Functional studies of purified transmembrane proteases, omptins, of Yersinia pestis and Salmonella enterica

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Title: Functional studies of purified transmembrane proteases, omptins, of Yersinia pestis and Salmonella enterica
Author: Lobo, Leandro
Contributor organization: University of Helsinki, Faculty of Biosciences, Department of Biological and Environmental Sciences, General Microbiology
Helsingin yliopisto, biotieteellinen tiedekunta, bio- ja ympäristötieteiden laitos
Helsingfors universitet, biovetenskapliga fakulteten, institutionen för bio- och miljövetenskaper
Publisher: Helsingin yliopisto
Date: 2006-10-19
Language: eng
Belongs to series: E-thesis - URN:ISSN:1795-7079
Thesis level: Doctoral dissertation (article-based)
Abstract: Surface proteolysis is important in migration of cells through tissue barriers. In the case of prokaryotes, surface proteolysis has been associated with invasiveness of pathogenic bacteria from the primary infection site into circulation and secondary infection sites in the host. This study addressed surface proteases of two important bacterial pathogens, Yersinia pestis which is the causative agent of the lethal systemic zoonosis, plague, and Salmonella enterica serovar Typhimurium which is an oral-faecal pathogen that annually causes millions of cases of gastoenteritis that may develop to septicaemia. Both bacterial species express an ortholog of the omptin family of transmembrane β-barrel, outer membrane proteases/adhesins. This thesis work addressed the functions of isolated plasminogen activator Pla of Y. pestis and the PgtE omptin of S. enterica. Pla and PgtE were isolated as His6-fusion proteins in denaturing conditions from recombinant Escherichia coli and activated by adding lipopolysaccharide (LPS). The structural features in LPS that enhance plasminogen activation by His6-Pla were determined, and it was found that the lack of O-specifi c chain, the presence of outer core oligosaccharide, the presence of phosphates in lipid A, as well as a low level of acylation in lipid A influence the enhancement of Pla activity by LPS. A conserved lipid A phosphate binding motif in Pla and PgtE was found important for the enhancement of enzymatic activity by LPS. The results help to explain the biological signifi cance of the genetic loss of the O-specifi c chain biosynthesis in Y. pestis as well as the variations in LPS structure upon entry of Y. pestis into the human host. Expression of Pla in Y. pestis is associated with adhesiveness to lamin of basement membranes. Here, isolated and LPS-activated His6-Pla was coated onto fluorescent microparticles. The coating conferred specifi c adhesiveness of the particles to laminin and reconstituted basement membrane, thus confi rming the intrinsic adhesive characteristics of the Pla protein. The adhesiveness is thought to direct plasmin proteolysis at tissue barriers, thus increasing tissue damage and bacterial spread. Gelatinase activity has not been previously reported in enteric bacteria. Expression of PgtE in S. enterica was associated with cleavage of porcine skin gelatin, denaturated human type I collagen, as well as DQ-gelatin. Purifi ed His6-PgtE also degraded porcine skin gelatin and human type I gelatin but did not react with DQ-gelatin, indicating that minor differences are seen in proteolysis by isolated and cell-bound PgtE. Pla was less effective in gelatin degradation. The novel gelatinase activity in S. enterica is likely to enhance bacterial dissemination during infection.
Subject: yleinen mikrobiologia
Rights: Julkaisu on tekijänoikeussäännösten alainen. Teosta voi lukea ja tulostaa henkilökohtaista käyttöä varten. Käyttö kaupallisiin tarkoituksiin on kielletty.

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