TWiM #305: The Marvel of MAC

1. Paper Abstracts

1.1. Snippet paper discussion starts at 7:53 minutes

Ubesie et al (2024) is not licensed for Creative Commons use, so the abstract cannot be copied here. Please see the article at the journal’s web page.

1.2. Main paper discussion starts at 14:50 minutes

Wu et al. (2024) is not licensed for Creative Commons use, so the abstract cannot be copied here. Please see the article at the journal’s web page.

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

3.  Potential Learning Objectives for the Podcast

¹Papers: Snippet (S) or Main (M)

²Learning 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

• Outbreak Epidemiology (8:13–11:56): There are epidemiological strategies to reduce the death rate due to cholera. These include increasing vaccine production, as well as increasing availability of oral rehydration salts.
• Crude Filtering (12:56–14:50): The use of a sari cloth has been used to filter water contaminated with amoeba inhabited with Vibrio bacteria and the parasitic Guinea worm. This filtering method can reduce the number of contaminants in water to limit the spread of disease.

4.2. Main Paper

• in vivo Microbiome Transfer Experiments (23:11–28:10): In this type of experiment germ-free mice are given gut microbiome communities from other sources.  Here, the germ-free mice were given transplants from age-matched specific pathogen-free mice and within two weeks the C3 levels of the recipients were comparable to those of the donors. Mice from different vendors had differing C3 levels, and germ-free mice that received the donor microbiome displayed fecal C3 levels similar to their specific donor. The microbiome of healthy human donors (who, similar to the non-germ-free mice, showed varying C3 levels) were transferred to germ-free mice, who again showed comparable levels of C3 to that of their human donor.
• in vivo Broad-Spectrum Antibiotic (BSA) Dose Response Experiments (25:34–26:28): This is an assay to determine the effects of antibiotics on gut microbiome.  Here, broad-spectrum antibiotics were given to specific-pathogen-free mice, and fecal C3 levels dropped to a comparable level to that of germ-free mice.
• C3 Reporter Mice (34:11–35:01): To determine the cell type responsible for the production of C3 in the gut, the authors used C3 reporter mice along with single-cell sequencing, flow-cytometry, and imaging to determine that stromal cells within the gut-associated lymphoid tissue were the primary producers of C3.
• in vivo Infection Experiments (37:37–39:32): In these experiments, mice or other living organisms are infected with a pathogen.  Here, C3-deficient and age-matched wild-type control mice were infected with Citrobacter rodentium. Half of the C3-deficient mice died, with many of the others losing weight, while all of the wild-type mice remained healthy. From this, the authors determined that gut C3 is important for surviving gut pathogens. The authors then infected wild-type mice with GFP-labeled C. rodentium and observed that after 7 days the bacteria were coated in C3.
• Cobra Venom Factor Assay (40:28–41:23): Cobra venom factor, which depletes C3 in the blood sera, was administered to wild-type mice prior to infection with C. rodentium, and no changes in susceptibility were observed in the mice. From this, the authors determined that it was gut C3, not sera C3, that was responsible for protecting the mice against gut pathogens.

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

5.1. Snippet Paper

• Disease Triangle & Public Health Emergency (8:13–8:54): Throughout Central and Southern Africa, there has been a rampant outbreak of cholera over the past several years. A combination of malnutrition and severe storms causing flooding has resulted in poor sanitation that reduces separation of wastewater from potable water. The African Centres for Disease Control and Prevention (Africa CDC) has reported over 250,000 cases and more than 4,000 deaths due to cholera in this region over the span of a year.
• Pathogenesis & Treatment (8:54–11:56): Oral Rehydration Salt Treatment to Reduce the Dangers of Dehydration.
• Those with cholera lose 20 to 30 liters of fluid per day. This not only puts the ill person in danger, but also poses a risk to the water supply, allowing for cholera to spread throughout a community. For rural populations, most get their water from natural bodies of water and are readily contaminated if pit toilets overflow and contaminate drinking water. Oral rehydration salts consist of sodium chloride, potassium chloride, trisodium citrate and glucose. These salts are extremely cheap and can reduce mortality rates to less than 1%.
• Vaccination & Disease Prevention (11:56–12:56): Two serotypes of cholera are responsible for the majority of outbreaks: O1 and O139. Further data about the period of effectiveness are needed to determine the viability of vaccines.
• Microbial Control by Filtration (12:56–14:50): Amoeba contaminated with Vibrio can be filtered using a sari cloth to reduce the number of Vibrio an individual may intake from contaminated water. A sari can also be used to filter parasites such as the Guinea worm that may enter the water system from individuals traveling through water.

