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The biology of filamentous phage infection - implications for display technology

Karlsson, Fredrik LU (2004)
Abstract (Swedish)
Popular Abstract in Swedish

Det finns ett stort behov av molekyler eller reagenser för korrekt och snabb diagnos och/eller behandling av sjukdom. På laboratorier används särskilda verktyg för att ta fram sådana reagenser. Verktygen utgörs ibland av levande organismer, som t ex bakterier och virus, och kallas bioteknologiska verktyg. Ett av dessa verktyg baseras på användandet av en speciell typ av trådlikt bakterievirus (fag). Om vi kan lära oss mer om samspelet mellan bakterie och fag, skulle detta verktyg kunna utvecklas så att processen att finna de eftersökta molekylerna för diagnos och terapi effektiviseras. I den här avhandlingen, som bygger på fyra vetenskapliga originalartiklar, har jag beskrivit den trådlika fagens... (More)
Popular Abstract in Swedish

Det finns ett stort behov av molekyler eller reagenser för korrekt och snabb diagnos och/eller behandling av sjukdom. På laboratorier används särskilda verktyg för att ta fram sådana reagenser. Verktygen utgörs ibland av levande organismer, som t ex bakterier och virus, och kallas bioteknologiska verktyg. Ett av dessa verktyg baseras på användandet av en speciell typ av trådlikt bakterievirus (fag). Om vi kan lära oss mer om samspelet mellan bakterie och fag, skulle detta verktyg kunna utvecklas så att processen att finna de eftersökta molekylerna för diagnos och terapi effektiviseras. I den här avhandlingen, som bygger på fyra vetenskapliga originalartiklar, har jag beskrivit den trådlika fagens biologi samt undersökt möjligheterna att utveckla den teknologi som heter “fag-display”. Detta har jag gjort dels genom att utvärdera en variant av fag-display-teknologin som benämns “selektiv infektion” och dels genom att studera fagens mekanism för infektion av bakterien. Mina resultat visar att verktyget “selektiv infektion” har egenskaper som gör det lämpligt för att hitta proteinlika molekyler som binder starkt till målmolekyler (exempelvis en markör för en sjukdom), något man med traditionell fag-display-teknologi inte alltid klarar av att göra. De övriga studierna har gällt fagens infektionsförlopp, som jag har studerat m h a tre olika metoder. I en studie dissekerade jag de två proteiner som är inblandade i infektionsförloppet. Det ena proteinet (pIII) härrör från fagen och det andra (TolA) är ett bakterieprotein. Resultaten visade på flertalet interaktioner mellan olika delar av de två proteinerna, varav några varit hittills okända. I en annan studie introducerades mutationer i bakterieproteinet TolA, varefter den kvarvarande funktionen i mutanterna mättes. De flesta mutanterna som isolerades hade behållit funktionen som fag-receptor men förlorat sin huvudsakliga funktion; att hålla samman cellmembranet. Därmed visades att TolA:s olika funktioner grundar sig i helt olika mekanismer. Slutligen studerades hur bakteriecellens genreglering förändrades i samband med fag-infektion. För detta användes mikromatriser som kan mäta genuttryck i hela organismer. Mikromatrisanalysen gav indikationer på att fagen påverkat uttrycket av vissa bakteriella gener som dittills inte satts i samband med fag-infektion. Generellt kan studier om fagens infektionsmekanism bidra till att öka förståelsen av de processer där bakterier fångar upp nya genetiska element och omvandlas till sjukdomsframkallande bakterier. (Less)
Abstract
Phage display technology is a biotechnological tool that can be used to obtain molecules/reagents able to discriminate between target molecules. Reagents with such features can be used in diverse settings like biological chemistry, diagnostics and in therapeutic applications. Phage display technology is based on the use of recombinant DNA technology, bacteria and a special kind of bacterial virus, the filamentous phage. However, the chance of obtaining high binding strength (affinity) reagents using conventional phage display is limited by e. g. the selection procedure. Therefore, protocols that increase the likelihood of recovering high affinity binding proteins from a pool of similar molecules, a so called library, are of interest. One... (More)
Phage display technology is a biotechnological tool that can be used to obtain molecules/reagents able to discriminate between target molecules. Reagents with such features can be used in diverse settings like biological chemistry, diagnostics and in therapeutic applications. Phage display technology is based on the use of recombinant DNA technology, bacteria and a special kind of bacterial virus, the filamentous phage. However, the chance of obtaining high binding strength (affinity) reagents using conventional phage display is limited by e. g. the selection procedure. Therefore, protocols that increase the likelihood of recovering high affinity binding proteins from a pool of similar molecules, a so called library, are of interest. One approach is to modify the selection procedure of phage display, which has generated a special application called selective infection. Another approach is to study the infection process of filamentous phage, in hope of revealing mechanistic events suitable to manipulation. In this thesis, which is based on four original papers, selective infection is evaluated and the infection mechanism of filamentous phage is assessed in conjunction to implications for phage display technology. By analysing the prerequisite for