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A Bacterial Protein as a Potential Therapeutic for Clostridioides difficile Infection (Presented by Dr. Prameet Sheth)

By Abbie Davis, MSc Candidate and TMED 801 Student

 

On Thursday, September 22nd our class, Profession of Medicine (TMED 801), was enthralled by Dr. Prameet Sheth’s medical grand rounds (MGRs) presentation. Dr. Sheth highlighted the roles of our bodies' bacterial communities (microbiota) and their genomes (microbiome) and his group’s work on a bacterial-derived protein that has the potential to translate into the treatment of humans with antibiotic-resistant bacterial infections.

It is widely recognized that the “good bacteria” in all our bodies play a role in competing with the disease-causing bacteria, and this balance is critical to human health.1 There are so many bacteria in your gut right now that experts do not even know what exactly the microbiome is made of.  Clostridiodes difficle (C. diff) is just one of these disease-causing bacteria that can cause problems from diarrhea and intestinal inflammation to whole-body infection (sepsis).1 Dr. Sheth mentioned that carrying C. diff is not enough to cause health disturbances, in fact, the asymptomatic establishment of C. diff colonies has been seen in 3–5% of healthy adults.2 Rather, the toxins (Toxin A and B) C. diff produces are the main culprits, which change the cell membrane, causing cell rounding and ultimately death.3

 

The key cause of C. diff disease is antibiotics, which disturb the balance of good-to-bad microbes. Infection is very difficult to treat, and reoccurrence of the disease is strikingly common (25-30%), which is largely owing to the spores, which provide a stable coating around the bacteria that can allow it to live for months or even years in even the harshest environments.4 A treatment called fecal microbiota transplantation has disease resolution rates of 85%–90% after just one treatment.4,5 Fecal transplants, as Dr. Sheth describes, “re-poop-ulate” the microbiota back to its natural state. However, there are two major limitations. The first involves the strict guidelines for fecal donors, who must pass a series of blood and stool tests6 in addition to not being able to travel. It is not a surprise that many donors fail this stringent screening.

 

To overcome these limitations, a brilliant paper aimed to administer a sterile fecal filtrate, which was described by Dr. Sheth as a “bacteria cocktail”. This led to the development of a defined microbial community that is now called SER-109.7 Despite the obvious efficacy of both fecal transplantations and SER-109, there is still a second limitation to these treatments; an unknown mechanism.

 

Cue doctor Sheth and his lab, who have been working tirelessly on the mechanism of a bacteria-derived protein called High-temperature Requirement A protein (HTRA-1). Dr. Sheth created a microbiota cocktail consisting of 18 bacterial members and measured their ability to preserve mouse weight, reduce cell rounding, and decrease toxin levels.  Of the 18, four were able to protect the mouse against Toxins A and B, and Parabacteroides distasonis was selected as the best target. Following a series of tests, Dr. Sheth determined that the bacteria was releasing a protein that was able to break down the toxin. Therefore, he separated the proteins and using mass spectroscopy and functional prediction techniques, Dr. Sheth was able to narrow down the protein to 9 candidates. One of these candidates, HTRA-1, was put into Escherichia coli, which reproduces at a much greater rate than Parabacteroides distasonis. Ultimately, Dr. Sheth found that HTRA-1 was able to break down more toxins with increased amounts, and that cell rounding was decreased. HTRA-1 was effective in both in virto and in vivo models. Further, HTRA-1 is a post-translational protein; therefore, this treatment target would not put the selective pressure on bacteria that results in resistance.

 

Dr. Sheth described the limitations of his research, which involved the use of a mouse model. The course of the disease is much more aggressive in mice and Dr. Sheth was not confident that the oral administration of the protein would withstand the human stomach. Despite this, to our best knowledge, Dr. Sheth and his group’s research was the first to show a mechanism by which the microbiota can restore and protect against C. diff. We are excited to see what is to come of the remaining 8 proteins that need to be investigated.

 

Following MGRs the TMED 801 class discussed with Dr. Sheth his work in the lab and as a medical microbiologist. A large area of discussion was the overuse of antibiotics for various treatments; it was reassuring to hear this issue is being combatted by medical microbiologists like Dr. Sheth, who ensure doctors are properly prescribing antibiotics. We also discussed Dr. Sheth's diverse background, from education to his time in the industry, and now, in academia. Dr. Sheth's key message to TMED students was “Don’t be afraid to make mistakes”.