5.2. Main Paper

• Complement (15:00–22:00): The complement system plays a crucial role in innate immunity by acting as a selective barrier helping to protect the body from infection while allowing essential microbiota to thrive, through differentiation between harmful pathogens and beneficial microbes in the gut, and marking pathogens for destruction or directly killing them through the membrane attack complex (MAC). MAC is activated through the conversion of classical, lectin, and alternative pathways of the complement system.
• Tolerance, Regulation, and Therapeutics (22:00–33:00): CD59 protein inhibits MAC on the body’s own cells to prevent accidental harm to host cells. Deficiencies in regulatory proteins like CD59 can cause diseases like paroxysmal nocturnal hemoglobinuria (PNH), as a result of uncontrolled complement activity. Therapies targeting MAC, like Eculizumab (a monoclonal antibody that inhibits C5), show promise for conditions such as PNH.

6. Podcast Questions

1. What is the primary mode of transmission for cholera?
   1. Direct touch contact
   2. Airborne droplets
   3. Vector-borne by insects
   4. Fecal-oral route
2. Which of the following best describes the features of cholera infection?
   1. A bacterial infection, primarily causing severe respiratory distress.
   2. A viral infection, primarily causing high fever and muscle pain.
   3. A bacterial infection, primarily causing severe diarrhea and dehydration.
   4. A parasitic infection, primarily causing fever and chills.
3. What environmental factors contributed to the cholera outbreak described in the podcast?
   1. Natural disasters
   2. Armed conflicts
   3. Malnutrition
   4. All of the above
4. How would a change in the water-based transmission route affect the success of the crude filtering by sari cloth to reduce cholera infection?
   1. It would not affect it because the bacteria cluster into large clumps which can be filtered.
   2. It would make it more effective because dehydrated bacteria are more like to be killed.
   3. It would make it less effective because the amoeba could not engulf the bacteria.
   4. It would make it more effective because the bacteria the metallic threads kills bacteria.
5. What is the role of complement proteins in the immune system?
   1. To produce antibodies against pathogens, regulate fever, and stimulate white blood cell production in the thymus.
   2. To tag pathogens for destruction, promote inflammation, and form membrane attack complexes to lyse infected cells.
   3. To engulf pathogens directly using phagocytosis, suppress inflammation, and repair damaged tissue in infected areas.
   4. To neutralize toxins in the bloodstream, activate platelets, and stimulate the bone marrow to increase red blood cell count.
   5. To inhibit double-stranded RNA viral replication, reduce antibody levels, and block cytokine signaling in immune cells.
6. Which of the following describes the mechanism that the alternative pathway activates the complement system?
   1. Pathogen associated molecular patterns are recognized.
   2. Pathogen specific antibodies bind to the pathogen.
   3. Mannose-binding lectin bind to pathogen polysaccharides.
   4. Activating the C1r and C1s proteases within the C1 complex.
7. The recent study by Wu et al. (2024) found fecal complement proteins (C3) in which of the following conditions?
   1. Absence of a gut microbiome
   2. Presence of a gut microbiome
   3. Treatment with cobra venom factor
   4. High abundance of gut Firmicutes
8. Based on the discussion of the paper’s results, what effect would you expect for pathogen load if mice were depleted for gut C3?
   1. You’d expect pathogen load to be the same as wild-type mice.
   2. You’d expect pathogen load to be lower than C3-deficient mice.
   3. You’d expect pathogen load to be much lower than wild-type mice.
   4. You’d expect pathogen load to be higher, similar to C3-deficient mice.

7. Figure Reading Exercises

The following are two figure reading exercises, both from the main paper (Figures 1A-C and 5A-C)

