Analysis of gene expression

BACKGROUND

Bacterial infections have serious impact on human- and animal health and welfare. To discover and develop new therapies there is a need for a more detailed knowledge of the fundamental molecular processes occurring in both host and pathogen during infections. Furthermore, a thorough understanding of the pathogenic variation between different strains (e.g. different serotypes) within the same bacterial species is essential.

The pathogenesis of bacterial infections is the combined results of prokaryotic gene expression and the gene expression of the host but many details of this interplay are still unknown. In many pathogenic bacteria, significant variations in virulence can be observed among different subpopulations (e.g. serotypes and genetic lineages) within the same species. This indicates that some serotypes may possess a higher virulence potential to infect eukaryotic organisms and cause disease than other serotypes. The genetic mechanisms behind these variations in virulence are largely unknown, and non-virulent strains often contain many of the virulence genes required for an infection.

Porcine pleuropneumoniae caused by Actinobacillus pleuropneumoniae (Ap) is an important health problem in the modern pig production with severe effects on animal welfare, ecology, and economy. This is in part due to difficulties in diagnosis, lack of knowledge of infection routes and lack of efficient epidemiological tools. Until now, 15 different serotypes of Ap have been described based upon differences in the capsular polysaccharides of the bacterium. The virulence of different serotypes of Ap has been experimentally determined and the difference in mortality and morbidity is considerable.

The overall hypothesis in this study is that subpopulation-specific virulence factors do exist and are reflected in the expression profiles obtained from both host and pathogen during an infection. Based on in vivo infections of pig by pathogen strains of low and high virulence, the link between bacterial virulence, genotype and gene expression will be determined and a correlation to the host expression “phenotype” and other host factors will be established. This will enable development of a new class of tools for control of infectious diseases in Denmark.

OBJECTIVES

The major objective of this project is to provide basis for the development of effective therapies against microbial infections currently affecting human and animal health. This will be achieved by an analysis of the molecular processes in vivo in both host and pathogen during infection. The porcine lung disease pleuropneumonia caused by Actinobacillus pleuropneumoniae (Ap) is used as a model for bacterial infection in a mammalian host.

Based on in vivo infections of pigs by pathogen strains of low and high virulence, the link between bacterial virulence, genotype and gene expression will be determined and a correlation to the host expression “phenotype” and other host factors (e.g. genetic background, immunological status, physiological status) will be established. This will enable development of new tools for control of infectious diseases in mammals. The specific objectives are:

1. Characterise two serotypes of Ap by full genome sequencing and annotation
2. Design and produce full genome microarrays targeting Ap strains of both high and low virulence.
3. Analyse the span of genetic variation between Ap strains of different serotypes by comparative genomic hybridisation and sequence analysis of variable regions.
4. Conduct in vitro infections of porcine lung epithelial cells with two serotypes of Ap and study the interplay between pig and Ap cells during infection and transcriptional variation between the two serotypes.
5. Conduct in vivo experimental infection of pigs with two serotypes of Ap and study the molecular processes occurring simultaneously in both pig tissues and Ap by microarray expression profiling and real-time quantitative PCR.
6. Combine the results and describe the fundamental molecular processes occurring in both host and pathogen during infections and thereby provide a scaffold for the development of effective therapies against microbial infections in mammals.

CURRENT STATUS OF PROJECT

Full genome sequencing has been performed for the Ap serotypes 2 and 6, annotation is in progress.

A pangenomic array targeting Ap serotypes, 1, 2, 3, 5, 6 and 7 has been designed and the first hybridisation experiments are under performance.

Two in vivo infection experiments with Ap serotype 2 and 6 has been conducted. Each experiment included 27 pigs.

PROJECT PARTICIPANTS

National Veterinary Institute-Technical University of Denmark:

Mette Boye, Ph.D, Professor, project manager

Kerstin Skovgaard, Senior Scientist

Kirstine Klitgaard Schou, Ph.D, senior scientist

Øystein Angen, Ph.D, senior scientist

Anastasia Ibrand, technician

Faculty of Agricultural Sciences, University of Aarhus:

Christian Bendixen, Ph.D. Research professor and Chief Scientist

Jakob Hedegaard, Ph.D. Project scientist

Frank Panitz, Ph.D, senior scientist

Henrik Hornshøj, Ph.D-student