TWiM #221: Weapon of Mucus Destruction, WMD

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 6:15 minutes

This article is not licensed for Creative Commons use. Thus, the abstract and figures cannot be copied here. Please see the article on the journal’s website.

1.2. Main paper; discussion starts at 31:38 minutes

“The human microbiome encodes extensive metabolic capabilities, but our understanding of the mechanisms linking gut microbes to human metabolism remains limited. Here, we focus on the conversion of cholesterol to the poorly absorbed sterol coprostanol by the gut microbiota to develop a framework for the identification of functional enzymes and microbes. By integrating paired metagenomics and metabolomics data from existing cohorts with biochemical knowledge and experimentation, we predict and validate a group of microbial cholesterol dehydrogenases that contribute to coprostanol formation. These enzymes are encoded by ismA genes in a clade of uncultured microorganisms, which are prevalent in geographically diverse human cohorts. Individuals harboring coprostanol-forming microbes have significantly lower fecal cholesterol levels and lower serum total cholesterol with effects comparable to those attributed to variations in lipid homeostasis genes. Thus, cholesterol metabolism by these microbes may play important roles in reducing intestinal and serum cholesterol concentrations, directly impacting human health.” (Kenny et al. 2020)

2. Vision and Change Core Concepts and 2024 ASM Fundamental Statements

3.  Potential Learning Objectives for the Podcast

¹ Papers: Snippet (S) or Main (M)

² Learning Objectives: Lower Order or Higher Order (H)

4. Techniques Described (with Time Stamps)

Here is a link to a bio-dictionary that has many, but not all definitions if you need a definition: Explore Biology Bio-Dictionary

4.1. Snippet Paper

• Delivery Systems (17:18–20:58; 30:05–30:28): In order for a drug to be effective it must be delivered efficiently.  Here, the researchers test two different types of delivery vehicles (chitosan and beta-cyclodextrin) for their effectiveness in delivering nitrous oxide and breaking up the biofilm formation. Chitosan effectively released nitrous oxide but hardened and compressed the biofilm, making it difficult for the nitrous oxide to break up the mucus. The presence of protons in an acidic environment allows beta-cyclodextrin (CD) to release nitrous oxide quickly in relevant concentration that can be sustained for hours, since this molecule is small and can easily invade the biofilm.
• Enrichment Media (19:09–19:26): Media is used in the laboratory to culture microbes based on their nutritional requirements.  It can be enriched with compounds that are expected to enhance cell or bacterial growth. In this scenario Pseudomonas aeruginosa‘s behavior was examined  when it was  grown on different media, i.e., TSA, sputum, porcine gastric mucins  and sputum filtrate medium (sputum was isolated from a CF patient) to evaluate its ideal conditions when forming biofilm
• Antimicrobial Susceptibility Test (23:55–24:25): This assay is also called disk diffusion assay or Kirby-Bauer susceptibility test.  It is a test for a bacterium’s antibiotic sensitivity and typically involves placing a paper disk impregnated with a compound onto a lawn of bacteria and looking for a zone of inhibited growth around the disk.  This experiment used three commonly used antibiotics currently used to treat cystic fibrosis which included tobramycin cyclodextrin, and colistin. Resistance to antibiotics would show continuous growth, while susceptibility would display extinction of the biofilm.

4.2. Main Paper

• Molecular and Biochemical Techniques (31:38–39:35): Utilizing DNA changes (mutagenesis) and the associated biochemical techniques of epitope tagging and metabolite level quantification, the researchers confirmed that ismA encodes a protein that can convert cholesterol to coprostanol, is oxygen-independent, requires NADP, is found in fecal samples when cholesterol metabolites are high, and homologs are present in a large number of species, particularly in the Clostridium genus.
• Metabolomics (38:50–39:38): This is a high throughput method for identifying and quantifying metabolites in a mixture.  Stool metabolomes were analyzed for coprostanol, the end product of cholesterol degradation, which allowed identification of cholesterol-relevant protein clusters. This was then used to identify putative genes for degrading cholesterol to coprostanol.
• Enzymatic/Tissue Lysate Preparation  (39:35–52:28): Lysates are cell-free homogenates made by lysing cells.  Here, the researchers utilized different lysate types to determine whether Eubacterium have the enzymes available to degrade cholesterol to cholestenone, allowing the genes to be narrowed down
• Bioinformatics (39:35–52:28): Bioinformatics uses computational approaches to analyze large datasets.  Here, it was used in a number of ways to identify species and genes in bacteria that can alter cholesterol and when present in gut bacteria influence host gut cholesterol and serum cholesterol levels.
• Metagenome Bioinformatics (48:10–52:00): Metagenome sequences are pools of DNA sequences isolated from an environmental source to identify the species present, such as in the gut microbiome.  Here, metagenomic sequences from fecal samples were used to identify bacteria that were correlated with the presence of known cholesterol metabolites.  Through a series of adjustments, the researchers were able to also the identify putative genes involved. These were then compared to available databases in order to identify homologous genes.

