR4667-5p and MIR4668-5p in regulation of autophagy during B. pseudomallei infection.4. To identify the mechanism responsible for the upregulation of MIR4458, MIR4667-5p and MIR4668-5p during B. pseudomallei infection in A549 cells.We used UCSC Genome Bioinformatics to screen the regulatory regions of MIR4458, MIR4667-5p and MIR4668-5p, and found there was a Cp G island located within the three mi RNAs promoters, respectively.【Results】1. Autophagy was inhibited in response to B. pseudomallei infection, and stimulation of autophagy could suppress the intracellular survival of B. pseudomallei infection.2. ATG10 expression is downregulated during B. pseudomallei infection. Enforced expression of ATG10 enhanced the colocalization of intracellular B. pseudomallei with autophagosomes, and significantly decreased the bacterial load of intracellular B. pseudomallei.3. The expression level of MIR4458, MIR4667-5p and MIR4668-5p were upregulated after B. pseudomallei infection. The results of luciferase assay, Western blot and q RT-PCR analysis showed that ATG10 is the target of MIR4458, MIR4667-5p and MIR4668-5p. Furthermore, transfection with the three mi RNAs mimic decreased MAP1LC3B-II conversion and the number of MAP1LC3 B puncta.4. The BSP result indicated that the methylation status of the Cp Gs within MIR4458, MIR4667-5p and MIR4668-5p promoters was decreased in response to B. pseudomallei infection. And we also that B. pseudomallei could reduce the DNMT activity in the nuclear extract. Furthermore, inhibition of promoter Cp G methylation by 5-Aza-Cd R treatment activated MIR4458, MIR4667-5p and MIR4668-5p expression in A549 cells in a dose-dependent manner.【Conclusion】This study shows the molecular basis involved in the impaired autophagic clearance of B. pseudomallei in human lung epithelium cells. Specifically, we found that the underlying mechanism for downregulated ATG10 expression after exposure to B. pseudomallei in A549 cells is through increased MIR4458, MIR4667-5p and MIR4668-5p expression, caused by reducing promoter methylation.【Significance】These insights increase our understanding of the host-pathogen relationship, lay the groundwork for strategies aimed at combating infectious diseases, and may provide useful information for developing potential therapeutic interventions against pathogens.