7.1. First Figure Reading Exercise

7.1.2. Questions

1. The schematic in panel A shows the relationship of the different mouse treatment groups.  Which statement best describes the relationships?
   1. There was a microbiome transfer from SPF mice to GF mice, and the SPF mice were treated with broad-spectrum antibiotics.
   2. There was a microbiome transfer from GF mice to SPF mice, and the SPF mice were treated with broad-spectrum antibiotics.
   3. There was a microbiome transfer between antibiotic-treated ABX mice to all other mouse groups (SPF and GF).
   4. There was a microbiome transfer between SPF mice to all other mouse groups (GF, SPF, and antibiotic treated).
2. Match the mouse with its microbiome type when serum and fecal material were assayed for C3 (panel A). [1 = GF; 2 = SPF; 3 = GF^SPF; 4 = SPF^ABX]
   1. ________ pathogen-free microbiome
   2. ________ no microbiome
3. C3 was measured in to generate the data in panels B and C.  The data are displayed by bar plots. Match the bar plot feature with its description. (1 = mean; 2 = median; 3 = not statistically significant; 4 = standard error of the mean; 5 = statistically significant; 6= full data range excluding outliers; 7 = standard deviation)
   1. ________ Bar height
   2.  ________ Asterisks
   3. _________Whiskers
   4. _________ nss
4. Based on the data provided, which mouse model has equivalent fecal C3 levels as GF mice (panel B)?  What is your evidence?
   1. SPF mice; these mice have no pathogens
   2. GF^SPF mice; they are also germ-free mice
   3. SPF^ABX mice; they also have low C3
   4. All are different from GF mice.
5. Which mouse treatment group(s) has/have higher fecal C3 levels and what does this indicate?
   1. None have high levels; fecal C3 levels are unaffected by the microbiota in mice.
   2. All have high levels; fecal C3 levels are unaffected by the microbiota in mice.
   3. GF and SPF^ABX ; the presence of a microbiota increases fecal C3 levels in mice.
   4. SPF and GF^SPF ; the presence of a microbiota increases fecal C3 levels in mice.
6. Which mouse treatment group(s) has/have higher serum C3 levels and what does this indicate?
   1. Neither has serum C3; serum C3 levels are unaffected by the microbiota in mice.
   2. Levels are equal; serum C3 levels are unaffected by the microbiota in mice.
   3. GF; the absence of a microbiota increases serum C3 levels in mice.
   4. SPF; the presence of a microbiota increases serum C3 levels in mice.

7.2. Second Figure Reading Exercise

7.2.2. Questions

1. What color and shape indicate the data for mice treated with cobra venom factor?
   1. red triangles
   2. black squares
   3. blue circles
   4. red lines
2. Which mice survive C. rodentium infection better (panel A)?
   1. wild-type mice
   2. complement-deficient mice
   3. wild-type mice treated with cobra venom factor
   4. None, they all survive equally well.
3. Weight loss, which is a measure of health, was quantified for each infection group to generate the data in panel B.  The data are displayed by line graphs. Match the line graph feature with its description. [1 = mean; 2 = median; 3 = not statistically significant; 4 = standard error of the mean; 5 = statistically significant; 6= full data range excluding outliers; 7 = standard deviation]
   1. ________ symbol location
   2.  ________ asterisks
   3. _________whiskers
   4. _________ ns
4. What can you conclude about the effect of serum and gut complement on the health of mice infected with C. rodentium (panel B)? What is your reasoning?
   1. Gut complement is protective since complement deficiency reduces weight and CVF treatment has no effect on weight.
   2. Serum complement is protective since complement deficiency reduces weight and CVF treatment has no effect on weight.
   3. Gut and serum complement both contribute to protection since complement deficiency and CVF treatment both reduce weight.
   4. Serum complement is inhibitory since complement deficiency reduces weight and CVF treatment has no effect on weight.
5. Pathogen load, which is a measure of infection, was quantified as colony forming units (CFU) for each infection group to generate the data in panel C.  The data are displayed by strip plots. Match the strip plot feature with its description. [1 = standard deviation; 2 = statistically significant; 3 = standard error of the mean; 4= full data range excluding outliers; 5 = standard deviation; 6 = mean; 7 = median; 8 = not statistically significant; 9 = individual data points]
   1. _________ ns
   2. _________ horizontal line
   3. _________ individual symbols
   4. ________ asterisks
   5. _________whiskers
6. What can you conclude about the effect of serum and gut complement on pathogen load of mice infected with C. rodentium (panel C)? What is your reasoning? [pick all that apply]
   1. Gut and serum complement both contribute to protection since complement deficiency and CVF treatment both increase pathogen load.
   2. Gut complement is dispensable for protection since CVF treatment has no effect on pathogen load and complement deficiency increases it.
   3. Serum complement protects health by inactivating the pathogen’s virulence, which shows as increased bacterial load but normal weight (panel B).
   4. Serum complement is necessary for protection since complement deficiency increases pathogen load and CVF treatment has no effect on it.

8. Paper Information and Licensing

8.1. Snippet paper

• Ubesie AC, Odimegwu CL, Ezeanolue EE. 2024. Cholera outbreaks among children in sub-Saharan Africa. Curr Opin Pediatr. 36(2):150-155. https://doi.org/10.1097/MOP.0000000000001329
• This article is not licensed for Creative Commons use; Thus, the abstract and figures cannot be copied here. Please see the article at the journal’s web page.

8.2. Main paper

• Wu M, Zheng W, Song X, Bao B, Wang Y, Ramanan D, Yang D, Liu R, Macbeth JC, Do EA, et al. 2024. Gut complement induced by the microbiota combats pathogens and spares commensals. Cell. 187(4):897-913. https://doi.org/10.1016/j.cell.2023.12.036
• This article is not licensed for Creative Commons use; Thus, the abstract and figures cannot be copied here. Please see the article at the journal’s web page.