Folding@home

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1. Bacteria reproduce by binary fission which produces clones. Offspring are genetically identical to their parent cells so if a parent is resistant and there are no random mutations occurring, the offspring will inherit the resistance and will pass it to all of their offspring. If there is a selection pressure for the resistance, those that already have it will survive and continue to pass it on. Bacteria are also able to exchange genetic material through a sex pilus in a process called conjugation. If this occurs, bacteria might gain resistance without inheriting it but would then pass it to any offspring clones it produces. It is also possible that bacteria could gain resistance by random mutation if the mutation occurs in the location of the bacteria that the antibiotic is targeting. If this occurs, the mutation would again be passed on to any offspring.

2. Bacteria reproduce by binary fission, which is an extreme form of inbreeding because all offspring are clones of their parental cells. If a colony of bacteria is treated with antibiotics, all of the bacteria will die unless there happens to be a few that are previously resistant. Once the non-resistant bacteria have been killed, only the resistant ones are left. If they are able to reproduce, then the entire colony, once it has been re-built, will then be resistant to that drug. In this situation, resistance has been passed on to all the given bacteria very quickly.

What We've Learned

• I think that it is interesting that by prescribing antibiotics that doctors can be killing patients ribosomes and in the long run making patients resistance to the antibiotics. Doctors can also be causing mutations when in fact they are attempting to treat the patient. Also doctors try to educate people about mutations which occur with UV rays as in tanning and try to “speak out” against it but may also be contributing to it.

• Although I’ve heard of diseases being caused by protein misfolding, and antibiotics that interfere with protein folding, I had never made any sort of connection between the two. It’s interesting to think that something potentially dangerous to humans (antibiotic resistance) could lead to a better understanding and ultimately, treatment, of other diseases. It’s scary to think about how bacteria is slowly becoming resistant to antibiotics, and one day we might not be treat certain bacterial infections. To me, it’s the most easily observable example of natural selection today.

• Something I learned and was interesting to me was, that not all bacteria are defenseless against the antibiotic producers. Many possess genes that encode proteins to neutralize the affects of antibiotics and prevent attacks on their cell machinery. These specific proteins protect ribosomes by binding them and changing their shape or conformation. The change in the ribosome shape prevents an antibiotic from binding and interfering with protein synthesis. So in essence they have their own way to resist antibiotic pressures and tactics in dodging the bullet.
  
  Thoughts for group:
  Doctors need to be aware of evolution especially because of antibiotic resistance. Doctors need to understand that if they over-prescribe antibiotics (e.g. for viral infections), they can be causing a person more harm that may not respond to antibiotics when they need it. Also, they need to be able to explain the importance of patients taking all of their antibiotics or else they might only knock out the less resistance bacteria.

Questions

1. Given this information and what you’ve learned from your project, explain why it might be important to study antibiotic resistance by looking at protein folding and mis-folding.

Translation occurs within a ribosome. Protein folding often begins at the N-terminus while the rest of the mRNA is still being translated. Since the ribosomes of bacteria are structurally different from those found in eukaryotic cells, antibiotics often target ribosomes which can inhibit protein synthesis of the bacteria, ultimately killing it. If bacteria are becoming resistant to antibiotics, there must be something occurring to stop the inhibition of the ribosome. Antibiotics can also lead to protein mis-folding, which would mean that antibiotic resistance is affecting correct folding (somewhat indirectly). If there are diseases that are caused by protein misfolding, perhaps studying antibiotic resistance would provide insight into how to treat these diseases.

2. Why would the sequences of the NBD₂ determinants (prokaryotic) be similar to the eukaryotic ABCF subfamily?

This suggests that these proteins have similar functions. Since the ABCF subfamily of proteins has been shown to be associated with ribosomes, it is logical that the NBD₂ proteins may also associate with ribosomes (to confer antibiotic resistance) although this has not been currently demonstrated.

3. Even the mechanism of the antibiotic resistance determinants comes down to proteins (or the cessation of their production). Which of the alternative hypotheses expressed in this paper (see text and Figure 1) do you consider the best explanation for this mechanism?

