1. Type III secretion effectors in bacterial genome 5 May 2010 Sūn Guăng Wén Biochemistry, NUS

2. Melioidosis Causative agent: Burkholderia pseudomallei Causative agent: Burkholderia pseudomallei Endemic area: SEA; northern Australia Endemic area: SEA; northern Australia Symptom: variable Symptom: variable Diagnosis: Serum test, culture of bacteria, PCR Diagnosis: Serum test, culture of bacteria, PCR Risk factor: Type II diabetes and kidney failure Risk factor: Type II diabetes and kidney failure Treatment: Ceftazidime Treatment: Ceftazidime Vaccine: not available Vaccine: not available

3. Burkholderia pseudomallei Facultative anaerobic, in soil and surface water Opportunistic pathogen Resistant to many antibiotics Huge genome: 4.1Mb +3.2Mb Many virulence factors: siderophore, flagella, T3SSs, T6SSs Able to invade cells and survive intracellularly

4. Type III secretion system (T3SS) SPI I of Salmonella

5. T3SS and flagella

6. Distribution of T3SSs among pathogenic bacteria Nat rev MCB 3:743

7. T3 Effectors modify host signaling pathways

8. Effectors mimic host protein function

9. Effectors can introduce covalent- modification

10. Effectors are less conserved

11. N-terminal of effector contains secretion signal Fusion assay N-ter signal reporter

12. Determination of secretion signal PLoS Pathogens April 2009 | Volume 5 | Issue 4 | e1000376 http://www.effectors.org/http://www.effectors.org/ ---very high false positive rate!

13. Signal is more defined within a species

14. Inv/mxi/spa-like T3S3 of B. pseudomallei Base and inner membrane complex Translocons Secreted effectors Molecular Microbiology (2002) 46(3), 649–659 Molecular Microbiology (2010) 76(3), 677–689 Regulator Chaperone

15. Burkholderia pseudomallei genome Burkholderia pseudomallei K96243: NC_006350; NC_006351,NC_006350NC_006351 http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi http://www.sanger.ac.uk/cgi-bin/blast/submitblast/b_pseudomallei

16. The challenge: To identify T3S3 effectors in bp genomes Define training sets, collect effector sequences Define training sets, collect effector sequences Find patterns in N-terminals Find patterns in N-terminals Search Burkholderia pseudomallei genomes Search Burkholderia pseudomallei genomes Blast top hits in protein databases Blast top hits in protein databases Verify through experiments Verify through experiments

17. Assembly of T3S needle