Question

You notice that there is an increasing prevalence of a bacterial infection at your hospital that

appears to be affecting people. The bacterium has been named T. routmania, and

microbiologists have found that it is a gram negative organism that is highly resistant to typical

antibiotics. Immunologists have found that the organism appears to target T-cells in the host.

Analysis of lymphatic fluid from infected patients has found an unusually low level of IL-2, yet

tissue samples appear to have normal levels measured inside of cells. Based on this it is

concluded that IL-2 is somehow being affected by the bacterium

a. So the bacterium appears to be changing levels of IL-2 produced by cells in the tissue,

but nothing is known beyond that. You begin to ask yourself: How does the organism

interact with this human signaling molecule? Does it consume IL-2? Does it modify IL-2

in the medium making it undetectable by standard means? Propose an experiment that

would allow you to address these questions. Note that IL-2 is available in pure form as a

control and several antibodies are available that detect a variety of epitopes on the

surface of the protein. You are also able to culture T. routmania. Name your techniques

and diagram them out. Please make it clear precisely what controls you will use.

b. Next you ask a technician to perform the experiment that you should have proposed

above, and she finds that the organism appears to consume up to 20 ug of IL-2 when it

is added to 1 mL cultures of the organism. Can you tell me how you might have done

that quantification?

c. You tell the technician to lyse the cells and use a similar detection method to see if the

IL-2 is present inside the bacterium. The technician does this and tells you that using

one antibody it appears that it does still have intact IL-2 inside the bacterial cell, but

using another antibody the IL-2 is undetectable. The tech has an idea about why this is.

What do you think the tech would suggest which is different from what we see in (d)?

d. Next, she thinks that maybe there is a protein in the bacteria that binds IL-2. Maybe this

interaction induces the production of something else, maybe a toxin or something that

affects a host. Suggest a method and experiment for her that would allow her to

determine whether there is a protein from T.routmania that does interact with IL-2.

Don’t forget to say how you will know. Draw some diagrams on what the data will look

like, and explain how it works.

e. The tech finds that there is something between 40 and 80 kDa that interacts with IL-2

based on analysis of isolated fractions from the above experiment. She wonders what

this could possibly be. She then decides that she really should identify what this protein

is, as it could be the link between IL-2 and virulence of the organism. She needs to know

more about the stuff that she has to work with. She knows that the protein she is

interested in is in there, she wonders how many other things are in the samples and

what properties they may have. Based on this what are the simplest next steps?

Answer 1

a) Western blot is the technique we will use to check whether this

• Culture the cells as well as the bacterium
• Keep a control where we do not do the infection
• Infect the cells with bacterium and collect the cells after 24hr
• lyse the cells and collect the protein lysate
• Denature the sample and do a Western blot
• Use antibody against the IL2
• The difference in the bands from the control will let us know that after infection there is a change in the amount of IL2
• Further doing IP we can understand how it helps the bacteria in its virulence

The diagram for Western blot is given below

b) Elisa is the method used for quantification of the amount of IL2 after adding to the culture.

• Culture 2 sets of bacterial culture
• To one set add IL2 pure form and let the bacteria grow and let the other be the control
• After one generation time, centrifuge both sets of bacteria and collect the supernatant
• Plate the ELISA plate with the IL2 antibody
• Add the supernatant of both the cultures to the antibody-coated plates
• Add substrate, detect the signal and quantify the protein using the standard curves

c) The reason could be the IL2 might be cleaved before taken into the bacterial cell. As different antibodies have different epitopes to bind, one of them could not bind to the IL2 cause that part of the IL2 protein would have been cleaved

d) We can do Immunoprecipitation of the IL2 protein to find its interacting proteins.

• Culture the cells as well as the bacterium
• Transfect the cells with tagged IL2 constructs
• Infect the cells with bacterium and collect the cells after 24hr
• lyse the cells and with the help of the tag present on the IL2 protein isolate them
• You will get the proteins interacting with IL2 in it
• Denature the sample and do a Western blot
• By analyzing the various bands present on the western blot from the control ( uninfected with bacteria) you can understand there might be proteins interacting with the IL2 from the bacteria

e) The simplest next step would be to do mass spec of the interacting proteins of IL2 from the previous experiments. This would help her to know what is that protein and also what are its properties.