Describe how you would verify that the putative virulence factors identified by your method are indeed involved in pathogenesis. 

Gene Therapy

Chapter questions about microbiology

The Power of Bacteria

The genomes of a number of bacterial pathogens have now been completely sequenced. How might the availability of this information affect the definition of virulence factors?

Why are in vivo expression technologies continuing to turn up housekeeping genes rather than the “virulence genes” the researchers who developed these methods originally envisioned?

If you have the genome sequence of a bacterial pathogen, would you still need to clone genes or does cloning become obsolete?

Suppose you perform an RNA-Seq experiment comparing RNA samples prepared from a bacterium isolated from the blood of an infected animal to that grown in rich tissue culture medium in vitro. You detect changes in the relative transcript amounts of several genes for the bacterium grown in blood versus culture medium.  How would you interpret the results?  What does it tell you about the virulence of this bacterium?

Solving Problems in Bacterial Pathogenesis

You are a researcher working for the USDA. After two years of effort, you have isolated in pure culture a new, highly virulent bacterium from duck feces that is responsible for several major outbreaks of death in mammalian wildlife from contaminated pond water in the South.

Based on 16S rRNA sequence comparison, you have determined that this new bacterium is distantly related to the Gram-negative bacterium, Vibrio cholerae and you have named this strain Vibrio birdsii.  You have subsequently determined that V. birdsii is sensitive to tetracycline (i.e., it cannot grow in its presence). But is resistant to ampicillin.  You obtained a Vibrio plasmid that you then used to construct a suitable E. coli. shuttle vector for V. birdsii which you could use for genetic manipulation in E. coli and then transfer into V. birdsii by transformation.

You have also determined that guinea pigs which have good, innate immune systems, are an excellent animal model for the disease, which results primarily from uptake through drinking contaminated water, followed by colonization of the gut and bloody diarrhea, followed by invasion of intestinal cells with spreading to lymph nodes and the spleen, and then death by dehydration and/or organ failure.

You are interested in identifying virulence factors associated with disease in guinea pigs.  You have decided to use Tn-Seq as a strategy to identify these virulence factors.  After infection and harvesting of the intestines, spleen, and lymph nodes of the guinea pigs, you have identified 10 genes that are likely involved in growth and another 6 that are likely involved in virulence.

A

Draw a diagram of the shuttle vector and transposon that you constructed.  Be sure to label all the key features (genes, promoters, etc.) that are necessary for them to work for V. birdsii in order to use in your studies.

How do you interpret the results from the initial screen?

Describe how you would verify that the putative virulence factors identified by your method are indeed involved in pathogenesis.  Be sure to state what specific criteria must be satisfied.

From infected rabbits, you have isolated a new, highly virulent Gram-positive bacterium related to Listeria monocytogenes, which you have named Listeria leporine. Pathogenic findings are most prominent in the intestine and spleen.  Clinical signs are generally mild or absent in adult, healthy animals, but you find that young and old animals have symptoms of increased thickness of the lining of the gut due to proliferation of epithelial cells and swelling of the lymph nodes and spleen.  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 of L. leporine.  But, because the results are rather urgent, you decide to use the available genome sequence of the related L. monocytogenes to conduct a comparative genome analysis.  Describe how you would go about conducting this study.  Be sure to include all of the reagents and steps you would need to accomplish this task.

From your comparative genome analysis, you identified seven genes encoding putative virulence factors, which have been 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 containing four genes, the gene llp5 was part of an operon containing five genes, and the other two genes (llp6 and llp7) were found on separate single-gene operons. Describe in detail how you would verify using a young rabbit infection model that those seven encode actual virulence factors.  Be sure to state the criteria that would need to be satisfied.

Be sure to include a description of all the reagents, conditions, and experimental procedures you would need to use, as well as your rationale.

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 would perform to determine this.