Natalie C. Marshall Finlay Lab University of British Columbia. How EPEC and EHEC modulate human cells via their type III secretion system

Transcription

1 Natalie C. Marshall Finlay Lab University of British Columbia How EPEC and EHEC modulate human cells via their type III secretion system

2 anthropomorphize verb (ăn thrə-pə-môr fīz ) : To attribute human form or personality to things not human Merriam-Webster Dictionary, 2015

3 appropriate meal-time conversation??? Kroen, 2013 Scientific American

4 The Finlay Lab The Giant Colon 2014, University of British Columbia

5 1 This talk EPEC and EHEC Type III secretion system (T3SS) How EPEC/EHEC use the T3SS to cause disease Current research + Where the field is going

6 Finlay lab: How do T3SS pathogens cause disease? Salmonella Typhimurium EPEC & EHEC NIAID Rosenshine et al., 1996 EMBO J.

7 Enterohemorrhagic E. coli (EHEC) Cause bloody diarrhea, potentially kidney failure Outbreaks from contaminated food and water E.g. O157:H7 Croxen and Finlay, 2010 Nat Rev Microbiol

8 Enteropathogenic E. coli (EPEC) Cause watery diarrhea Mostly affect children in the developing world Kill several hundred thousand children each year Croxen and Finlay, 2010 Nat Rev Microbiol Walker et al., 2013 Lancet Lond. Engl.

9 Attaching and effacing (A/E) pathogens E.g. EPEC, EHEC, rabbit EPEC, Citrobacter rodentium Similar histopathology & virulence mechanisms Dean et al., 2013 PLoS Pathogens

10 A/E Discovery Timeline : Identified a region in the EPEC genome required to form A/E lesions : Discovered a pathogenicity island specific to A/E pathogens: Locus of Enterocyte Effacement (LEE) Identified the first 5 EPEC secreted proteins Some were protease-resistant Identified putative T3SS s in EPEC + EHEC Adapted from Iguchi et al., 2009

11 What is a type III secretion system?

12 Many important global pathogens have one Disease Typhoid fever Bubonic and pneumonic plague Sexually-transmitted disease Conjunctivitis Diarrheal disease Pulmonary infections T3SS pathogen Salmonella Typhi Yersinia pestis Chlamydia spp. Chlamydia spp. Salmonella Typhimurium Yersinia enterocolitica Shigella spp. EHEC EPEC Pseudomonas aeruginosa Bordetella spp. Adapted from Marshall & Finlay, 2014 Expert Opin. Ther. Targets

13 It is essential for virulence ΔT3SS ΔT3SS + empty plasmid Complement Wild-type Yersinia pestis Bozue et al., 2012 FEMS Microbiol. Lett.

14 Adapted from Marshall & Finlay, 2014 Expert Opin. Ther. Targets The T3SS INNER MEMBRANE Gram-negative pathogen OUTER MEMBRANE Eukaryotic host cell HOST PLASMA MEMBRANE

15 Adapted from Marshall & Finlay, 2014 Expert Opin. Ther. Targets Galkin et al., 2010 J Mol Biol The T3SS Gram-negative pathogen Rosenshine et al., 1996 EMBO J. Eukaryotic host cell

16 Step 1: Build Step 2: Inject T3SS effectors Step 3: Control the host cell Marshall & Finlay, 2014 Expert Opin. Ther. Targets

17 HOW DO EPEC/EHEC USE THEIR T3SS TO CAUSE DISEASE? Let s take a look

18

19 In real life: Attaching/Effacing lesions Dean et al., 2013 PLOS Pathogens

20 In real life: A/E lesions Dr Paul Dean, Newcastle Uni.

21 In real life: Pedestals Finlay lab Rosenshine et al Finlay S. Schüller lab, UBC Science Photo Library J. Girón

22 How did we get from this to this? Marshall & Finlay, 2014 Expert Opin. Ther. Targets

23 Citrobacter rodentium ( Citro ): An A/E pathogen and in vivo model Natural mouse pathogen A/E lesions Pedestals Diarrheal disease Uninfected + Citrobacter (10 dpi)

24 Citro translocates Tir into the intestinal epithelium Tir Actin DNA Deng et al., 2003 Mol. Microbiol

25 Screened all 41 LEE gene mutants Deng et al., 2004 Proc. Natl. Acad. Sci.

26 Screened for secretion Deng et al., 2004 Proc. Natl. Acad. Sci.

27 for effects on pedestals + virulence Etc Basic model of LEE protein functions Deng et al., 2004 Proc. Natl. Acad. Sci.

28 Most T3SS effectors: Esp_ (E. coli secreted protein) Some effectors: Nle_ (Non-LEE-encoded) CAN WE IDENTIFY ALL OF THE T3SS EFFECTORS?

