Burkholderia pseudomallei survival in lung epithelial cells benefits from miRNA-mediated suppression of ATG10

Abstract

(2015) Burkholderiapseudomallei survival in lung epithelial cells benefits from miRNA-mediated suppression of ATG10, Autophagy, 11:8, 1293-1307, DOI: 10.1080/15548627.2015 Keywords: ATG10, autophagy, Burkholderia pseudomallei, DNA methylation, MIR4458, MIR4667-5p, MIR4668-5pAbbreviations: 3-MA, 3-methyladenine; 3 0 UTR, 3 0 -untranslated region; 5-Aza-CdR, 5-aza-2 0 -deoxycytidine; ACTB, actin, b; ATG4C, autophagy-related 4C, cysteine peptidase; ATG5, autophagy-related 5; ATG10, autophagy-related 10; ATG12, autophagyrelated 12; B. pseudomallei, Burkholderia pseudomallei; CFU, colony forming unit; CQ, chloroquine; DNMT1, DNA (cytosine-5-)-methyltransferase 1; DRAM2, DNA-damage regulated autophagy modulator 2; MAP1LC3B, microtubule-associated protein 1 light chain 3 b; miRNAs, microRNAs; MOI, multiplicity of infection; MSP, methylation-specific PCR; PBS, phosphate-buffered saline; p.i., postinfection; Rapa, rapamycin; siRNA, small interfering RNA; SQSTM1, sequestosome 1; TEM, transmission electron microscopy.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 0 -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 autophagymediated elimination of B. pseudomallei by targeting ATG10, and provide potential targets for clinical treatment.

Bacterial Pathogens versus Autophagy: Implications for Therapeutic Interventions

“…Another pathogen, Burkholderia pseudomallei promotes its replication in the cytosol of murine macrophages by evading LC3 targeting through an undefined process involving the type III secretion system ATPase BpscN[38]. In addition, infection of macrophages and epithelial cells with B. pseudomallei or Burkholderia cenocepacia leads to downregulation of certain autophagy genes, resulting in decreased levels of autophagy[39,40]. Group B streptococcus (GBS) induces autophagy during infection of EC, but still resides primarily in single membrane-bound compartments, suggesting that GBS evades targeting to autophagosomes[41].…”

Differential Effects of Autophagy-Related 10 Protein on HCV Replication and Autophagy Flux Are Mediated by Its Cysteine44 and Cysteine135

“…Autophagy-related 10 gene ( ATG10 ), which encodes an E2-like enzyme ( 25 ), interacts with ATG7 to receive an ubiquitin-like protein ATG12, recognizes both ATG12 and ATG5 directly and catalyzes their conjugation reaction ( 26 – 28 ). ATG10 has been shown to play an important role in the invasion and proliferation of cancer cells ( 8 , 29 ), and bacterial infections ( 30 ). At present, the research of the function of ATG10 alone is lacking, and few studies have examined the interdependency between the structure and function of ATG10.…”

Transcriptome Analysis Reveals Dynamic Changes of Inflammation and Stress Responses during Different Infected Stages with Burkholderia pseudomallei

“…Bacterial invasion of A549 cells followed our previously described strategy [10], with minor modifications. A549 cells were diluted to 1.5 × 10 5 /mL, and 1 mL cell suspension was plated in each well of a 12-well plate containing glass coverslips (NEXT, China).…”

“…It has been reported that B. pseudomallei can escape into the cytoplasm by inducing rearrangement of host actin cytoskeleton and with the help of its type III secretion system (T3SS) effector proteins, such as BopE and BopA [7][8][9]. Our team previous research found that B. pseudomallei could evade autophagy by 3 novel miRNAs of MIR4458, MIR4667-5p, and MIR4668-5p targeted regulation of ATG10 in A549 cells [10]. Macrophages could up-regulate Rab32 GTPase through reducing miR-30b/30c to accelerate B. pseudomallei-containing phagosomes maturation [11].…”