Characterization of the fimbrial adhesin of type 3 fimbriae in Klebsiella pneumoniae

Stefanie Mørk Christensen

Studenteropgave: Speciale

Abstrakt

Klebsiella pneumoniae is an opportunistic pathogenic bacterial species. It is commonly associated with acquired hospital infections where it mostly causes pneumonia and urinary tract infection. Recently emergence of multidrug resistant K. pneumoniae has created a big problem in treatment of these infections which is reflected in increasing mortality. Several virulence factors contribute to the pathogenicity of K. pneumoniae. Among them, type 3 fimbriae mediate bacterial adherence to host surfaces thereby helping to maintain the infection. Type 3 fimbriae are also involved in formation of biofilm which is a problem in patients with indwelling devices. Biofilm is very difficult to treat because of its resistance toward both antibiotics and host immune response. However the specific receptor of type 3 fimbriae remains still unknown. Therefore, identification would provide valuable information regarding the binding of type 3 fimbria to the host. This would help in developing new antimicrobial agents, which could be used for treatment of K. pneumoniae infections. The aim of the thesis was to characterize the fimbrial adhesin of type 3 fimbria in K. pneumoniae in an attempt to identify the receptor of the fimbria. The fimbrial adhesin MrkD was expressed in E. coli BL21(DE3) cells and purified with Immobilizing Metal-ion Affinity Chromatography (IMAC). However, refolding of the protein after IMAC purification was not possible. Instead a type 3 fimbriae expressing E. coli strain was used to perform binding assays in order to examine the binding of MrkD to a variety of different ligands. MrkD was binding to all tested ligands with exception of type 1 fimbriae from E. coli, which is remarkable. The binding of MrkD to type 1 fimbriae were investigated further by examining the binding to different type 1 fimbriae variants. There was a difference in binding of MrkD to the different type 1 fimbria variants. To examine whether the structure of type 1 fimbriae was responsible for the reduced binding potency of MrkD, the fimbriae were treated with various agents that can potentially incur structural changes upon them, such as HCl, SDS and glycerol. Glycerol-treatment of type 1 fimbriae did not influence MrkD binding to the fimbria. However, an increase in MrkD binding was seen to HCl- and SDS-treated fimbriae. SDS-PAGE showed that HCl-treated fimbriae were depolymerized into monomeric subunit. Interestingly, the SDS-PAGE also showed that SDS did not depolymerize the fimbriae. Additionally, transmission electron microscopy of SDS-treated type 1 fimbriae showed no structural change of the fimbriae. Though, SDS has been shown to be bind to proteins and increase the negatively charge of the proteins. Therefore the increase in MrkD binding to SDS-treated fimbriae might be due to electrostatic interactions caused by the binding of negatively charged SDS to type 1 fimbriae. To examine whether the blocking potency toward MrkD binding was unique to type 1 fimbria, the binding of MrkD was examined on CFA/I and CS17 pili. It was found that CS17 pili had similar blocking potency to MrkD binding as type 1 fimbriae. Meanwhile CFA/I pili showed no blocking of MrkD binding. It seems that MrkD binding to the ligands is caused by electrostatic interactions. However, a lot still remains unknown about type 3 fimbriae and the receptor.

UddannelserMolekylærbiologi, (Bachelor/kandidatuddannelse) KandidatMedicinalbiologi, (Bachelor/kandidatuddannelse) Kandidat
SprogEngelsk
Udgivelsesdato12 feb. 2016
VejledereHåvard Jenssen & Karen Angeliki Krogfelt

Emneord

  • MkrD
  • Type 3 fimbriae
  • Klebsiella pneumoniae