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Identification and Characterization of the Moraxella catarrhalis IgD-binding Protein MID

Möllenkvist, Andrea LU (2005)
Abstract (Swedish)
Popular Abstract in Swedish

Moraxella catarrhalis är en human luftvägsbakterie som ofta koloniserar de övre luftvägarna och som orsakar mellanöroninfektion hos barn. Bakterien associeras även med bihåleinfektion, laryngit och nedre luftvägsinfektioner främst hos vuxna med underliggande sjukdomar som kronisk obstruktiv lugninflammation. Forskningen fokuserar främst på moraxellas ytexponerade antigener för att kunna tillverka ett framtida vaccin.



MID är en adhesin som binder både membranbundet och lösligt IgD på ett icke-immunt sätt. Membranproteinet MID isolerades genom solubilisering i den zwitterjoniska detergenten Empigen och renades sedan med hjälp av jonbyteskromatografi och gelfiltrering. Mid genen... (More)
Popular Abstract in Swedish

Moraxella catarrhalis är en human luftvägsbakterie som ofta koloniserar de övre luftvägarna och som orsakar mellanöroninfektion hos barn. Bakterien associeras även med bihåleinfektion, laryngit och nedre luftvägsinfektioner främst hos vuxna med underliggande sjukdomar som kronisk obstruktiv lugninflammation. Forskningen fokuserar främst på moraxellas ytexponerade antigener för att kunna tillverka ett framtida vaccin.



MID är en adhesin som binder både membranbundet och lösligt IgD på ett icke-immunt sätt. Membranproteinet MID isolerades genom solubilisering i den zwitterjoniska detergenten Empigen och renades sedan med hjälp av jonbyteskromatografi och gelfiltrering. Mid genen klonades och det 2139 aminosyror långa proteinet betedde sig som en multimer under nativa förhållanden.



Vi har undersökt förekomsten av MID i 91 kliniska isolat och 7 referens-stammar. Den konserverade mid genen fanns i alla stammar, men proteinuttrycket varierade något oberoende av varifrån stammarna var isolerade eller deras geografiska ursprung. Vi har också visat att mid genen reglerades av fasvariation styrd av en poly(G)-box inom genens läsram.



Vi lyckades kartlägga det membranbundna MID proteinet med hjälp av guldinmärkta specifika antikroppar i transmissions elektronmikroskopi (TEM). MID har en 100 nm lång fibrillär struktur med en kompakt globulär domän längst ut på stjälken som svarar för både IgD-bindning och adhesiva egenskaper. Vi har dessutom visat att MID är en multimer som består av trimerer, tetramerer och pentamerer. Genom sekvensanalys har vi även visat att MID är ett autotransport protein som tillhör den Oligomeriska coiled-coilade adhesin (Oca) familjen.



Slutligen har vi identifierat adducin, ett membranprotein som finns i humana röda blodkroppar, som receptor för den adhesiva domänen MID764-913. Denna binding är grundläggande för den hemagglutinerande förmågan hos M. catarrhalis.



Identifikation och karakterisering av yttermembranproteinet MID är ett betydelsefullt bidrag rörande mikrobiell patogenes. Artiklarna inkluderade i denna avhandling beskriver utbredning, genreglering, domänstrukturella uppbyggnaden och receptor-specificiteten av MID. (Less)
Abstract
Moraxella catarrhalis is now an established human pathogen that frequently colonizes the upper respiratory tract causing most importantly otitis media in children. It has also been associated with sinusitis, laryngitis and lower respiratory tract infections in adults with predisposing conditions such as chronic obstructive pulmonary disease (COPD). Current research mainly focuses on surface antigenic determinants of M. catarrhalis as it progresses towards a future vaccine.



MID is an adhesin that binds both soluble and membrane-bound IgD in a non-immune fashion. The membrane protein MID was isolated by solubilization using the zwitterionic detergent Empigen and then purified by ion exchange chromatography and gel... (More)
Moraxella catarrhalis is now an established human pathogen that frequently colonizes the upper respiratory tract causing most importantly otitis media in children. It has also been associated with sinusitis, laryngitis and lower respiratory tract infections in adults with predisposing conditions such as chronic obstructive pulmonary disease (COPD). Current research mainly focuses on surface antigenic determinants of M. catarrhalis as it progresses towards a future vaccine.



MID is an adhesin that binds both soluble and membrane-bound IgD in a non-immune fashion. The membrane protein MID was isolated by solubilization using the zwitterionic detergent Empigen and then purified by ion exchange chromatography and gel filtration. The 2139 amino acids long protein ? as deduced from the cloned mid gene ? behaved as a multimer under native conditions.



