Microbial Ecology

TWiM #241: What Does Flu Do to Your Poo?

Podcast and Annotation Information
  • Annotation by Emily Hundley, Laura Lopez Salinas, Huynh Nguyen, Gina Orjuela, Rebecca Seipelt-Thiemann, Maia Larios-Sanz
  • Podcast audio by TWiM: Listen to TWiM #241 Podcast
  • Podcast transcript by Otter.ai and edited by Emily Hundley, Laura Lopez Salinas, Huynh Nguyen, Gina Orjuela, Maia Larios-Sanz, and Isabelle Norris: Access Podcast Transcripts
  • Papers Discussed:
    • Bridges AA, Bassler BL. 2021. Inverse regulation of Vibrio cholerae biofilm dispersal by polyamine signals. Elife. 10:e65487. doi: 10.7554/eLife.65487.
    • Groves HT, Higham SL, Moffatt MF, Cox MJ, Tregoning JS. 2020. Respiratory Viral Infection Alters the Gut Microbiota by Inducing Inappetence. mBio. 11(1):e03236-19. doi: 10.1128/mBio.03236-19.

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 3:48 minutes

The Most Interesting Things (according to students)

  • Biofilm formation and dispersal are highly regulated by small molecules that disperse in the environment, and transport of these signal molecules into the cell helps regulate the process as well.
  • Norspermidine promotes biofilm formation while spermidine represses biofilm formation, and these molecules only differ by a methylene group.

“The global pathogen Vibrio cholerae undergoes cycles of biofilm formation and dispersal in the environment and the human host. Little is understood about biofilm dispersal. Here, we show that MbaA, a periplasmic polyamine sensor, and PotD1, a polyamine importer, regulate V. cholerae biofilm dispersal. Spermidine, a commonly produced polyamine, drives V. cholerae dispersal, whereas norspermidine, an uncommon polyamine produced by vibrios, inhibits dispersal. Spermidine and norspermidine differ by one methylene group. Both polyamines control dispersal via MbaA detection in the periplasm and subsequent signal relay. Our results suggest that dispersal fails in the absence of PotD1 because endogenously produced norspermidine is not reimported, periplasmic norspermidine accumulates, and it stimulates MbaA signaling. These results suggest that V. cholerae uses MbaA to monitor environmental polyamines, blends of which potentially provide information about numbers of ‘self’ and ‘other’. This information is used to dictate whether or not to disperse from biofilms.” (Bridges and Bassler 2021)

1.2. Main paper; discussion starts at 32:47 minutes

The Most Interesting Things (according to students)

  • Viral infections result in changes in the bacterial composition of the microbiome, and these changes are similar to those seen in mice that have had food withheld.
  • Lack of appetite during a viral infection is actually a result of the immune system – both TNF-alpha and CD8+ cells are implicated in this response.

“Respiratory viral infections are extremely common, but their impacts on the composition and function of the gut microbiota are poorly understood. We previously observed a significant change in the gut microbiota after viral lung infection. Here, we show that weight loss during respiratory syncytial virus (RSV) or influenza virus infection was due to decreased food consumption, and that the fasting of mice altered gut microbiota composition independently of infection. While the acute phase tumor necrosis factor alpha (TNF-α) response drove early weight loss and inappetence during RSV infection, this was not sufficient to induce changes in the gut microbiota. However, the depletion of CD8+ cells increased food intake and prevented weight loss, resulting in a reversal of the gut microbiota changes normally observed during RSV infection. Viral infection also led to changes in the fecal gut metabolome, with a significant shift in lipid metabolism. Sphingolipids, polyunsaturated fatty acids (PUFAs), and the short-chain fatty acid (SCFA) valerate were all increased in abundance in the fecal metabolome following RSV infection. Whether this and the impact of infection-induced anorexia on the gut microbiota are part of a protective anti-inflammatory response during respiratory viral infections remains to be determined.” (Groves et al. 2020)

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

Snippet Main
Vision and Change Topics
  • Metabolic Pathways (V&C_MP)
  • Microbial Ecology (V&C_ME)
  • Microbial Ecology (V&C_ME)
ASM Fundamental Statements
  • Fundamental Statement 7 (ASM_7): . Microbes have evolved structures adapted for specific functions that are often associated with a fitness advantage in a particular environment.
  • Fundamental Statement 13 (ASM_13):  Intrinsic factors, such as genotype, metabolism, and cell structures, impact the survival and growth of microbes.
  • Fundamental Statement 18 (ASM_18): The regulation of gene expression is influenced by external and internal molecular cues and signals.
  • Fundamental Statement 21 (ASM_21): Microbes and the environment interact with and affect each other.
  • Fundamental Statement 29 (ASM_29):  The extent of microbial damage can be minimized by host-derived and external factors, including the microbiome, antibiotics, and immunity.

