Establishment of a high content assay for the identification and characterisation of bioactivities in crude bacterial extracts that interfere with the eukaryotic cell cycle.

High content microscopy as a screening tool to identify bioactive agents has provided researchers with the ability to characterise biological activities at the level of single cells. Here, we describe the development and the application of a high content screening assay for the identification and characterisation of cytostatic bioactivities from Myxobacteria extracts. In an automated microscopy assay Sf9 insect cells were visualised utilising the stains bisbenzimide Hoechst 33342, calcein AM, and propidium iodide. Imaging data were processed by the ScanR Analysis-software to determine the ploidy and vitality of each cell and to quantify cell populations. More than 98% of the Sf9 cells were viable and the culture consisted of diploid ( approximately 30%), tetraploid ( approximately 60%), polyploidic (<10%) and apoptotic (<5%) cells. Treatment with the reference substances blasticidin, colchicine, paclitaxel, and cytochalasin D induced changes in ploidy and vitality, which were characteristic for the respective bioactive substance. Furthermore, crude extracts from the chivosazole producing Myxobacterium Sorangium cellulosum So ce56 induced an increase of polyploid cells and a decrease in total cell count, while a mutant producing nearly no chivosazole triggered none of these effects. Purified chivosazole induced the same effects as the wild type extract. Similar effects have been observed for the reference compound cytochalasin D. On the basis of this assay, crude extracts of ten different Myxobacteria cultures were screened. Three extracts exhibited strong cytotoxic activities, further five extracts induced weak changes in the ploidy distribution, and two extracts showed no detectable effect within the assay. Therefore, this robust assay provides the ability to discover and characterise cytotoxic and cytostatic bioactivities in crude bacterial extracts.

Endocytosis of Xanthomonas campestris pathovar campestris lipopolysaccharides in non-host plant cells of Nicotiana tabacum.

The specific recognition of phytopathogenic bacteria by plant cells is generally mediated by a number of signal molecules. The elicitor-active lipopolysaccharides (LPS) of the phytopathogenic bacterium Xanthomonas campestris pv. campestris (X.c.c) are recognized by its non-host plant Nicotiana tabacum (N.t.). This LPS was purified and labelled with fluorescein isothiocyanate (FITC) for monitoring the fate of these signal molecules in intact plant cells of tobacco. In this study we were able to show that the so-labelled LPS rapidly bound to the cell wall and was then internalized into the cells in a temperature- and energy-dependent way. This uptake of LPS could be outcompeted by the addition of an excess of unlabelled LPS. Furthermore, it was blocked by amantadine, an inhibitor of receptor-mediated endocytosis of mammalian cells. Immunolocalization experiments showed for the first time a significant co-localization of the LPS-elicitor with endosomal structures using an anti-Ara6 antibody. These observations suggest specific endocytosis of LPS(X.c.c.) into tobacco cells. The possibility for a receptor-mediated endocytosis comparable to the mammalian system will be discussed.