Understanding the immune response to bacterial infections
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1 Understanding the immune response to bacterial infections A Ph.D. (SCIENCE) DISSERTATION SUBMITTED TO JADAVPUR UNIVERSITY SUSHIL KUMAR PATHAK DEPARTMENT OF CHEMISTRY BOSE INSTITUTE 2008
2 CONTENTS Page SUMMARY Chapter 1 1. INTRODUCTION The genus Mycobacterium \ 1.2. Interaction of mycobacteria with host macrophages Mycobacterial entry into host macrophages Salient characteristics of the mycobacterial phagosome The interactions between dendritic cells (DCs) and mycobacteria Interaction of mycobacteria with other innate immune cells Immunomodulatory molecules of mycobacteria Toll-like receptors Negative regulation of TLR signaling The role of Toll-like receptors in mycobacterial signaling The RD-1 region and ESAT-6 family proteins Host cell signaling induced by mycobacteria Rationale of the study 24 Chapter 2 2. METHODS Cell culture techniques Culture of the murine macrophage-like cell line RAW Culture of the human embryonic kidney cell line HEK Trypan blue dye exclusion method 27
3 2.2. Limulus Amoebocyte Lysate (LAL) test Protein estimation Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE) Western blotting using nitrocellulose and polyvinylidene 34 fluoride (PVDF) membrane 2.6. General molecular biology technique Agarose gel electrophoresis of DNA Polyacrylamide gel electrophoresis of PCR amplification of genes Purification of DNA fragments from PCR reactions Restriction enzyme digestion of DNA fragments and plasmids Purification of DNA fragments from agarose gel Insertion of DNA into plasmid vectors by ligation Preparation of competent. coli cells Transformation of competent E. coli Isolation of plasmid DNA Small-scale preparation Preparation of plasmid DNA on a large scale RNA isolation and Reverse Transcription RNA isolation Reverse transcription Plasmid constructs Transient transfections Transfection of plasmid constructs in RAW264.7 cells 47
4 Transfection of plasmid constructs in HEK293 cells Transfection of sirnas in RAW264.7 or HEK293 cells (3-Galactosidase assay Co-immunoprecipitation of TRAF6 and IRAK Western Blotting for Phospho-IicB-a Study of binding of nuclear NF-KB c-rel or p50 subunitsand c-fos 51 to their respective DNA-binding consensus sequence by ELISA Western blot for Irak-M Generation and transfection of small interfering RNA constructs 54 for Irak-M Procedure for transfection of RNA interference expression vectors 55 using Lipofectamine Luciferase reporter assays Generation of bone marrow derived macrophages (BMDM) Generation of mutants of ESAT Expression and purification of His-ESAT Cloning, expression and purification of Flag-ESAT Expression and purification of His-CFP Cloning, expression and purification of His-tagged extra cellular 59 domain of TLR Enzyme-linked immunoassay for cytokines Nuclear extract preparation IRAK-4 kinase assay In Vitro IKK kinase assay 64
5 2.28. Akt kinase assay TLR2/ESAT6 ELISA -like binding assay Study of interaction of MyD88 and IRAK Study of the in vitro interaction of ESAT-6 with TLR Fluorescence activated cell sorting (FACS) to study the 66 interaction of ESAT-6 with TLR Fluorescence microscopy Small interfering RNA-dependent down-regulation of Akt kinase Analysis of MAPK activation Study of IKB-O, phosphorylation by ELISA 68 Chapter 3 3. Understanding the mechanism of LAM-mediated suppression of interleukin-12 (IL-12) release in macrophages 3.1. Man-LAM inhibits LPS-induced IL-12p40 mrna expression and 70 protein release in RAW264.7 cells 3.2. Inhibition of LPS-induced IL-12p40 induction by Man-LAM is 71 independent of IL Man-LAM inhibits LPS-induced IL-12p40 promoter activity Inhibition of DNA binding of Rel family proteins by Man-LAM Man-LAM inhibits IKB-CC phosphorylation in LPS-treated 74 RAW264.7 cells 3.6. Man-LAM attenuates the interaction of IRAK-1 with TRAF Man-LAM induces Irak-Mexpression 75