 

Thank you, Dr. Sheth, for the engaging lecture and discussion.

 

References:

 

1.         Sehgal K, Khanna S. Gut microbiome and Clostridioides difficile infection: a closer look at the microscopic interface. Ther Adv Gastroenterol. 2021;14:1756284821994736. doi:10.1177/1756284821994736

2.         Sheth PM, Douchant K, Uyanwune Y, et al. Evidence of transmission of Clostridium difficile in asymptomatic patients following admission screening in a tertiary care hospital. PLOS ONE. 2019;14(2):e0207138. doi:10.1371/journal.pone.0207138

3.         Di Bella S, Ascenzi P, Siarakas S, Petrosillo N, di Masi A. Clostridium difficile Toxins A and B: Insights into Pathogenic Properties and Extraintestinal Effects. Toxins. 2016;8(5):134. doi:10.3390/toxins8050134

4.         van Nood E, Vrieze A, Nieuwdorp M, et al. Duodenal Infusion of Donor Feces for Recurrent Clostridium difficile. N Engl J Med. 2013;368(5):407-415. doi:10.1056/NEJMoa1205037

5.         Ott SJ, Waetzig GH, Rehman A, et al. Efficacy of Sterile Fecal Filtrate Transfer for Treating Patients With Clostridium difficile Infection. Gastroenterology. 2017;152(4):799-811.e7. doi:10.1053/j.gastro.2016.11.010

6.         Bibbò S, Settanni CR, Porcari S, et al. Fecal Microbiota Transplantation: Screening and Selection to Choose the Optimal Donor. J Clin Med. 2020;9(6):1757. doi:10.3390/jcm9061757

7.         McGovern BH, Ford CB, Henn MR, et al. SER-109, an Investigational Microbiome Drug to Reduce Recurrence After Clostridioides difficile Infection: Lessons Learned From a Phase 2 Trial. Clin Infect Dis. 2021;72(12):2132-2140. doi:10.1093/cid/ciaa387

 

Comments

Name
Isaac Emon

Tue, 09/27/2022 - 15:18

Hi Abbie,

Well done! You provide an excellent review of Dr. Sheth's talk and the current work that is being done on C. diff in his lab. These novel protein candidates are super exciting, specifically the HTRA-1, though it's understandable that research in mice is not directly translatable to research in human patients. Hopefully research on these other 8 proteins can help us gain a deeper understanding of the mechanisms by which the bacteria help protect from Toxins A and B. In your opinion, do you think that the proteins that prove to be effective could be used synergistically in some sort of combination therapy to target C. diff? Would there be any concerns with that? Further, how could we develop a delivery method to help these proteins withstand our stomach pH? A study published this spring in nature identified polyzwitterionic complexes, or pZCs, as a new oral drug delivery method, which withstands the stomach but doesn't break down until the intestine (1). Do you think we could identify a way to release these proteins in the stomach directly from the pZCs to C. diff with little pH stomach exposure? Interested to hear your thoughts!

Isaac

Reference paper:

Margossian, K. O., Brown, M. U., Emrick, T., & Muthukumar, M. (2022). Coacervation in polyzwitterion-polyelectrolyte systems and their potential applications for gastrointestinal drug delivery platforms. Nature communications, 13(1), 1-11.

Name
Isaac Emon

Hi Isaac,

Thank you for the kind words and for your insight!

I think Dr. Sheth's results are exciting, and we should be optimistic about the future of C. diff treatment. Unfortunately, Dr. Sheth’s results have not been published yet, so it is difficult to comment on the potential synergy of HTRA-1 protein with other proteins without the context of their composition and targets. I can mention, however, that these proteins come from the same “bacterial cocktail”. As we saw in fecal transplants and SER-1091,2, these treatments also used whole microbiota communities and were very effective. I think since Dr. Sheth’s proteins all come from the same community, it can be inferred that at least some of these proteins work together and provide a synergistic effect. This is exciting because this research could also shed some light on fecal transplant and sterile fecal filtrate mechanisms as well.

For potential concerns, I would be worried about the potency of this treatment. Here I would like to pose the question: can the treatment be too good? As I mentioned, the balance of “good” to “bad” bacteria is key to patient health, and the purpose of treatment is to maintain the natural state of the gut microbiome. I would be keen to ensure that the proteins used together are highly specific at targeting the toxins and do not disrupt the balance of the microbiota by tipping the scale the other way. I think there is a lot of work ahead to test these proteins together and determine the best combination.

