From infected rabbits, you have isolated a new, highly virulent gram-positive bacterium related to Listeria monocytogenes, which you have named Listeria leporine. Pathologic findings are most prominent in the intestine and spleen. Clinical signs are generally mild or absent in healthy adult animals, but you find that young and old animals have symptoms of increased thickness of the lining of the gut due to overproliferation of epithelial cells, and swelling of lymph nodes and spleen, and those animals often succumb to systemic infection, including brain lesions and death in about 70% of cases. You wish to better understand the pathogenesis of the disease and to identify potential virulence factors. However, because the need for these results is rather urgent, instead of developing a new approach, you decide to use an existing IVET approach based on reagents that have already been developed by other researchers for L. monocytogenes, which includes a temperature-sensitive plasmid (i.e., it integrates into the chromosome when the temperature is shifted from 37C to 42C for a brief time). The plasmid, which can be electroporated into L. leporine, contains an erythromycin resistance gene (ermr ) with a constitutively ‘‘on’’ promoter and a promoterless listeriolysin O gene, hly, downstream of a BglII site, but no other genes. From your IVET screening using a young-rabbit infection model, you identified seven genes encoding putative virulence factors, which you have named llp1 through llp7. Two of the genes (llp1 and llp2) were found adjacent to each other on a two-gene operon, the genes llp3 and llp4 were part of an operon consisting of four genes, the gene llp5 was part of an operon consisting of five genes, and the other two genes (llp6 and llp7) were found on separate single-gene operons.
A. Describe in detail how you identified those six genes as genes encoding putative virulence factors using this IVET approach. Be sure to include a description of all the reagents, conditions, and experimental procedures used, as well as your rationale. (Hint: it might be helpful to include one or more diagrams showing the plasmid with all of its features, indicating how the plasmid integrates into the chromosome, showing how you would distinguish between in vivo and in vitro virulence gene expression, and describing how you determined in which operons the genes were located.)
B. You would like to know at which point during the in vivo infection process each of these putative virulence genes gets turned on. Describe an experiment that you could perform to determine this.