THE YIN AND YANG OF A BACTERIAL TOXIN

Transkript

1 THE YIN AND YANG OF A BACTERIAL TOXIN Fooling host Immunity and delivering T cell vaccines P. Šebo PRAGUE Institute of Microbiology Academy of Sciences of the CR

2 Bordetella pertussis The agent of whooping cough (pertussis)

3 Pertussis used to be a major cause of infant morbidity and death in the pre-vaccine era (slide dr. Fabianova NIPH) Died of pertussis according to age groups in a 15 million population of Czechoslovakia Age (months) Total Total Unspec Total

4 We thought the problem was solved, but in CR we expect this year 3000 cases Pertussis incidence in CR in per 100,000 wp vaccine introduced wp production deteriorated ap vaccine Introduced in CR

5 Thanks to democracy and introduction of acellular vaccines the whooping cough is back to the most developed countries.

6 the 2010 and 2014 California outbreaks (we all wake up when something happens in the US ) 10 infants < 2 month old died in 2010

7 No. cases We`ve got a problem again. Confirmed clinical pertussis in in CR (10 million people) We had 2,518 cases in 2014 (like in 1961 ) year

8 So, does pertussis resemble this?

9 Ministerstvo zdravotnictví Palackého nám Praha Doporučení pro očkování těhotných proti pertusi v České republice Doporučení pro těhotné: Těhotné je doporučeno očkovat jednou dávkou kombinované vakcíny proti pertusi, difterii a tetanu (Tdap) během (každého) těhotenství, ideálně mezi 28. a 36. týdnem těhotenství. Tdap - vakcína se sníženým množstvím difterického toxoidu, s tetanickým toxoidem a acelulární pertusovou složkou. Registrované očkovací látky: Adacel výrobce Sanofi Pasteur, vakcína proti difterii, tetanu a pertusi (acelulární), (adsorbovaná se sníženým obsahem antigenů) Boostrix výrobce GSK, vakcína proti difterii, tetanu a pertusi (acelulární komponenta) se sníženým obsahem antigenů Hlavním cílem u očkování v těhotenství je chránit nejmenší děti prostřednictvím posílení mateřských protilátek. Hladina mateřských protilátek je považována za nejdůležitější faktor ochrany kojenců před onemocněním. Většina žen byla v dětství proti pertusi očkována nebo pertusí onemocněla. Očkování ani prožité onemocnění však neposkytuje celoživotní ochranu. Očkování v posledním trimestru těhotenství proti pertusi dočasně zvýší ochranné mateřské protilátky, které přechází od matky přes placentu jejímu nenarozenému dítěti. Přenesené mateřské protilátky pasivně chrání dítě v prvních dvou měsících života, než může být očkováno proti pertusi. Nebylo prokázáno zvýšené riziko vedlejších reakcí po Tdap vakcinaci u těhotných žen ve třetím trimestru a ani u jejich dětí.

10 B. pertussis is armed with numerous parallel and redundant virulence systems (cytotoxins, immunomodulators and adhesins) Slide: Courtesy of C. Locht, Institut Pasteur de Lille

11 B. pertussis makes two of the smartest toxins that subvert cell signaling fools cells by camp the second messenger! TII TIV Cya AC Sec TI Cholera toxin Pertussis toxin ATP camp B. pertussis AC B. anthracis EF TTSS Gs a ADPR Gi a ADPR P. aeruginosa Exo Y AC AC ATP camp AC ATP camp ATP camp Slide: Courtesy of S. Lory, Harvard Medical School

12 Adenylate cyclase toxin - cytolysin AC domain RTX hemolysin moiety T25 T18 Principal CD11b/CD18 binding segment N C I II CBS III Hydrophobic segments 42 calcium binding repeats X-(L/I/F)-X-G-G-X-G-(D/N)-D Secretion signal Palmitoylation on K860 and K983 T25 T18 C-CaM Guo Q. et al. (20005) EMBO J. 24,

13 A conserved block of C-proximal residues is required also for activities of other RTX toxins *T * +XWF - a conserved motif

14 Láďa ACT is an RTX protein secreted by a type I system >100 mm Ca 2+ Outer membrane Inner membrane <100 nm Ca 2+ Need to unfold and refold on the way to target

15 Calcium-dependent folding of CyaA starts from the C-terminus and proceeds towards the N-terminus of the RTX domain

16 Calcium-driven formation of an intramolecular ratchet directs movement of large RTX proteins through type I secretion system conduits