We believe that the ‘cuckoo’ hypothesis is currently the best explanation. It has the most direct evidence to support it. One study using co-localization experiments showed Vga(A) to be associated with the cell membrane. Another study showed a reduction of radiolabelled erythromycin when transformed by the NBD₂ encoding gene msr(A). The ‘ribosome protection’ hypothesis has only indirect evidence supporting it thus far.

1. The authors refer in the introduction to their capacity to “fine-tun[e] the selective pressures in ways not permitted by the cellular environment.” To what selection pressures might they be referring?

There are several pressures that they might be referring to. The first, and most relevant to this article, are unnatural substrates (such as antibiotics). Other selection pressures might be dramatically changed temperatures and a selection that deletes certain translation components. These pressures may not be possible to do in vivo but may give insight into what is occurring in living animals.

2. The discussion focuses on two mutations (one transition and one transversion) that confer resistance to the MLS family of antibiotics (the same family discussed in the other paper). How could these two little mutations change the ribosome sufficiently to confer antibiotic resistance?

These two mutations might seem insignificant but are capable of changing the 3-D structure of the ribosome. In this case, the mutations seem to alter the part of the ribosome to which the MLS family of antibiotics binds to inhibit the function of the ribosome, without altering the function of the ribosome itself. If the antibiotic can’t bind to the ribosome, the ribosome will continue to function, and antibiotic resistance has just occurred.

3. Why might the single-mutant ribosomes be less common in the population than the double-mutant ribosomes?

The authors suggest that there may be subtle differences in activity between the two mutants that cannot be seen in the in vitro assays. This seems to be the most likely explanation. It would be interesting to look the 3-D version of both mutant ribosomes. It is possible that the double-mutant is more conducive to antibiotic resistance or that the single-mutant is less likely to survive for some reason. Other possible explanations are that the double-mutant ribosome amplified better during PCR for some reason, a skewed original population, or that selection occurred during the induction period.

Interview

Our group interviewed Mark Clements M.D., PhD. at Children's Mercy Hospital, whom Kelli has worked closely with since Summer 2007 conducting research.

Q: What are some of the differences between mammal and bacterial ribosomes?

Dr. Clements: Bacterial ribosomes are smaller in size, they also have different sequence structures than mammalian ribosomes. They also differ in that they are not attached to endoplamsic reticulum in bacteria, they are free in the cytoplasm.


Q:What is the function of a ribosome?

Dr. Clement: The function of a ribosome is translation of messenger RNA into protein.


Q:What is a specific example of an antibiotic that targets ribosomes and its mechanism of action?

Dr. Clement: Azithromycin antiobiotic, which is used to treat certain bacterial infections, most commonly middle ear infections, tonsillitis, throat infections, and pneumonia. This antibiotic
blocks the peptide transferase activity of the ribosome.



Q:How else do antibiotics work, besdies trageting ribosomes?

Dr. Clement: Antibiotics also inhibit the production of the cell wall, inhibit DNA synthesis, block transcription, and also block folate production.


Q: How does anitbiotic resistance develop?

Dr. Clement: Resistance can develop in many ways: mution, acquired plasmid and natural defense.


Q: Antibiotics tend to be associated with only positive effects, are there any side effects of them?

Dr. Clement: There are side effects. To name a few there is: gastrointestinal upset, diarrhea, drug rash, allergy, electrolyte abnormalities and also weakness

Intro to Project

For our project, we have decided to use the Folding@home distributed computing site. http://folding.stanford.edu/English/Main According to the site, their goal is "to understand protein folding, misfolding, and related diseases." Proteins play many integral roles in our survival. As enzymes, they drive many biochemical reactions. As structural elements, they make up our bones, muscles, skin, hair, and blood vessels. They also function in the immune system as antibodies. To be functional as a protein, a string of amino acids must fold itself into a correct 3-D structure. Protein misfolding is believed to be the basis for many diseases. The website is geared towards understanding the proteins implicated in Alzheimer's disease, Huntington's Disease, cancer (including p53), Osteogenesis imperfecta, Parkinson's, and those targeted by antibiotics, specifically ribosomes. Each disease is described in the page http://folding.stanford.edu/English/FAQ-Diseases Our group is interested in learning more about the bacterial ribosome which is targeted by antibiotics. The initial picture is of a ribosome. It is from the above site.