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

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

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

Type III secretion system (T3SS) SPI I of Salmonella

T3SS and flagella

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

T3 Effectors modify host signaling pathways

Effectors mimic host protein function

Effectors can introduce covalent- modification

Effectors are less conserved

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

Determination of secretion signal PLoS Pathogens April 2009 | Volume 5 | Issue 4 | e very high false positive rate!

Signal is more defined within a species

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

Burkholderia pseudomallei genome Burkholderia pseudomallei K96243: NC_006350; NC_006351,NC_006350NC_

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

Assembly of T3S needle