29 Intensity Intensity SILAC analysis of EPEC and C. rodentium type III secretomes WT, grown in heavy amino acids 13 C 6 -Arg, 2 H 4 -Lys T3S conditions ΔT3SS mutant, grown in light amino acids Precipitate proteins Trypsinize LC-MS/MS Peptide from non-type III proteins Peptide from type III-secreted proteins m/z m/z Collab. Foster lab (UBC)

30 T3SS effector repertoire of prototypical A/E pathogens Genes C. rodentium EHEC EPEC T3SS effectors Pseudogenes Strain DBS100 O157 E2348/69 Deng et al. 2004, 2010; Tobe et al. 2006; Iguchi et al. 2008, 2009; Ogura et al. 2009; Petty et al. 2010

31 What do T3SS effectors do in host cells? Transfect effectors into 293T cells Effectors + GST tag Co-immunoprecipitation with anti-ha antibody Incubate effector with 293T cell lysate Complex (Effector : Host protein) Trypsinize Fourier-Transform mass spectrometry Identify host protein(s)

32 T3SS effectors manipulate the host EspZ EspZ Colonization Ion/water channels Gene expression Intracellular trafficking Host inflammation Host cell death Host immune response Adapted from Law & Finlay, 2013 CSH Perspectives in Medicine

33 Pathogenesis 101 RULE #1 Don t get caught

34 CHALLENGE #1 EPEC vs. Host inflammation

35 NF-κB pathway Pro-inflammatory cytokines E.g. IL-8 Phagocytosis Gerondakis et al., 2014 Nat. Rev. Immunol.

36 EPEC vs. Inflammation Glycosyltransferase Kinase Methylase Protease EspG Santos & Finlay, 2015 Cell. Microbiol.

37 Turco et al., 2014 Biochemistry

38 Pathogenesis 101 RULE #2 Don t kill your host

39 CHALLENGE #2 EPEC vs. Host apoptosis

40 EPEC should activate apoptosis Challenge accepted! Rudel et al., 2010 Nat. Rev. Microbiol. Shames & Finlay, 2010 PLoS Pathogens

41 Extrinsic apoptosis Glycosyltransferases Protease Santos & Finlay, 2015 Cell. Microbiol.

42 Intrinsic apoptosis Santos & Finlay, 2015 Cell. Microbiol.

43 NleF: Caspase kryptonite Blasche et al., 2013 PLoS ONE

44 Intrinsic apoptosis Santos & Finlay, 2015 Cell. Microbiol.

45 CURRENT PROJECTS IN THE FINLAY LAB

46 T3SS effectors in mitochondria EspZ Adapted from Law & Finlay, 2013 CSH Perspectives in Medicine

47 T3SS effectors are imported into mitochondria Nucleus Nucleus EspF Map EspZ TOM OMM MPP Icp55 TIM IMS IMM Matrix Apoptosis

48 Mitochondrial membrane potential plummets during EPEC infection Uninfected ΔΨ m EPEC wild-type Time post-infection Shames et al., 2011 Infect. Immun.

49 The Citro T3SS alters mitochondria Wild-type ΔT3SS Ma et al., 2006 Cell. Microbiol. What s happening in mitochondria during EPEC infection?

50 Proteolysis is a vital cellular process and a potent virulence mechanism Host Apoptosis IL-1β processing Pro-enzyme activation Pathogen Clostridial tetanus + botulinum toxins HIV-1 protease Proteolysis can tell us a lot about what s happening inside cells

51 WHERE IS THE FIELD GOING? We re addressing our weaknesses

52 Host-pathogen interaction (Not to scale!) -microbiota + metabolites + mucus Croxen & Finlay, 2010 Nat. Rev. Microbiol. Adapted from Włodarska and Finlay, 2010 Mucosal Immunol.

53 Different mouse strain = different susceptibility to C. rodentium infection NIH Swiss (resistant) GUT MICROBIOTA HeJ (susceptible) 129S1 BALB/c C57BL/6 Does the gut microbiome contribute to host resistance/susceptibility to infection? Vallance et al., 2003 Infect. Immun.

54 Vallance et al., 2003 Infect. Immun. Microbiota control host susceptibility! Survival Weight

55 Microbiota affect mucosal immunity Microbiota transfer Antibacterials Probiotics Adapted from Włodarska and Finlay, 2010 Mucosal Immunol.

56 Microbial signals drive production of the inner mucus layer Adapted from Włodarska and Finlay, 2010 Mucosal Immunol.

57 MUCUS FUNCTION

58 Can we manipulate the inner mucus layer to affect the outcome of enteric infection?? EUGENOL METRONIDAZOLE Włodarska et al., 2015 Sci. Rep.

59 Where else is the field going? HOT TOPICS: All of the omics Microbiome Metabolome Proteome - Phosphoproteome - Terminal proteome? T3S inhibitors

60 THANK YOU Prof. Leonard Foster Nick Stoynov Jason Rogalski Nichollas Scott Prof. Richard Flavell Prof. Eran Elinav Prof. José Puente Prof. Christopher Overall Theo Klein Ulrich Eckhard Nikolaus Fortelny Prof. Brett Finlay Wanyin Deng Marta Włodarska Andrew Santos Stephanie Shames Maichael Thejoe Roland Scholz Niklas von Krosigk Lisa Thorson Supervisory committee Brett Finlay Chris Overall Rachel Fernandez Laura