Thank you for sharing the polyzwitterion-polyelectrolyte paper; this was an interesting read. I am certainly not a chemist, but this looks promising!

Thanks again for your thoughtful reply.

Abbie

References:
1. van Nood E, Vrieze A, Nieuwdorp M, et al. Duodenal Infusion of Donor Feces for Recurrent Clostridium difficile. N Engl J Med. 2013;368(5):407-415. doi:10.1056/NEJMoa1205037

2. Ott SJ, Waetzig GH, Rehman A, et al. Efficacy of Sterile Fecal Filtrate Transfer for Treating Patients With Clostridium difficile Infection. Gastroenterology. 2017;152(4):799-811.e7. doi:10.1053/j.gastro.2016.11.010

3. Sehgal K, Khanna S. Gut microbiome and Clostridioides difficile infection: a closer look at the microscopic interface. Ther Adv Gastroenterol. 2021;14:1756284821994736. doi:10.1177/1756284821994736

Name
Abbie Davis

Name
Tarrah Ethier

Thu, 09/29/2022 - 18:01

Hi Abbie!
You put together a great summary of the MGR lecture and the smaller scale discussion that we had with Dr. Sheth. Something that you highlighted in your blog post, and that I found incredibly interesting in Dr. Sheth's lecture, was the process in which Dr. Sheth and his team used to narrow down the toxin-degrading protein to 9 candidates and that the introduction of new microbes can have therapeutic benefits. I was curious whether other diseases can be treated in a similar manner, with the introduction of other microbes, and found that some labs are trying to treat Alzheimer's disease (AD) using the re-"poop"ulate method. I thought this was interesting as it links the previous week's MGR topic to this one! Obviously trying this in AD comes with the same challenges as it does for treating C Diff (donors, screening, etc). However, I thought Alzheimer's would come with greater challenges. For example, patients with AD can be easily agitated, potentially making it harder to administer treatments. What are your thoughts on using the re-"poop"ulate method, or fecal microbiota transplants, in other diseases? Do you think there are some diseases in which this treatment is more feasible?

Looking forward to hearing your opinion,
Tarrah

Reference:

Cai, J. (2022). Exploring Probiotics and Fecal Microbiota Transplantation: Two Potential Therapeutics for Alzheimer's Disease in a Mouse Model.

Name
Tarrah Ethier

Hi Tarrah,

Thank you for your comments and input!

I agree that fecal transplants have the potential to treat various diseases since the microbiome has been associated with the pathogenesis of endless diseases, especially those involving the gastrointestinal tract. You may find it interesting that fecal transplantation is not a new concept- in fact, this treatment was first documented in China in the fourth century. Ge Hung coined the term “yellow soup, " which was used to treat severe diarrhea and food poisoning cases.1

As you described in AD, fecal microbiota transplants could also be used as an intervention in various neurologic and psychiatric conditions due to the brain-gut axis and its implications on neurologic development and disease progression.1 Specifically, there has been research conducted on autism spectrum disorders, multiple sclerosis, and Parkinson's disease.1 I was also interested to read about the gut-joint axis, which has linked dysbiosis to inflammation in autoimmune disorders such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE).1 Although to my knowledge there have not been any studies to date that have specifically looked at fecal transplants in these disorders, I think this is a really important avenue to explore. Recent studies have clearly shown dysbiosis of the gut microbiota in patients with RA, and it is thought that gut dysbiosis occurs before the clinical onset of RA and influences the development of arthritis.2

Due to the strict guidelines for fecal donors, I do not think these treatments are feasible at a population level. Additionally, many diseases linked to dysbiosis are extremely complex, and the treatment of these diseases will likely need a multi-directional approach. I do not think fecal transplants are feasible or are the solution to all these diseases. However, I do think there is a future for sterile fecal filtrates, and the use of this treatment will become an important approach in the future, especially when used in combination with other disease-modifying treatments.

Thanks again Tarrah for your thoughtful reply.