successful selective infection, we found a correlation between the affinity of the interacting pairs and infection efficiency and that a phage format allowing multiple display of binding proteins was superior to one that does not allow such multiple display. Thus, selective infection has characteristics that make it suitable for retrieving mainly high affinity binding proteins from phage display libraries. The infection mechanism of filamentous phage was dissected by analysing the molecular interactions between phage coat protein pIII and the bacterial co-receptor protein of phage infection, TolA. The binding affinities between these proteins and their different domains were characterised and novel interactions were detected, allowing for a refined hypothetical model of the infection mechanism of filamentous phage. Furthermore, TolA mutants were created for the analysis of its phage receptor and outer membrane integrity functions. It was found that these two TolA functions could be segregated, thus emphasising the mechanistic differences between the two functions of TolA. Finally, the changes in gene expression of phage infected Escherichia coli, was monitored by global transcription analysis and it was demonstrated that several host genes were co-ordinately affected. In conclusion, these studies have provided a basis for the development of phage display technology, as well as insights into several different aspects of the infection process of filamentous phage. (Less)
Please use this url to cite or link to this publication:
author
opponent
  • Prof Ståhl, Stefan, Stockholm
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Biotechnology, Bioteknik, infection mechanism, selective infection, TolA, Escherichia coli, pIII, Filamentous phage, Phage display technology
pages
136 pages
publisher
Fredrik Karlsson Dept. of Immunotechnology P.O. Box 7031 SE-22007 Lund,
defense location
Lundmarksalen Astronomy Department, Sölveg. 27, Lund
defense date
2004-11-19 10:15
ISBN
91-7422-064-0
language
English
LU publication?
yes
id
1567c469-2b05-43aa-b026-0e7e871f5156 (old id 467514)
date added to LUP
2007-09-10 11:25:59
date last changed
2016-09-19 08:45:14
@phdthesis{1567c469-2b05-43aa-b026-0e7e871f5156,
  abstract     = {Phage display technology is a biotechnological tool that can be used to obtain molecules/reagents able to discriminate between target molecules. Reagents with such features can be used in diverse settings like biological chemistry, diagnostics and in therapeutic applications. Phage display technology is based on the use of recombinant DNA technology, bacteria and a special kind of bacterial virus, the filamentous phage. However, the chance of obtaining high binding strength (affinity) reagents using conventional phage display is limited by e. g. the selection procedure. Therefore, protocols that increase the likelihood of recovering high affinity binding proteins from a pool of similar molecules, a so called library, are of interest. One approach is to modify the selection procedure of phage display, which has generated a special application called selective infection. Another approach is to study the infection process of filamentous phage, in hope of revealing mechanistic events suitable to manipulation. In this thesis, which is based on four original papers, selective infection is evaluated and the infection mechanism of filamentous phage is assessed in conjunction to implications for phage display technology. By analysing the prerequisite for successful selective infection, we found a correlation between the affinity of the interacting pairs and infection efficiency and that a phage format allowing multiple display of binding proteins was superior to one that does not allow such multiple display. Thus, selective infection has characteristics that make it suitable for retrieving mainly high affinity binding proteins from phage display libraries. The infection mechanism of filamentous phage was dissected by analysing the molecular interactions between phage coat protein pIII and the bacterial co-receptor protein of phage infection, TolA. The binding affinities between these proteins and their different domains were characterised and novel interactions were detected, allowing for a refined hypothetical model of the infection mechanism of filamentous phage. Furthermore, TolA mutants were created for the analysis of its phage receptor and outer membrane integrity functions. It was found that these two TolA functions could be segregated, thus emphasising the mechanistic differences between the two functions of TolA. Finally, the changes in gene expression of phage infected Escherichia coli, was monitored by global transcription analysis and it was demonstrated that several host genes were co-ordinately affected. In conclusion, these studies have provided a basis for the development of phage display technology, as well as insights into several different aspects of the infection process of filamentous phage.},
  author       = {Karlsson, Fredrik},
  isbn         = {91-7422-064-0},
  keyword      = {Biotechnology,Bioteknik,infection mechanism,selective infection,TolA,Escherichia coli,pIII,Filamentous phage,Phage display technology},
  language     = {eng},
  pages        = {136},
  publisher    = {Fredrik Karlsson Dept. of Immunotechnology P.O. Box 7031 SE-22007 Lund,},
  school       = {Lund University},
  title        = {The biology of filamentous phage infection - implications for display technology},
  year         = {2004},
}