17 THE YIN Fooling host Immunity

18 ACT/cAMP signaling breakes the hell loose and supresses TLR signaling of the bug on the mucosa mucosa signal transduction events: NF-κB, MAPK p38, ERK, JNK expression and upregulation of TLR: TLR1-6, 9, TLR4, TLR2 AEC other cells LPS ACT mucin: MUC2, MUC5AC ciliary beating defensins and other antimicrobial peptides: hβdefensin2, βdefensin1, cathelicidin other soluble factors: NO, PGE2 camp cytokine and chemokines: IL-1α, IL-1β, IL-6, IL-8, IL-10, TNFα, IFNβ, TGF-β, GM-CSF, MCP-1, MIP-1α, RANTES,.. expression of costimulatory x inhibitory molecules: CD80, CD86, CD40, CD54, B7-H2, B7-H3 x FasL, PD-L1, PD-L2

19 the Yin: ACT as a SWIFT SABOTEUR low ACT (CyaA) concentrations make a difference on respiratory mucosa Pertactin Capsule Periplasm DNT PT CyaA FHA TCT fimbriae IL-6 invasion Macrophage Mucus layer Respiratory epithelium Ciliated cells Nonciliated cells (Goblet cells, Absorptive cells) Cilliated cells Type I CD11b + B.pertussis intraepithelial secreting CyaA DC Submucosa Immature dendritic cell? IL-6 IL-8 Macrophage CyaA IL-12, TNFα, CCL3 IL-10, IL-6, IL-1beta, IL-17 (Semi-mature state of DC?) T regulatory response? ATP camp CD11b/CD18 Intoxication Phagocytic functions Phagocytosis Superoxid production Apoptosis Cell lysis Cations Channel formation? IL-1β Dendritic cell Neutrophil Macrophage Neutrophil B cell T cell Osičková et al., (1999) J. Biol. Chem. 274, 37644) Vojtová et al., 2006, Curr. Op.. Microbiol. 9, 69-75

20 AC AC The three cytotoxic activities of ACT adenylate cyclase toxin & pore-forming hemolysin/cytolysin Repeats Repeats Intoxication CR3 a b = -integrin Mac-1 M 2 receptor CD11b/ CD18 Cytoplasmic membrane Na + Ca 2+ Na + ATP AC CaM camp Sebo et al. (1991) Gene 104:19 Sakamoto et al. (1992) J. Biol. Chem. 267, Benz et al. (1994) J. Biol. Chem. 269, Hackett et al. (1995) J. Biol. Chem. 270, Gray et al. (1998) J. Biol. Chem. 273, Osickova et al. (1999) J. Biol. Chem. 274, Basler et al. (2007) J. Biol. Chem. 282, Fiser R. et al. (2007) J. Biol. Chem. 282, 2808 Osickova et al. (2010) Mol. Microbiol. 75: Translocation precursor K + Channel precursor K + Oligomeric Cation-selective Channel

21 AC AC The toolbox: A panel of mutations characterized that block ACT activity at each individual step of toxin action Repeats Repeats E509K+E516K Intoxication b 2 -integrin receptor (CD11b/ CD18) E570Q+K860R E570Q E581P C + AC camp CaM ATP E570P E570K+E581P Translocation precursor Channel precursor E570K+E581P E509K+E516K Oligomeric Cation-selective (hemolytic) Channel Basler et al., (2007) J. Biol. Chem. 282, drop of channel cation selectivity Marek

22 AC AC AC - Hly - toxoid under evaluation as pertussis vaccine candidate Repeats Intoxication Ca 2+ b 2 -integrin receptor (CD11b/ CD18) Cytoplasmic membrane AC CaM ATP camp Translocation precursor Osickova et al. (2010) Mol. Microbiol. 75: Osickova et al., (1999) J. Biol. Chem. 274, Basler et al., (2007) J. Biol. Chem. 282, Fiser R. et al.(2007) J. Biol. Chem. 282, 2808

23 Bordetella adenylate cyclase toxin hijacks its β 2 integrin receptor into lipid rafts to accomplish membrane translocation in two steps Bumba et al. (2010). PLoS Pathog 6(5): e

24 CyaA-induced morphological rearrangements Mouse macrophage-like cell line J774 A.1: Buffer, 5 min CyaA, 10 ng/ml, 5 min CyaA-AC -, 10 ng/ml, 5 min db-camp, 2mM, 10 min Kamanova et al. (2008) J. Immunol. 181,

