A group of researchers at the university veterinary diagnostics laboratory isola

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A group of researchers at the university veterinary diagnostics laboratory isolated a new bacterium from the blood and stool of several horses that became ill and died at a local farm. Prior to death, symptoms included disorientation, loss of motor function, and flaccid paralysis, so the researchers suspected central nervous system involvement and possible production of neurotoxin. Based on 16S rRNA comparison, they found that the new bacterium was related to the gram-positive bacterium Clostridium botulinum, and they named it Clostridium equiniae. The researchers subsequently developed a mouse model of infection, in which bacteria are injected into the bloodstreams of mice, and the mice are monitored for paralysis and death. Using this animal model, they have conducted signature-tagged mutagenesis to identify genes involved in virulence. From these studies, they have isolated three avirulent mutants (CeMut1 through CeMut3) that have genes encoding putative virulence factors deleted. They have conducted a series of experiments to determine the role of each of these virulence factors in pathogenesis. Interestingly, the researchers subsequently found that CeMut1, while avirulent, still produces and secretes the proteins encoded by the genes deleted in CeMut2 and CeMut3. Sequence comparison with protein databases revealed that the gene deleted in CeMut2 encodes a protein of 50 kDa and has significant sequence similarity to the catalytic N-terminal domain of botulinum neurotoxin (therefore, they named this protein BltA for botulinum-like toxin A part). The gene deleted in CeMut3 encodes a protein of 100 kDa and has significant sequence similarity in its N terminus to cholesterol-dependent cytolysins and in its C terminus to the B subunit of cholera toxin (therefore, they named this protein CptB for cytolysin plus toxin B part). Based on this, they suspect that the two proteins BltA and CptB form an A-B-type neurotoxin and decide to test this idea. They make antibodies against the culture filtrate from wild-type C. equiniae and then use the antisera in Western blots to compare the localization of the putative toxin proteins in the wildtype and CeMut1 bacteria and the neuronal cells treated with each bacterium. The results are shown in Figure 2.

A. Provide a detailed interpretation (with rationale) of the results shown in Figure 2. Were the researchers correct in their hypothesis? Provide your rationale.

B. Provide (with rationale) a possible function for the protein encoded by the gene deleted in CeMut1. Provide an experiment that could be performed to confirm your prediction.

C. Draw a clearly labeled schematic diagram depicting a possible model that accounts for all of the experimental observations, i.e., show how BltA and CptB are delivered to host cells and show the mode of action of the putative toxin on neuronal cells. Be sure your model is consistent with your answers to parts A and B.

Figure 2 Western blot of cellular fractions from the wild type (left) and CeMutl (right) using poly- clonal antibodies against secreted Bacterial Culture Neuronal cells medium cells proteins from C. equiniae. 000 kDa 0 kDa Bacterial Culture Neuronal cell medium cells

Explanation / Answer

a) The left gel photograph suggests that wild type bacteria do not have virulent protein because we still get neuronal cells’ protein. If we look on right gel photograph, there is neuronal cells’ protein absent, which suggests that virulent protein from bacterial cells has performed the task. That means Mutant CeMut1 is responsible for generating an A-B-type neurotoxin, BltA-CptB.

b) That protein may haul function of neurotransmitters. Neurotoxins paralyze patients by decreasing the effect of neurotoxins. That can proof by performing invivo membrane potential experiment.

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