5. Connections to General Microbiology Processes/Concepts (with Time Stamps)

5.1. Snippet Paper

5.2. Main Paper

1. Which factors contribute to the severity of cystic fibrosis? [pick all that apply]
   1. Quorum sensing of P. aeruginosa
   2. Charge of the gut environment
   3. Biofilm formation in the lung
   4. Blocking host endocytosis
2. In what ways does the presence of extracellular DNA and other negatively charged molecules present in the biofilm contribute to antibiotic resistant P. aeruginosa in cystic fibrosis patients? [pick all that apply]
   1. They increase biofilm stiffness.
   2. They physically protect bacteria.
   3. They increase the local pH level.
   4. They destroy cationic antibiotics.
   5. They cause bacteria to mutate.
3. In the podcast, Michael called nitric oxide a molecular grenade for treatment of P. aeruginosa biofilms. What properties is he talking about?
   1. It adjusts the pH of the environment, killing bacteria.
   2. It inhibits production of flagella just in P. aeruginosa.
   3. It disrupts the biofilm structure and kills via oxidation.
   4. It targets P. aeruginosa by receptor binding and kills it.
4. The main point of the paper was to test delivery of nitric oxide using cyclodextrin (CD-NO).  To do this, the researchers made a variety of thick, viscous media and compared the ability of P. aeruginosa to survive exposure to typical antibiotics or CD-NO in these media.  Survival was lower for CD-NO than typical antibiotics, particularly in the thicker medium.  If the results had instead shown that CD-NO protects P. aeruginosa, what would the results have looked like?
   1. CD-NO would have had higher survival than antibiotics.
   2. CD-NO would have had equal survival with antibiotics.
   3. CD-NO would have had lower survival with antibiotics.
   4. CD-NO would have had no survival in any medium.
5. Cholesterol is an important biomolecule because it is used to produce ________.
   1. Pancreatic salts
   2. steroid hormones
   3. NADP and oxygen
   4. Vitamins D and E
6. Homologs of ismA were found to be prevalent in which bacterial genus?
   1. Actinobacillus
   2. Prevotella
   3. Clostridium
   4. Brachyspira
7. Genomic, metagenomic, and metabolomic datasets contributed greatly to this work.  Match the description and its role in this study.

8. How could you find an ismA homolog in a newly sequenced genome?
   1. Compare an ismA-containing genome to your new genome sequence.
   2. Culture the bacterium; look for the presence of high NADP levels.
   3. Culture the bacterium; test for cholesterol production using metabolomics.
   4. Search the sequence of the new genome using a known ismA sequence.

7. Figure Reading Exercises

The following are two figure reading exercises, both from the main paper (Figures 4B and 5AB).

7.1. First Figure Reading Exercise

7.1.2. Questions

1. Metabolomic data are presented for cultures of eight stool samples from healthy donors.  What is the notation that indicates where to expect the coprostanol peak?
   1. It is the “4” at the top of the panel.
   2. It is the peak in the metabolomic data.
   3. It is notated by a bright red arrow.
   4. It is not notated as there is no peak.
2. Which stool sample culture produces the highest level of coprostanol?  What is your evidence?
   1. LD04; the widest peak
   2. LD11; the highest peak
   3. LD09; the narrowest peak
   4. LD20; the smoothest peak
3. Which color indicates the abundance of bacterium msp_0196 in the metagenomic data for the stool sample cultures?
   1. gray
   2. orange
   3. Medium red
   4. Dark red
4. Which color(s) denote(s) species in the metagenomic data that is/are IsmA+? [pick all that apply]
   1. gray
   2. orange
   3. Medium red
   4. Dark red
5. Which stool sample culture has the largest abundance of IsmA+ bacteria?  What is your evidence?
   1. LD04; the largest non-gray bar
   2. LD11; the largest dark red bar
   3. LD09; most “red” species present
   4. LD20; the most balanced bar
6. What can you conclude about the relationship of coprostanol production and abundance of IsmA+ bacteria in the stool sample cultures?
   1. Coprostanol is produced regardless of the presence of IsmA+ bacteria.
   2. Coprostanol production correlates with abundance of IsmA+ bacteria.
   3. When IsmA+ bacteria are present, we also see coprostanol is present.
   4. Coprostanol production occurs only in the absence of IsmA+ bacteria.

7.2. Second Figure Reading Exercise 

7.2.2. Questions