M. Sc. Thesis
Matej Kociper (Author), Andreja Nataša Kopitar (Reviewer), Marjanca Starčič Erjavec (Mentor), Reinhard Würzner (Co-mentor), Alojz Ihan (Co-mentor)

Abstract

Escherichia coli (E. coli) is a Gram-negative motile facultative anaerobic bacterium. It is part of the normal intestinal microbiota in humans and other warm-blooded animals. Usually, it has a mutualistic relationship with its host, but some E. coli strains have acquired specific virulence factors and can cause a variety of infections. E. coli strains involved in extraintestinal infections are known as extraintestinal pathogenic E. coli (ExPEC). Such E. coli strains have characteristic virulence factors involved in adhesion to epithelia, crossing of the body barriers, iron uptake and resistance to the immune system. In the study we investigated whether E. coli strains from the healthy human gut microbiota that have ExPEC virulence factors are able to trigger complement system response. Lipopolysaccharide (LPS) structure was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining. Selected E. coli strains had smooth LPS profile, except for the strains BJ33, MG1655 and DH5α that were rough. LPS profile of the studied E. coli strains did not necessarily correlate with the strength of the complement response. Smooth strains of bacteria were very resistant to complement lysis, even if they were detected by the complement (determined by binding particle C7), and rough strains were complement sensitive. The outer part of O-core in LPS was determined with specific polymerase chain reaction (O-core PCR). Most of the studied strains exhibited R1 type of the outer O-core region. Further, it was assessed, if E. coli strains differ in their ability to activate complement system (binding of complement particle C7), and binding of specific complement regulatory protein factor H to the surface. Specific IgG antibodies against factor H or particle C7 were bound to E. coli strains and the indirect immunofluorescence analysis was performed on the flow cytometer. E. coli strains were capable of triggering the complement activation in different intensity, some strains even to the extent of uropathogenic strains. Some of the E. coli strains could be able to avoid the immune system activation by binding regulatory factor H. To our knowledge, this is the first research revealing different complement factor H binding abilities of E. coli strains.

Keywords

Escherichia coli;lipopolysaccharides;O-core;complement system;complement regulatory factor H;complement particle C7;SDS-PAGE;polymerase chain reaction;flow cytometry;indirect immunofluorescence;

Data

Language: English
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL BF - Biotechnical Faculty
Publisher: [M. Kociper]
UDC: 579.23:577.27.083.3
COBISS: 4946040 Link will open in a new window
Views: 846
Downloads: 413
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Other data

Secondary language: Slovenian
Secondary title: Struktura lipopolisaharida in odgovor komplementnega sistema na izbrane seve bakterije Escherichia coli
Secondary abstract: Escherichia coli (E. coli) je po Gramu negativna gibljiva fakultativno anaerobna bakterija. Je del normalne črevesne mikrobiote ljudi in drugih živali s stalno telesno temperaturo. Običajno je v mutualističnem odnosu s svojim gostiteljem, vendar so nekateri sevi pridobili specifične virulentne dejavnike in lahko povzročijo raznotere okužbe. Sevi E. coli, ki povzročajo zunajčrevesne okužbe, so poznani kot zunajčrevesni patogeni sevi E. coli (ExPEC). Ti sevi imajo značilne virulentne dejavnike, ki bakterijam omogočajo vezavo na epitelij, prehod telesnih pregrad, privzem železa in izogibanje imunskemu sistemu gostitelja. V študiji smo raziskovali, če sevi E. coli iz črevesne mikrobiote zdravih ljudi, ki imajo virulentne dejavnike, značilne za ExPEC, aktivirajo komplementni sistem. Struktura lipopolisaharida preučevanih sevov je bila določena z natrijev dodecilsulfat-poliakrildamidno gelsko elektroforezo (SDS-PAGE) in barvanjem s srebrom. Izbrani sevi E. coli so imeli gladki profil LPS, razen sevov BJ33, MG1655 and DH5α, ki so bili hrapavi. Lipopolisaharidni profil raziskovanih sevov ni vedno koreliral z jakostjo komplementnega odziva. Gladki sevi so bili zelo odporni proti lizi s komplementom, čeprav jih je komplement zaznal (določeno z vezavo odlomka C7), medtem ko so hrapavi sevi bili občutljivi za komplement. Zunanji del O-sredice v LPS je bil analiziran z verižno reakcijo s polimerazo, specifično za O-sredico (PCR za O-sredico). Večina raziskovanih sevov je imela zunanjo O-sredico tipa R1. Nadalje smo preverjali, če se sevi E. coli razlikujejo v sposobnosti aktivacije komplementnega sistema (vezava odlomka C7), in vezavi specifičnega komplementnega regulacijskega proteina faktorja H na površino. Specifična IgG protitelesa proti faktorju H in odlomku C7 so bila vezana na seve E. coli in s pretočno citometrijo izvedena analiza indirektne imunofluorescence na pretočnem citometru. Sevi E. coli so bili sposobni aktivirati komplement v različni jakosti, nekateri celo do jakosti značilne za uropathogene seve. Nekateri analizirani sevi E. coli bi se lahko bili sposobni izogniti aktivaciji komplementnega sistema z vezavo regulatornega faktorja H. To je prva raziskava, ki je pokazala različne vezavne sposobnosti komplementnega faktorja H pri E. coli sevih.
Secondary keywords: Escherichia coli;lipopolisaharidi;O-sredica;komplementni sistem;komplementni regulacijski faktor H;odlomek komplementa C7;SDS-PAGE;verižna reakcija s polimerazo;pretočna citometrija;indirektna imunofluorescenca;
Type (COBISS): Master's thesis/paper
Study programme: 0
Thesis comment: Univ. v Ljubljani, Biotehniška fak., Študij mikrobiologije
Pages: XII, 73 f., [8] f. pril.
ID: 10959425