6 3.8. Specific down-regulation of Irak-Mabolishes the inhibitory 77 effect of Man-LAM on LPS-induced IL-12 production 3.9. Effect of mannan on IRAK-M production in RAW264.7 cells DISCUSSION 80 Chapter 4 4. Understanding the mechanisms of ESAT-6 mediated attenuation of TLR signaling 4.1. ESAT-6 inhibits TLR signaling Inhibition of LPS-induced IL-12p40 induction by ESAT-6 is 83 independent of IL MyD88-dependent TLR signaling is inhibited by ESAT ESAT-6 inhibits activation of NF-KB IRAK-4-MyD88 interaction is inhibited by ESAT TLR ligand induced IRAK-4 kinase activity and TRAF6 88 ubiquitination is inhibited by ESAT ESAT-6 does not induce expression of known negative 90 regulators of TLR signaling 4.8. ESAT-6 binds to the cell surface of RAW264.7 cells ESAT-6 signals through TLR ESAT-6 interacts with TLR ESAT-6-mediated attenuation of IL-12p40 release does not require 96 TLRl,TLR6orCD Inhibition of PI-3K/Akt signaling attenuates ESAT-6-mediated 97 dampening of IL-12 p40 production
7 4.13. ESAT-6 activates PI-(3)K and Akt in a TLR2-dependent manner Knockdown of Akt prevents ESAT-6 activity Inhibitory effect of ESAT-6 is lost in bone marrow derived macrophages (BMDM) obtained from TLR2" mice ESAT-6 abrogates TLR-dependent activation of IRF proteins LPS-induced activation of ASKl and p38 is inhibited by ESAT Residues in ESAT-6 critical for interaction with TLR DISCUSSION REFERENCES APPENDIX Plasmid constructs used for transfection Abbreviations Materials Publications
8 SUMMARY Nearly a third of the world's population is infected with Mycobacterium tuberculosis, the causative agent of human tuberculosis (TB). The organism evades the host's immune response by manipulating host cell signaling pathways. To design novel therapeutics and to rationalize vaccination strategies against mycobacterial diseases, it is of foremost importance to understand the mechanisms by which mycobacteria subvert the protective immune response. This dissertation focuses on how two effectors from M. tuberculosis dampen the innate immune response. Interleukin-12 (IL-12) is critical for the generation of Thl responses against M. tuberculosis infection. Mannose-capped lipoarabmomannans (Man-LAMs) are members of the repertoire of M. tuberculosis modulins which the bacillus uses to subvert the host innate immune response. Mycobacterial Man-LAMs modulate the immune response by dampening DL-12 production in macrophages and dendritic cells and attenuating apoptotic signaling. We have demonstrated that Man-LAM from M. tuberculosis suppresses LPS-induced IL-12p40 protein production, mrna expression and promoter activity in murine RAW264.7 macrophages by inhibiting LPS-induced IRAK-1-TRAF6 interaction, IicB-a phosphorylation and nuclear translocation of c-rel and p50. Man-LAM induces IRAK-M expression which is a negative modulator of TLR4 signaling that is restricted to monocytes and macrophages. Silencing of IRAK-M by RNA interference supports our view that Man-LAM dampens IL*12p40 production through an IRAK-M-mediated pathway. Our studies provide mechanistic insight into how a mycobacterial modulin functions to dampen TLR signaling. Early secreted antigenic target protein 6 (ESAT-6) is a member of a unique family of proteins present in the genus Mycobacterium. We present evidence that ESAT-6 or a peptide derived from it dampens TLR signaling by preventing assembly of the cytosolic MyDS8-dependent signaling scaffold. ESAT-6 binds to TLR2, activates signaling dependent on phosphatidylinositol- 3-OH kinase (PI3K)-Akt kinase without activating signaling dependent on MyD88-IRAK-IKK (inhibitor of NF-KB (IKB) kinase), and prevents ligand-mediated recruitment of IRAK-4 to MyD88, leading to inhibition of NF-KB activation. We provide evidence demonstrating that direct interaction on the extracytosolic side of the plasma cell membrane between a single TLR (TLR2) and a single peptide (from ESAT-6) inhibits the assembly of TLR signaling molecules inside the cell required for activating innate immune responses. We also show that attenuation of MyD88- dependent TLR signaling by ESAT-6 requires Akt kinase and ESAT-6 also inhibits TLRdependent activation of IRF1, IRF3 and IRF5. i
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