1. Burkholderia pseudomallei inhibits autophagy to benefit its intracellular survival in lung epithelial cells by suppressing ATG10 Qian Li Graduate student May 17 th, 2016

2. liver abscesspulmonary abscess brain abscessgangrene Burkholderia pseudomallei ， Bp Melioidosis Background (multiple abscesses)

3. Wiersinga WJ, Currie BJ, Peacock SJ. N Engl J Med. 2012, 367(11):1035-44. Background prevalent in tropical regions of the world high pathogenicity difficult to diagnose high mortality a long latency period inside host easy to obtain (water, soil) easy to spread (aerosol)

4. Background Schematic representation of selected steps in the intracellular life style of Bp Elizabeth MA, Rodney JD, John DB. Front Microbiol. 2011, 8(2):37-56.

5. Autophagy pathway: Background Yang Z, Klionsky DJ. Nat Cell Biol. 2010, 12(9):814-822.

6. Methods for Monitoring Autophagy: Background  Transmission electron microscopy (TEM)  LC3 detection and quantification 1. Western blotting and ubiquitin-like protein conjugation systems 2. Turnover of LC3II–PE 3. GFP-LC3 lysosomal delivery and proteolysis 4. GFP-LC3 fluorescence microscopy 5. Tandem mRFP/mCherry-GFP fluorescence microscopy 6. Autophagic flux determination 7. Immunohistochemistry  SQSTM1/p62 and related LC3 binding protein turnover assays …… Daniel JK, Fabio CA, Hagai A, et al. Autophagy. 2016, 12(1):1-222.

7. 1. Intracellular survival and replication of B. pseudomallei in human lung epithelium cells. uninfected 2 h p.i. (postinfection) 12 h p.i.24 h p.i. A549 cells

8. 2. Autophagy is induced in early response to B. pseudomallei infection, but then decreased in the later infection phase. (1) Western blot (2) GFP-LC3 puncta assay Autophagic response detection CQ ： Chloroquine, block autophagic flux;

9. (3) Intracellular survival of bacteria Rapa ： autophagy enhancer; 3-MA ： autophagy inhibitor ； 2. Autophagy is induced in early response to B. pseudomallei infection, but then decreased in the later infection phase.

10. 3. Down-regulation of ATG10 expression in response to B. pseudomallei infection. 6 h p.i. GeneChipsqRT-PCR, WB ATG10 ： qRT-PCR, WB ATG10

11. overexpressed ATG10 WB, immunofluorescence, intracellular survival assay 3. Down-regulation of ATG10 expression in response to B. pseudomallei infection.

12. 4. MIR4458, MIR4667-5p and MIR4668-5p are upregulated after B.pseudomallei infection. miRNA microarrays qRT-PCR bioinformatics tools

13. 5. MIR4458, MIR4667-5p and MIR4668-5p suppresse expression of ATG10 by binding with its 3’UTR. miRNA target validation methods ： 1. Luciferase assay2. WB A549 cells 3. qRT-PCR HEK293 cells

14. 5. MIR4458, MIR4667-5p and MIR4668-5p suppresse expression of ATG10 by binding with its 3’UTR. miRNA target validation methods ： 1. Luciferase assay2. WB3. qRT-PCR

15. 6. MIR4458, MIR4667-5p and MIR4668-5p inhibit autophagy and promote the intracellular survival of B.pseudomallei. 1. Autophagy response （ WB, GFP-LC3 puncta assay ） 2. intracellular survival assay (1) WB CQ ： Chloroquine, block autophagic flux;

16. (2) GFP-LC3 puncta assay 6. MIR4458, MIR4667-5p and MIR4668-5p inhibit autophagy and promote the intracellular survival of B.pseudomallei. 1. Autophagy response （ WB, GFP-LC3 puncta assay ） 2. intracellular survival assay

17. 6. MIR4458, MIR4667-5p and MIR4668-5p inhibit autophagy and promote the intracellular survival of B.pseudomallei. 1. Autophagy response （ WB, GFP-LC3 puncta assay ） 2. intracellular survival assay

18. Schematic figure ：

19. Acknowledgments Prof. Xuhu Mao Dr. Bing Tang Dr. Yao Fang Pan Zhu Chunyan Ren Kun Wang Yinping Jia

20. Thanks for your attention!