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Peptidoglycan regulation of Drosophila immunity
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology. (Håkan Steiner)
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Innate immunity is an ancient defense system that distinguishes between self and non self and is present in both vertebrates and invertebrates. Peptidoglycan (PGN), a cell wall component shared by both gram-negative and gram-positive bacteria, is the major recognition molecule for the detection of bacteria in Drosophila. Peptidoglycan Recognition Proteins (PGRPs) are conserved from insect to mammals and bind PGN with high affinity. In Drosophila, distinct PGRPs provide essential signals upstream of the Toll and Imd pathways. This thesis concerns the recognition of PGN by PGRPs and the expression of antibacterial peptides from the Imd pathway.

The PGRP-LC locus encodes three splice forms, a, x, and y. PGRP-LCx and PGRP-LCa form heterodimers in the presence of monomeric PGN. We propose a model for activation of Imd where PGRP-LCx binds to monomeric PGN leading to dimerization with PGRP-LCa and activation of the Imd pathway. With polymeric PGN, PGRP-LCx dimers activate the Imd pathway.

Drosophila PGRP-SC1B has amidase activity and cleaves the PGN lactamyl bond between the glycan strand and the peptide. The digested material is less immunogenic than intact PGN or PGN digested by egg white lysozyme. We suggest PGRP-SC1B to be a scavenger of immunogenic PGN. Amino acid sequence homology predicts six of the Drosophila PGRPs as well as two vertebrate PGRPs to be amidases. Murine PGRP-L is expressed in the liver and was shown to have PGN degrading activity. We found that an earlier purified human serum amidase is in fact identical to PGRP-L.

Although PGN is a potential trigger of the insect immune system, the actual detection of PGN in vivo poses problems. In gram-negative bacteria, PGN is covered by an outer membrane. Using a cell line approach we demonstrate that bacteria can be recognized by the Imd pathway without the need of phagocytosis or contact between bacteria and cells. Instead growing bacteria release immune eliciting fragments of PGN nature.

Place, publisher, year, edition, pages
Stockholm: Department of Genetics, Microbiology and Toxicology, Stockholm University , 2009. , 50 p.
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Genetics
Identifiers
URN: urn:nbn:se:su:diva-29828ISBN: 978-91-7155-938-8 (print)OAI: oai:DiVA.org:su-29828DiVA: diva2:235363
Public defence
2009-10-23, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Submitted.Available from: 2009-10-01 Created: 2009-09-15 Last updated: 2009-09-17Bibliographically approved
List of papers
1. A mammalian peptidoglycan recognition protein with N-acetylmuramoyl-L-alanine amidase activity
Open this publication in new window or tab >>A mammalian peptidoglycan recognition protein with N-acetylmuramoyl-L-alanine amidase activity
2003 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, Vol. 306, no 4, 988-994 p.Article in journal (Refereed) Published
Abstract [en]

The family of peptidoglycan recognition proteins (PGRPs) is conserved from insects to mammals. Recently, Drosophila PGRP-SC1B was demonstrated to be an N-acetylmuramoyl- -alanine amidase (NAMLAA), an enzyme that cleaves the lactylamide bond between muramic acid and the peptide chain in peptidoglycan (PGN). We now show an M.mPGRP-L mRNA to be expressed in the liver. The recombinant M.mPGRP-L protein has NAMLAA activity and degrades PGN from both Escherichia coli and Staphylococcus aureus; however, the Gram-positive PGN was a better substrate after lysozyme treatment. The activity of M.mPGRP-L was further analysed using Bordetella pertussis tracheal toxin as a substrate. Cleavage products were separated on HPLC and identified using mass spectrometry. From these results we conclude that M.mPGRP-L has activity and other properties identifying it as the NAMLAA protein present in mammalian sera.