Best,

Abbie

References

1. Stripling J, Rodriguez M. Current Evidence in Delivery and Therapeutic Uses of Fecal Microbiota Transplantation in Human Diseases-Clostridium difficile Disease and Beyond. Am J Med Sci. 2018;356(5):424-432. doi:10.1016/j.amjms.2018.08.010
2. Zaiss MM, Joyce Wu HJ, Mauro D, Schett G, Ciccia F. The gut-joint axis in rheumatoid arthritis. Nat Rev Rheumatol. 2021;17(4):224-237. doi:10.1038/s41584-021-00585-3

Name
Abbie Davis

Name
Abhishek Shastry

Mon, 10/03/2022 - 16:25

Hi Abbie,

Your summary of Dr. Sheth's lecture and research was fantastic. I was very interested in his narrowing of possible candidate bacteria from the 'bacteria cocktail' to four possible candidates, and finally choosing Parabacteroides distasonis as a model to produce the HTRA-1 protein, which breaks down the Toxins generated by the pathogen, Clostridioides Difficile. Are there alternative methods to introducing microbes like P. distasonis to native gut microbiota, other than the fecal transplantation method described in lecture? In addition, it was discussed numerous times that results found in mice, though being a convenient and useful model for studying infectious disease, are limited by translatability to humans. Hugenholtz and de Vos (2018) outlined in their work that the abundance of gut bacteria and the variance in strains is significantly different in humans and mice, and that in total, only 4% of the bacterial genes are considerably shared. In addition, as was mentioned in lecture, the pH of human stomachs is around 2-3 whereas the murine stomach pH rests between 3-4 (McConnell et al, 2008). This could significantly change how the HTRA-1 protein survives after passing through a highly acidic environment. Could introducing P. distasonis to the gut microbiota, as a common vector between mice and humans, be useful for introducing HTRA-1 to the body rather than through HTRA-1 consumption? Thank you!

Best,
Abhishek

Reference List:
Hugenholtz, F., & de Vos, W. M. (2018). Mouse models for human intestinal microbiota research: a critical evaluation. Cellular and molecular life sciences : CMLS, 75(1), 149–160. https://doi.org/10.1007/s00018-017-2693-8

McConnell EL, Basit AW, Murdan S. Measurements of rat and mouse gastrointestinal pH, fluid and lymphoid tissue, and implications for in-vivo experiments. J Pharm Pharmacol. 2008 Jan;60(1):63-70. doi: 10.1211/jpp.60.1.0008. PMID: 18088506.

Name
Abhishek Shastry

Hi Abhishek,

Thank you for your reply.

As Dr. Sheth highlighted in his lecture, I think sterile fecal filtrates are a useful technique to bypass fecal transplant limitations.

Regarding your question about introducing only P. distasonis, I would be weary of overloading a patient with one type of bacteria. I would like to remind you of two key points. First, it is not the bacteria itself that is bad- it is the toxins the bacteria produce. Many people are living with C. diff right now, and this is not problematic for their own health because the toxins are not being produced. Second, the goal of treatment is to return the gut microbiome to its natural state. I would be cautious about introducing one type of bacteria, as this could make the disease worse depending on the environment and species it competes with. Fecal transplants and sterile fecal filtrates aim to introduce many bacteria to treat C. diff. I think the advantage of using HTRA-1 is that the bacterial component is gone, and we are introducing a potent bacterial toxin degrader to the patient to tackle the symptoms of C. diff directly.

Thank you for your thoughtful insight and for contributing to the discussion!

Abbie

Name
Abbie Davis

Name
Martha

Mon, 10/03/2022 - 16:40

Hi Abbie,
Great summary of last week's MGR given by Dr. Sheth, I appreciated your honesty when describing the wide ranging effects of C. diff infection and the limitations to the research currently trying to combat CDI. Something that really stuck out to me from the lecture was the risk factors of CDI including antibiotic therapy, old age, and hospital or nursing home stay. These populations tending to be adults with both asymptomatic and symptomatic types but then another population with high incidence rates of CDI are babies. Do you think that there could be some sort of insight gained to compare these two widely different populations in terms of their intestinal microflora, fecal bacteria or bacterial protein?
Czepiel, J., Drozdz, M., Pituch, H., Kuijper, E.D., Perucki, W., Mielimonka, A., Goldman, S., Wultanska, D., Garlicki, A., and Biesida, G. (2022). Clostridium difficile infection: review. Eur J Clin Microbiol, 38, 1211-1221.

Name
Martha

Hi Martha,

Thank you for your reply!

