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Interaction Between Drosophila Melanogaster Mbn-2 Cells and Bacteria Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 9 Interaction Between Drosophila melanogaster mbn-2 Cells and Bacteria KARIN JOHANSSON ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6214 UPPSALA ISBN 91-554-6140-9 2005 urn:nbn:se:uu:diva-4772 ! "##$ %#&## ' ' ' ( ) * + ) , -) "##$) . + /" ) 0 ) 1) !% ) ) .23 1%/$$!/4%!#/1 . ' / / ' ) * ' ' 5 ' 6 '' ' ' + / ' ) * ' ' + ' ' ) . + ' + 3/7 89/ + ' ) . ' + 5 + ' ' ' ' + ' ) 5 ' ' ) * ' ' /5 6 /") : + + + '' + ) / ! " # ! " ! $%& '()! ! *+,-./0 ! ; - , "##$ .223 %4$%/4"%! .23 1%/$$!/4%!#/1 & &&& /!<<" = &88 )5)8 > ? & &&& /!<<"@ List of Papers This thesis is based on the following papers, which will be referred to in the text by their Roman numerals: I Lindmark, H., Johansson, K. C., Stöven, S., Hultmark, D., Eng- ström, Y. and Söderhäll, K. Enteric bacteria counteract lipopoly- saccharide induction of antimicrobial peptide genes. J. Immunol. 2001. 167: 6920-6923. II Johansson, K. C., Metzendorf, C. and Söderhäll, K. Microarray analysis of immune challenged Drosophila hemocytes. Exp Cell Res (in press). III Johansson, K. C., Söderhäll, K. and Cerenius, L. Diptericin expression in bacteria infected Drosophila mbn-2 cells – effect of infection dose and phagocytosis. Manuscript IV Johansson, K. C., Lind, M. I. and Söderhäll, K. Pefabloc – a sulfonyl fluoride serine protease inhibitor blocks induction of the diptericin gene in Drosophila mbn-2 cells. Manuscript, Short communication Reprints were made with permission from the publishers. © The American Association of Immunologists, Inc., 2001 © 2005 Elsevier Ltd Skaparen har klätt hela världen till en blomtapet och därpå satt människan att spatsera, leva och sig förnöja. Carl von Linné 1707-1778 Contents Introduction.....................................................................................................7 The immune response of Drosophila melanogaster and other arthropods 8 Humoral responses .....................................................................................9 Antimicrobial peptides...........................................................................9 Melanization ........................................................................................10 Cellular responses ....................................................................................11 Phagocytosis ........................................................................................12 Encapsulation.......................................................................................12 Clotting ................................................................................................13 Recognition of infection...........................................................................14 PGRPs..................................................................................................14 GNBPs.................................................................................................15 Signalling pathways regulating immune responses in Drosophila ..........16 Toll-pathway........................................................................................16 Imd-pathway........................................................................................17 JAK/STAT-pathway............................................................................18 Host – microbe interactions......................................................................19 Microbial strategies for overcoming host innate immune responses...19 Objectives .....................................................................................................21 Results and discussion..............................................................................21 AMP induction in mbn-2 cells is reduced by live bacterial infection (Paper I) ...............................................................................................21 Microarray analysis of immune challenged mbn-2 cells (Paper II).....23 Infection dose and bacterial growth rate affects Diptericin expression (Paper III) ............................................................................................25 Inhibition of Diptericin expression by a commercial serine protease inhibitor (Paper IV)..............................................................................27 Svensk sammanfattning (Summary in Swedish) ..........................................29 Acknowledgements.......................................................................................31 References.....................................................................................................33 Abbreviations acyl-HSL Acylated homoserine lactone AI Autoinducer AMPs Antimicrobial peptides ȕGBP ȕ-1, 3-glucan recognition protein DAP-PGN Diaminopimelic acid-type peptido- glycan DIF Dorsal related immunity factor EMSA Electrophoretic mobility shift assay EST Expressed sequence tag FACS Fluorescence activated cell sorting GNBP Gram-negative binding protein Imd Immune deficiency JAK/STAT Janus kinase/signal transducer and activator transcription JNK Jun N-terminal kinase LPS Lipopolysaccharide LTA Lipoteichoic acid Lys-PGN Lysine-type peptidoglycan mAb Monoclonal antibody MOI Multiplicity of infection NF-țB Nuclear factor-kappa B PGN Peptidoglycan PGRP Peptidoglycan recognition protein PO Phenoloxidase PPAE Prophenoloxidase activating enzyme proPO Prophenoloxidase PRR Pattern Recognition Receptor RNAi RNA interference RT-PCR Reverse transcriptase polymerase chain reaction SDS/PAGE Sodium dodecylsulphate/ Polyacryl- amide gel electrophoresis Tep Thioester-containing protein Introduction Survival of all multicellular organisms depend on mechanisms that can tell the difference between self and potentially harmful non-self (antigens) and then activate the appropriate response that targets antigen-bearing entities while respecting self-cells and tissues. The immune system is classically divided in two major branches, named innate and adaptive immunity. Innate immunity is an ancient collection of protective mechanisms that appeared early in the evolution of multicellular organisms. It is common to all metazoans and recognizes molecular patterns present in microorganisms but absent from eukaryotic cells by germ line-encoded non-rearranging re- ceptors referred to as pattern recognition receptors (PRRs) (Janeway, 1989). Innate immune responses include phagocytosis and encapsulation, synthesis of antimicrobial peptides (AMPs) and activation of proteolytic cascades that lead to melanization, blood coagulation, release of stress-responsive proteins and molecules believed to function in opsonization and iron sequestration. Adaptive immunity is younger in origin and appeared some 500 million years ago in the ancestors of cartilaginous fish (e. g. sharks). Today, adaptive immunity is restricted to Gnathostomes, including around 45 000 vertebrate species where it co-exists with innate immune defences (Kimbrell and Beut- ler, 2001; Mayer et al. 2002). Adaptive immunity is based on random gen- eration of antigen receptors in lymphocytes through somatic gene rear- rangements and a subsequent clonal expansion of activated lymphocytes that gives the potential to recognize virtually any antigen that may be encoun- tered. In vertebrates the innate immune system serves as the first line of de- fence and interprets the ‘biological context’ of antigens and instructs the adaptive immune system to make the appropriate antibody or T-cell response (Medzhitov and Janeway, 1999). Adaptive immunity also allows for immu- nological memory, mediating long-term immunity through persistence of selected antibody-producing lymphocytes. A functionally similar feature in invertebrate immunity – ‘immunological priming’ has been discussed (Kurtz and Franz, 2003), but remains controversial as functional evidence for this is still scarce. It refers to observations that past experience with a pathogen can provide individual invertebrates with enhanced resistance in a second expo- sure. The protective action of immunological priming should not be con- fused with the antibody-mediated specificity of adaptive immunity, but may instead act through a prolonged general upregulation of innate immune fac- 7 tors that lingers on after the first encounter with a pathogen (Little and Kraaijveld, 2004). Although invertebrates lack the tremendous complexity of the adaptive immune system and rely solely on innate immunity their amazing diversity, abundance and evolutionary success argue for a highly efficient defence system against infections. Research on the common fruit fly Drosophila melanogaster has for over 100 years generated a vast amount of data on genetics and development not only applicable to insects and other invertebrates but also to mammals. Completion of the genome sequence (Adams et al. 2000) in combination with powerful genetic tools that facilitate fast generation of mutants with distinct phenotypes, easy and cost effective maintenance of fly stocks and lack of adaptive
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