Project leader: Professor AIRI PALVA,
Faculty of Veterinary Medicine, P.O.Box 66, 00014 University of Helsinki; +358 9 191 57058; airi.palva@helsinki.fi

Researcher:
Dr. Erja Malinen, Faculty of Veterinary Medicine, University of Helsinki

Results
Importance of the gut microbiota for the host physiological status and health has become generally acknowledged but molecular backgrounds underlying the mucosal host-microbe interactions are poorly understood. Despite being capable of distinguishing between self and nonself, the host tolerates the dense bacterial population present in the intestinal tract. Indeed, these indigenous bacteria are beneficial through affecting the nutritional status, maturation of immune system and colonisation resistance of the host. Host-microbe interactions in the gastrointestinal tract (GI-tract) and other mucosal surfaces are of importance also in many diseases of the host, since most pathogens use mucosal membranes as the portal of entry to the host tissues and as the first site of infection by triggering specific host responses, which result in altered cell functions that enable the bacterial translocation. The increasing knowledge of genome sequences is allowing more detailed studies on the determination of virulence factors of microbial pathogens and host responses to these pathogens. Probiotic organisms, on the other hand, have been claimed to possess many beneficial effects, such as increased resistance to infectious diseases, decreased duration of diarrhoea, reduction of allergy, modulation of immune system and adjuvant effects. The exact mechanisms behind these phenomena have remained largely unknown, and probiotic preparations are still widely used without knowing their real effects and targets in the gut. In principle, microarray analysis enables measurement of the gene expression changes taking place in host target cells, as well as gene expression of the bacterial counterpart. Host-microbe interaction studies, based on the use of DNA microarrays, are still lacking for most of the intestinal pathogens and even less is known about the effects of the normal intestinal microbiota on the gene expression of the host.

An abstract of the research plan (January 2003) 

In this project, we applied microarray technology to study gene expression patterns elicited in the host intestinal cells upon exposure to pathogenic or normal microbiota bacteria. The ground work included optimization of binding conditions for enterohaemorrhagic Escherichia coli (EHEC) and a non-pathogenic, intestinal E. coli strain ECOR 9. Human cancer cell lines Caco-2 and HT-29, resembling morphologically intestinal epithelial cells, were used as model cell lines in the binding tests. Binding of bacteria on the surface of intestinal epithelial cells is needed to introduce gene expression changes in the host cells; therefore, binding conditions (culture media and phase, cell-bacteria ratio) of the test bacteria were first optimized with Caco-2 and HT-29 cells in order to achieve good genomic responses. The interaction assays were performed by incubating the cells and bacteria together under optimized conditions and by harvesting cell samples for RNA isolation from various time points. Control samples were collected from cell cultures not exposed to bacteria. Microarray hybridizations were performed using the Hum16K arrays provided by the Finnish DNA Microarray Centre. Preliminary results from the hybridizations indicate that exposure of Caco-2 cells to either of the tested bacteria resulted in gene expression changes. Many of the altered genes seem to be involved in cell signaling or protein synthesis. However, the observed expression level changes were small and must therefore be verified with other methods, such as the real-time RT-PCR. This work is still in progress.

During the project, E. Malinen also participated in a microarray project in the University of Wageningen Laboratory of Microbiology. The work involved analysis of the gene expression changes of bifidobacteria when a complex carbohydrate is added in the growth medium as a sole carbon source. Differentially expressed genes were searched using microarray hybridizations as a tool to locate genes involved in the carbohydrate metabolism. During her visit in Wageningen, E. Malinen participated in isolation of RNA samples from bifidobacterial cultures and making of the microarray hybridizations. The work is still being continued in Wageningen. 

This project gives highly valuable insight into the expression level interactions occurring between intestinal bacteria and the host epithelial cells. We expect to find differences in the host gene expression patterns depending on the bacteria and host cell lines studied. Also, close contact with the host cells is expected to bring forth bacteria-specific responses. On the other hand, we believe that some expression patterns, regarding both host cells and the bacteria, are of universal nature. Comparison of different bacteria types (pathogenic, normal microbiota or probiotic bacteria) enables subtraction of truly specific responses from the general background data. Based on the results obtained, further interaction studies will be designed to search the mechanisms of host - microbe interactions at the functional level. In the future, we aim to shift the focus of our research towards analysis of bacterial gene expression changes, especially regarding the genetic background of the beneficial effects described for probiotic bacteria.