[BMB411 READINGS IN BMB 09/01/17] 1

Paper: Spaulding, et al. (2017). Selective depletion of uropathogenic E. coli from

the gut by a FimH antagonist." Nature 546:528-532 + supplemental online material and
an optional News and Views commentary.

Please fashion preliminary answers to the following questions before our class meeting
on Friday 10/13/17 and email me your responses (rmyers@miami.edu); this will help
direct your reading of the paper. Write your answers directly into this Pre-Discussion
File following each question; do not modify the file in any other way. Save your answers
to a file named exactly like this:

Pre-Discussion BMB411 Fall 2017 Paper Five YOUR NAME

(where YOUR NAME is your namee.g. RIK MYERS)

Send this file as an attachment to rmyers@miami.edu with the following subject heading
(exactly as written below):

Pre-Discussion BMB411 Fall 2017 Paper Five

After the meeting on 10/20/17, revisit your answers and modify them to suit your
evolved understanding. Mark your altered text using yellow highlights. Return these re-
answered questions via email to rmyers@miami.edu before 5 PM on Sunday 10/22/17.
Save the second set of answers as before, except replace Pre-Discussion with Post-
Discussion. Also send your email with the subject as I said before except again replace
Pre-Discussion with Post-Discussion. I want two sets of answers from you. If your
understanding remains the same after the discussion, submit your answers again with
the appropriate change to the file name. I use this information to see how your thinking
is altered by the in-class discussion.

Q1: What is the overall hypothesis of the paper?

- Targeting FimH with M4284, a high-affinity inhibitory mannoside, reduces
intestinal colonization of genetically diverse UPEC isolates, while
simultaneously treating UTI, without notably disrupting the structural
configuration of the gut microbiota.
Q2: In general terms, what is the plan Spaulding et al. came up with to test
their hypothesis in Q1? For example: Did they create mutants, knock-down
expression of a key gene and examine phenotypes, look for protein-protein
interactions, etc.
-Deletion of certain genes, creating mutants

Q3: What experimental techniques were used to carry out the plan in Q2?
- Red Recombinase system, immunofluorescence, phylogenetic analysis
and sequence alignment.
Q4: Why did the authors choose to use multiple E. coli UPEC isolates,
Caco-2 colorectal carcinoma cells, female C3H/HeN and female C57BL/6
mice? What unique features led the investigators to use these organisms? What
experimental limits did this choice of research material create?
- Eliminate male aggression, to examine effects of treatment across a wide
spectrum, treatment might be different in males due to higher
testosterone in males.

Q5: What were the overall conclusions from these studies?

The conclusions support the hypothesis.

Q6: What topic or question do you think the authors will take up next based
on this paper? What are the important next questions raised?
Can this affect the natural E.coli present in the digestive system.

Q7: Identify three different experiments that the authors used to come to the
conclusion you stated in answer to Question 5. State what you perceive to be a
limitation to one or more of these experiments that might weaken their
conclusions.

Q8: How do pili promote higher levels of UPEC in the gut? How do they
contribute (if at all) to UTIs?
Pili are adhesive and allow the bacteria to attach (better?, not sure what
word to use) to the lining of the digestive tract.
Q9: What are the implications the restricted phylogenetic distribution of
F17-like pili genes in E. coli?
-E.coli would be less able to cling to digestive lining (no adhesive protein
expression in pili)

Q10: The discovery and application of antibiotics has greatly extended

human lifespan and improved quality of life. Antibiotics select against sensitive
bacteria and enriches for resistant bacteria that repopulate the niche in a treated
individual. Briefly compare the mechanism by which M4284 works to the way a
bacteriacidal antibiotic works. Do you anticipate that M4284-resistant bacteria will
emerge and repopulate the gut of treated individuals? Answer this question in
comparison to emergent antibiotic resistance.
It probably will, in one way or another. Natural selection will always eliminate
the susceptible individuals, causing populations to be better adapted.