You raise a very interesting point. Up until more recently, microbiome studies have been mainly limited to adult populations. It is important to investigate the pediatric microbiome as well because it has been shown that the intestinal microbiota populations undergo dynamic changes during a human’s life. Literature suggests that the most substantial changes are likely to occur throughout childhood.1 A recent study that investigated the microbiomes of 2,111 children (ages 9-12) and 1,427 adults (ages 46-88) found a clear distinction between the two, including differences in microbiota diversity and Firmicutes and Bacteroidetes abundances.1

As you know, most therapeutics are approved in adults prior to being used in children. From a therapeutic standpoint, it is important to continue investigating these differences, as they could impact the treatment regimens in these two populations.

Thank you again.

Abbie

Reference:

1. Radjabzadeh D, Boer CG, Beth SA, et al. Diversity, compositional and functional differences between gut microbiota of children and adults. Sci Rep. 2020;10(1):1040. doi:10.1038/s41598-020-57734-z

Name
Abbie Davis

Name
Sam Delios

Mon, 10/03/2022 - 21:50

Hello Abbie!

Well done on your post! You did a great job at reviewing Dr. Sheth's talk. We had the privilege to look into Dr. Sheth’s research on bacterial protein as a potential therapeutic for clostridioides difficile (C. diff) infection. Dr. Sheth discussed how C. diff is the number 1 cause of infectious diarrhea & colitis and a large risk factor for this disease can be antibiotic use. Therefore, do you think there would be any alternative options to using antibiotics or do you believe there are any ways we can prevent antibiotics from causing C. diff?

I was also wondering if you could comment on the fecal microbiota transplants (FMT) that Dr. Sheth discussed. He shared how there is a very extensive screening process that goes with the FMT, and therefore this limits the number of donors for the procedure. Therefore, to overcome this (as you mentioned), he shared how he would aim to administer a sterile fecal filtrate (bacterial cocktail), but this also has an unknown mechanism behind it. Within Dr. Sheth’s lab, they are focusing on understanding such a mechanism by looking at this in mice. My question would be for you to comment on how you think the research he has done on mice thus far would translate into human testing, and what you believe could possibly be done to overcome the limitations he has shared. Overall, great post, and this was an amazing presentation done by Dr. Sheth.

Thanks!
Sam

Name
Sam Delios

Hi Sam,

Thank you for your reply!

As far as alternative treatments available, there are many in development. This includes probiotic/microbial therapies, phage therapy, fecal transplant, vaccines, immunotherapies, CRISPR-Cas/editors, stem cell AMPs, etc.1 I encourage you to read this Kumar et al. paper if you are interested. Unfortunately, there are limitations to these therapies, and antibiotics are still our best course of action in most cases, so I believe there are other things we can focus on in the meantime.

I believe a huge issue is misinformation. I think both patient and physician education would benefit the healthcare system immensely and decrease the occurrence of antibiotic-resistant bacteria. I think the reality is that antibiotics do not need to be used nearly as often as they are prescribed. Antibiotic overprescribing is a particular problem in primary care, where approximately 90% of all antibiotics are prescribed.2 Further, I think this paper by Llor & Bjerrum poses a great question: “ If respiratory tract infections are mostly self-limiting, why do we treat between 52% and 100% of the cases, with a median of 88%, with antibiotics? How is it possible that 88% of patients with acute bronchitis are special?”.2 Therefore, educating this population and auditing to ensure they follow proper protocols (e.g. swabs and other laboratory tests, not prescribing for an infection that is likely to resolve itself) would be a great place to start. Further, I think patient education is vital as well. This could be as simple as informing them that many infections are viral, which do not require treatment with antibiotics, or emphasizing that it is important to finish the prescribed antibiotics even if they are feeling better.

I commented on fecal transplants in the blog, and cited a great paper that you could read if you would like additional information ( van Nood et al., 2013). Regarding your question concerning translatability in mice, I will point you toward the idea that Isaac suggested (polyzwitterion-polyelectrolyte) which could have the potential to target the issue of stomach pH. I think the next step in terms of drug development would be to test the proteins in a non-rodent model (particularly one that may have similar stomach pH as humans).

Thanks again Sam for your reply!

Abbie

References:

1. Kumar M, Sarma DK, Shubham S, et al. Futuristic Non-antibiotic Therapies to Combat Antibiotic Resistance: A Review. Front Microbiol. 2021;12. Accessed October 6, 2022. https://www.frontiersin.org/articles/10.3389/fmicb.2021.609459
2. Llor C, Bjerrum L. Antimicrobial resistance: risk associated with antibiotic overuse and initiatives to reduce the problem. Ther Adv Drug Saf. 2014;5(6):229-241. doi:10.1177/2042098614554919

Name
Abbie Davis

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