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Research > Medical Microbiology and Hospital Epidemiology > WG Prof. S. Schubert > Research > 

A)    Horizontal Transfer and Evolution of the High-Pathogenicity Island (HPI)

The fyuA-irp gene cluster is definitely associated with virulence expression in highly pathogenic Yersinia species (Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica 1B). This 35-kb chromosomal DNA region meets all criteria of a pathogenicity island and it encodes for an iron uptake system mediated by the siderophore yersiniabactin.

Using PCR and southern hybridization techniques we could demonstrate that this Yersinia High-Pathogenicity Island (HPI) is distributed among extraintestinal pathogenic E. coli strains (ExPEC), predominantly in E. coli isolates from blood cultures and urine sample. By means of a bioassay we were able to detect the siderophore yersiniabactin in culture supernatants of HPI-positive isolates of E. coli and other Enterobacteriaceae grown under iron starvation. This indicates the functional conservation of the Yersinia-HPI in those strains and raises questions about the reason for this iron uptake system in presence of other high-affinity iron uptake systems (e.g. enterochelin). We could further demonstrate that the HPI contributes to virulence in ExPEC and shows the highest association with virulence in extraintestinal pathogenic E. coli. Ongoing studies are directed towards the exact mechanisms of HPI in the virulence process.

Diversification of bacterial species and pathotypes is largely due to horizontal transferof diverse DNA elements such as plasmids, phages and genomic islands (e.g. pathogenicity islands, PAIs). A PAI called high-pathogenicity island (HPI) carrying genes involved in siderophore-mediated iron acquisition (yersiniabactin system) has previously been identified in and Y. enterocolitica IB strains, and has been characterized as an essential virulence factor in these species. Strikingly, an orthologous HPI is a widely distributed virulence determinant among Escherichia coli and other Enterobacteriaceae which cause extraintestinal infections. The HPI of E. coli strain ECOR31 is distinct from all other HPIs described to date since the ECOR31 HPI comprises an additional 35-kb fragment at the right border compared to the HPI of other E. coli and Yersinia species. This part encodes for both a functional mating pair formation system and a DNA-processing region related to plasmid CloDF13 of Enterobacter cloacae. Upon induction of the P4-like integrase, the entire HPI of ECOR31 is precisely excised and circularized. The HPI of ECOR31 resembles integrative and conjugative elements termed ICE. It may represent the progenitor of the HPI found in Y. pestis and E. coli, revealing a missing link in the horizontal transfer of an element that contributes to microbial pathogenicity upon acquisition.

B)    Characterization of virulence factors and virulence associated factors of ExPECs

The aim of this project is to characterize yet unknown virulence-associated DNA fragments of extraintestinal pathogenic E. coli (ExPEC). Together with DNA derived from well characterized virulence determinants of UPEC, these fragments are used to design a DNA-microarray for diagnostic and epidemiological purpose. Moreover, the detection and characterization of unknown virulence determinants will give potential new targets for antimicrobial chemotherapy as well as for prevention strategies, e.g. vaccinations.
In order to isolate virulence-associated DNA fragments we performed the suppressive subtractive hybridization (SSH) technique. In brief, the genomic DNA of different clinical E. coli isolates obtained from patients with urinary tract infections or septicaemia was isolated and “subtracted” from the genomic DNA of the well characterized UPEC isolate CFT073. The resulting DNA-Fragments were sequenced and compared with nucleotide databases (GenBank, EMBL). Up to now, we were able to isolate 400 yet unknown DNA fragments, which were further characterized with regard to the association to virulence. For this, all 400 DNA fragments were labelled and used as probes for macroarray hybridization assay with a genomic DNA library of each 25 virulent and non virulent E. coli isolates. Out of the 400 unknown DNA-Fragments we could detect about 50 fragments with a clear cut relation to virulent E. coli isolates. For two of these fragments, we have already characterized the neighbouring DNA-regions by screening of a cosmid library a subsequent sequencing the entire border region. One of the fragments is located on a self transmissible plasmid and represents a part of a gene cluster with high homology to a virulence associated iron uptake system of E. coli. All unknown virulence associated DNA fragments will be included into a DNA-microarray for diagnostically typing as well as epidemiological investigations of clinical E. coli isolates.

C)    Host response to infections caused by extraintestinal pathogenic E. coli (ExPEC)

ExPEC infections of the urinary tract start with colonization of the bladder. It is known for several decades that adhesion molecules (e.g. type I fimbriae and P fimbriae) are involved in the attachment of ExPECs to urothelial cells. Recently, it has been shown that ExPEC exhibit an invasive phenotype, which leads to intracellular bacterial colonies (IBCs, “intracellular bacterial communities”). One project is dedicated to the characterization of ExPEC factors, which are involved in the invasive phenotype leading to formation of IBCs.

A second project regards ExPEC factors, which are produced during infection and subvert the innate immune response of the host. Previous studies of others have shown that ExPECs are able to modify or inhibit Toll-like receptor (TLR) mediated signalling pathways resulting in a decrease production of proinflammatory cytokines by host cells. Together with the group of Thomas Miethke (TU München) we could recently identify an ExPEC protein, which mimics the cytoplasmic TIR domain of TLRs leading to an inhibition of the MyD88 dependent signalling pathways of host cells.

D)    Clinical Microbiology

The clinical microbiology projects are directed towards the establishing of novel diagnostic methods, mostly based on molecular techniques (e.g. diverse real-time PCRs). Part of the work is dedicated to the molecular typing of clinical relevant bacterial isolates from patients’ samples in order to unravel nosocomial transmission (patient to patient transmissions) of infective agents.
A further project deals with the use of MALDI-TOF techniques (Matrix Assisted Laser Desorption/Ionisation - Time of Flight) analyses for rapid differentiation of bacterial and fungi isolates.