We investigated the prevalence of MID in 91 clinical isolates and 7 culture collection strains. The conserved mid gene was found in all strains, however the protein expression varied independently of anatomical site of isolation or geographical origin of the strains. We also showed that the mid gene was regulated by phase variation controlled by a poly(G) box within the open reading frame.



Mapping the membrane-bound MID with gold-labeled specific antibodies in transmission electron microscopy (TEM) revealed a double-folded 100 nm long fibrillar structure with a globular distal domain responsible for both the IgD-binding and adhesive capacities of MID. In addition, we demonstrated that MID is a multimer and consists of trimers, tetramers and pentamers. Furthermore, sequence analysis also showed that MID is an autotransporter that belongs to the Oligomeric coiled-coil adhesin (Oca) family.



Finally, the human erythrocyte membrane protein adducin was identified as a putative receptor for the adhesive domain MID764-913. This is the underlying interaction for the hemaglutinating activity of M. catarrhalis.



The isolation and characterization of a major novel outer membrane protein designated MID (Moraxella IgD-binding protein) has been a significant contribution to the field. The papers in this thesis describe the prevalence, gene regulation, domain structural organization and receptor specificity of MID. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • professor Sundqvist, Tommy, Avd f Medicinsk mikrobiologi, Universitetssjukhuset i Linköping
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Microbiology, receptor, structure, phase variation, MID, IgD, Moraxella catarrhalis, bacteriology, virology, mycology, Mikrobiologi, bakteriologi, virologi, mykologi
pages
142 pages
publisher
Medical Microbiology, Lund University
defense location
Patologiska Institutionens föreläsningssal, ingång 78, U-MAS, Malmö.
defense date
2005-10-06 09:15
ISSN
1652-8220
ISBN
91-85439-80-0
language
English
LU publication?
yes
id
eb5f460b-4485-4f89-bd3f-d5a922c451f9 (old id 24890)
date added to LUP
2007-06-05 09:57:10
date last changed
2016-09-19 08:44:54
@phdthesis{eb5f460b-4485-4f89-bd3f-d5a922c451f9,
  abstract     = {Moraxella catarrhalis is now an established human pathogen that frequently colonizes the upper respiratory tract causing most importantly otitis media in children. It has also been associated with sinusitis, laryngitis and lower respiratory tract infections in adults with predisposing conditions such as chronic obstructive pulmonary disease (COPD). Current research mainly focuses on surface antigenic determinants of M. catarrhalis as it progresses towards a future vaccine.<br/><br>
<br/><br>
MID is an adhesin that binds both soluble and membrane-bound IgD in a non-immune fashion. The membrane protein MID was isolated by solubilization using the zwitterionic detergent Empigen and then purified by ion exchange chromatography and gel filtration. The 2139 amino acids long protein ? as deduced from the cloned mid gene ? behaved as a multimer under native conditions.<br/><br>
<br/><br>
We investigated the prevalence of MID in 91 clinical isolates and 7 culture collection strains. The conserved mid gene was found in all strains, however the protein expression varied independently of anatomical site of isolation or geographical origin of the strains. We also showed that the mid gene was regulated by phase variation controlled by a poly(G) box within the open reading frame.<br/><br>
<br/><br>
Mapping the membrane-bound MID with gold-labeled specific antibodies in transmission electron microscopy (TEM) revealed a double-folded 100 nm long fibrillar structure with a globular distal domain responsible for both the IgD-binding and adhesive capacities of MID. In addition, we demonstrated that MID is a multimer and consists of trimers, tetramers and pentamers. Furthermore, sequence analysis also showed that MID is an autotransporter that belongs to the Oligomeric coiled-coil adhesin (Oca) family.<br/><br>
<br/><br>
Finally, the human erythrocyte membrane protein adducin was identified as a putative receptor for the adhesive domain MID764-913. This is the underlying interaction for the hemaglutinating activity of M. catarrhalis.<br/><br>
<br/><br>
The isolation and characterization of a major novel outer membrane protein designated MID (Moraxella IgD-binding protein) has been a significant contribution to the field. The papers in this thesis describe the prevalence, gene regulation, domain structural organization and receptor specificity of MID.},
  author       = {Möllenkvist, Andrea},
  isbn         = {91-85439-80-0},
  issn         = {1652-8220},
  keyword      = {Microbiology,receptor,structure,phase variation,MID,IgD,Moraxella catarrhalis,bacteriology,virology,mycology,Mikrobiologi,bakteriologi,virologi,mykologi},
  language     = {eng},
  pages        = {142},
  publisher    = {Medical Microbiology, Lund University},
  school       = {Lund University},
  title        = {Identification and Characterization of the Moraxella catarrhalis IgD-binding Protein MID},
  year         = {2005},
}