Identifiers
urn:nbn:se:su:diva-20035 (URN)10.1016/S0006-291X(03)01096-9 (DOI)12821140 (PubMedID)
Available from: 2007-11-21 Created: 2007-11-21 Last updated: 2010-01-11Bibliographically approved
2. Growing bacteria shed elicitors of Drosophila humoral immunity
Open this publication in new window or tab >>Growing bacteria shed elicitors of Drosophila humoral immunity
(English)Article in journal (Refereed) Submitted
Abstract [en]

Peptidoglycan (PGN) is a well-characterized cell wall component from bacteria that is recognized by Peptidoglycan Recognition Receptors (PGRPs) in the Drosophila immune system. It has long been a forum of debate how the immune system is able to recognize this elicitor since the PGN is hidden by the outer membrane in gram-negative bacteria and by teichoic acids in gram-positive bacteria. We show here that bacteria separated from Drosophila S2 cells by a semi permeable membrane are able to up-regulate the Imd pathway. Studies with supernatants from exponentially growning bacterial cultures show that bacteria shed elicitors in sufficient amounts to potentially induce the Imd pathway. However, when employing supernatants from bacteria in stationary phase, no stimulatory effect of the Imd pathway was detected. The elicitor effect was much reduced when the supernatans was treated with the N-acetylmuramoyl-Lalanine amidase M.mPGRP-L, which is a known scavenger of PGN. Our studies therefore indicate that bacteria in growth phase shed elicitors of PGN nature that can induce a humoral immune response in Drosophila S2 cells.

Identifiers
urn:nbn:se:su:diva-29783 (URN)
Available from: 2009-09-15 Created: 2009-09-14 Last updated: 2010-01-11Bibliographically approved
3. Ligand-induced dimerization of Drosophila peptidoglycan recognition proteins in vitro
Open this publication in new window or tab >>Ligand-induced dimerization of Drosophila peptidoglycan recognition proteins in vitro
Show others...
2005 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, Vol. 102, no 18, 6455-6460 p.Article in journal (Refereed) Published
Abstract [en]

Drosophila knockout mutants have placed peptidoglycan recognition proteins (PGRPs) in the two major pathways controlling immune gene expression. We now examine PGRP affinities for peptidoglycan. PGRP-SA and PGRP-LCx are bona fide pattern recognition receptors, and PGRP-SA, the peptidoglycan receptor of the Toll/Dif pathway, has selective affinity for different peptidoglycans. PGRP-LCx, the default peptidoglycan receptor of the Imd/Relish pathway, has strong affinity for all polymeric peptidoglycans tested and for monomeric peptidoglycan. PGRP-LCa does not have affinity for polymeric or monomeric peptidoglycan. Instead, PGRP-LCa can form heterodimers with LCx when the latter is bound to monomeric peptidoglycan. Hence, PGRP-LCa can be said to function as an adaptor, thus adding a new function to a member of the PGRP family.

Identifiers
urn:nbn:se:su:diva-20028 (URN)10.1073/pnas.0407559102 (DOI)15843462 (PubMedID)
Available from: 2007-11-21 Created: 2007-11-21 Last updated: 2010-01-11Bibliographically approved
4. A scavenger function for a Drosophila peptidoglycan recognition protein
Open this publication in new window or tab >>A scavenger function for a Drosophila peptidoglycan recognition protein
2003 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, Vol. 278, no 9, 7059-7064 p.Article in journal (Refereed) Published
Abstract [en]

Recent studies of peptidoglycan recognition protein (PGRP) have shown that 2 of the 13 Drosophila PGRP genes encode proteins that function as receptors mediating immune responses to bacteria. We show here that another member, PGRP-SC1B, has a totally different function because it has enzymatic activity and thereby can degrade peptidoglycan. A mass spectrometric analysis of the cleavage products demonstrates that the enzyme hydrolyzes the lactylamide bond between the glycan strand and the cross-linking peptides. This result assigns the protein as anN-acetylmuramoyl-l-alanine amidase (EC3.5.1.28), and the corresponding gene is thus the first of this class to be described from a eukaryotic organism. Mutant forms of PGRP-SC1B lacking a potential zinc ligand are enzymatically inactive but retain their peptidoglycan affinity. The immunostimulatory properties of PGRP-SC1B-degraded peptidoglycan are much reduced. This is in striking contrast to lysozyme-digested peptidoglycan, which retains most of its elicitor activity. This points toward a scavenger function for PGRP-SC1B. Furthermore, a sequence homology comparison with phage T7 lysozyme, also an N-acetylmuramoyl-l-alanine amidase, shows that as many as six of the Drosophila PGRPs could belong to this class of proteins.

Identifiers
urn:nbn:se:su:diva-20032 (URN)10.1074/jbc.M208900200 (DOI)12496260 (PubMedID)
Available from: 2007-11-21 Created: 2007-11-21 Last updated: 2010-01-11Bibliographically approved

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