25 Phagocytosis (% of control) pmol camp/6x10 5 J744A.1 cells ACT at low doses ablates complement-mediated phagocytosis (through RhoA inactivation) A camp B ng/ml 100 ng/ml Time (minutes) FcR-mediated phagocytosis CR3-mediated phagocytosis phagocytosis Buffer control 0 CyaA (ng/ml) CyaA-AC - (ng/ml) db-camp (mm) * * * * * * * * * * Kamanova et al. (2008) J. Immunol. 181,

26 camp signaling wipes out receptor signaling through Syk, blocking bactericidal functions of phagocytes Syk activation (%) Syk activation (%) ic3b- Zymosan ic3b-zymosan IP: anti-py, IB: anti-syk Lyzate, IB: anti-syk Syk Syk IP: anti-py, IB: anti-syk Lysate, IB: anti-syk Syk Syk ic3b- Zymosan Toxin added with ic3b zymosan ic3b-zymosan Toxin added after Syk signaling has been pre-activated with ic3b zymosan

27 CyaA-produced camp signaling through PAK activates SHP-1 tyrosine phosphatase that blocks inos gene transcription Cell-invasive Adenylate cyclase AC? Hydrophobic domain RTX-hemolysin repeats RTX? Palmitoylated Lys860 and Lys983 Translocation Ca 2+ a b Potassium efflux K + Plasma membrane RTX AC RTX RTX RTX RTX ATP AC camp Calcium influx Cation selective pores camp Reg PKA Cat SH2 SH2 Cat SHP-1 active conformation Černý et al. (2015) J. Immunol 194: AP1 c-fos X inos

28 Relative phosphorylation SHP-1 activity (A.U.) a a inos expression NO production [%] CyaA-produced camp signaling through PKA activates SHP-1 tyrosine phosphatase that blocks inos gene transcription 3 Upper band 2,5 Lower band 2 a 1,5 a a a 1 0,5 P-c-Fos 0 actin LPS CyaA NSC ** 2 1,5 1 0,5 0 inos actin LPS CyaA NSC ** ** LPS 0,7 0,6 0,5 0,4 0,3 0,2 ** ** LPS + CyaA LPS sirna: ** + LPS + LPS + CyaA ** ** 0,1 SHP min 10 min 30 min 60 min SHP-2

29 THE YANG OF A BACTERIAL TOXIN" delivering T cell vaccines Drugs from bugs

30 AC AC dact as a novel antigen delivery tool epitope insert Ag Repeats Ag Repeats epitope insert dact b 2 integrin receptor CD11b/ CD18 dact Ca + AC Translocation into cytosol Channel formation in plasma membrane Lysosome Endosome Antigenic peptides Proteasome Endogenous antigen integrin (CD11b CD18) Transporter associated with antigen procesing Antigenic peptides Endoplasmatic reticulum MHC II peptide MHC I Golgi MHC I peptide MHC II Proteasome processing TAP transport to ER MHC I binding Epitope presentation on MHC class I molecules CD8+ T cell Receptor mediated endocytosis endosomal processing MHC II binding Epitope presentation on MHC class II molecules CD4+ helper T cell

31 Vaccines that are built with the CyaA vector are chimeric recombinant proteins consisting of the CyaA protein and the antigen of choice. 1: Sebo P, Fayolle C, d'andria O, Ladant D, Leclerc C, Ullmann A. (1995) Infect Immun. 63(10): : Fayolle C, Sebo P, Ladant D, Ullmann A, Leclerc C. (1996) J Immunol. 156(12): Saron MF, Fayolle C, Sebo P, Ladant D, Ullmann A, Leclerc C. Proc Natl Acad Sci U S A. 1997;94(7):

32 September Genticel S.A. completed Phase I clinical trial for HPV16/18-induced cervical carcinoma Using a cgmp batch of the adenylate cyclase (CyaA-AC - ) toxoid for delivery of HPV E7 antigen as immunotherapeutic vaccine safe, immunogenic, inducing CD8 + CTLs and HPV 16/18 virus load reduction demonstrated Entered phase II trial = will be of interest to see pertussis incidence in CyaA-E7 toxoid treated woman IPO on April 4 at Euronext Paris and Brussels - 34 millions Euro

33 Knowledge is useful We still have 20 years of work on ACT ahead of us

34 Occasionally it is more fun

35 Acknowledgments Institut Pasteur: teams: Claude Leclerc Laleh, Catherine, Gilles Daniel Ladant Nicole Guiso Imperial College Robert Wilkinson Katalin Wilkinson VLA Surrey Martin Vordemeier Institute of Microbiology Lída Tučková Marek Kovář and their teams University Wurzburg Roland Benz and his team Bernhard Nocht Institut: Thomas Jacobs Susanne Tartz MH Hannover Ingo Just Harald Genth