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • Define biofilm.
  • Order the stages of biofilm formation.
  • Recall how norspermidine and spermidine regulate biofilm formation in Vibrio cholerae.
 S L
  • Predict the effect of a hypothetical small molecule inhibitor on biofilm formation using the researchers’ model.
 S H
  • Define microbiome.
  • Recall the effects of respiratory syncytial virus infection on mouse behavior and microbiome composition.
  • Identify the biological pathways of the gut-derived metabolites that are altered in respiratory syncytial virus infection in mice.
 M L
  • Design an experiment to test a hypothesis generated based on these data.
 M H

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

  • Lifecycle Microscopy (8:11): This process uses video and microscopy to show the lifecycle in a compressed time frame.  It was used to show the effects of mutating different genes on biofilm formation.
  • Classic Mutagenesis (9:40): This technique involves mutating genes in a strain, identifying those with a specific phenotype, identifying the gene that was mutated, then trying to restore function by re-introducing a wild-type gene.  Strains that failed to properly disperse were used to identify genes that are involved in the biofilm/motile behaviors.
  • Fluorescent Reporter (12:14): In this technique a fluorescent molecule is produced in response to the presence and level of a specific molecule.  The fluorescence can be visualized using microscopy or quantified using a detector.  Here, it was used to measure cytoplasmic concentrations of the cyclic-di-GMP to determine how levels of this messenger impact biofilm formation or dispersal.

4.2. Main Paper

  • Mouse Model (37:38): This involves experimenting on mice as a model for human infection to further understand the potential effect of viruses on humans.
  • Gut Microbiome Composition Studies (42:13): These are methods using next-generating sequencing technologies to characterize species composition.  Here it was used to determine differences of microbial composition of mice with and without viral infection.
  • Metabolomics and Pathway Analysis (50:58): Mass spectrometry is a technique to identify and quantify molecules in a mixture and when used with proteins it can be called metabolomics.  Here it was used to identify the molecules present in the mouse gut with and without viral infection.  Pathway analysis, which is identifying the biological pathways the molecules act in using computational methods, was then used to identify which biological processes were likely affected (enriched).  Here, they found that lipid metabolism is affected.

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

5.1. Snippet Paper

  • Biofilm Formation (3:35): The non-motile stage (biofilm) of Vibrio cholerae is a carefully regulated survival strategy that is also central to disease transmission since it alternates between marine and estuarine environments.
  • Cholera (5:16–7:50): This is an acute diarrhea disease caused by Vibrio cholerae that has affected human populations in seven documented pandemics throughout history.
  • Quorum Sensing (16:69): This  molecular communication allows cells to distinguish “self” from “others” and regulate gene expression based on cell density and environmental cues.
  • Membrane Transport and Signal Transduction (18:21):  Norspermidine interacts with membrane sensors to stop c-di-GTP degradation, promoting biofilm formation. Spermidine prevents binding to the sensor, which will then promote degradation of c-di-GTP, inducing cell motility (and thus dispersal).

5.2. Main Paper

  • Microbiome and Health (33:07): Microbiota can influence disease. The changes in our microbiome can also influence our immune system response. Here, the researchers study how a respiratory virus also impacts the microbiota.
  • Immune Response to a Viral Infection (43:33): Response to a virus involves production of cytokines, which then travel throughout the body. TNF alpha (tumor necrosis factor alpha) is one type which damages virus infected cells to restrict infection.
  • CD8+ T Cells (47:14): These are specialized immune cells that respond to viral infection.  Blocking their action also reduces weight loss associated with infection.

6. Podcast Questions

  1. What is a biofilm?
    1. The lab environment built to mimic a natural environment.
    2. A complex surface-attached stable bacterial community.
    3. A group of nonrelated species coexisting in an environment.
    4. An environmental structure where a single species lives.
  2. Order the steps in biofilm formation with 1 being first and 4 being last.
    1. ______ growth, division, and extracellular matrix
    2. ______ environmental conditions are detected
    3. ______ cells disperse from the biofilm
    4. ______ a founder cell attaches to a substate
  3. The snippet paper’s overall model for dispersal and biofilm formation is based on norspermidine and spermidine signals. In this model,   ______ induces biofilm formation and represses dispersal while ______ represses biofilm formation and induces dispersal.
    1. High norspermidine; low norspermidine
    2. Presence of both; absence of both
    3. Low norspermidine; high spermidine
    4. High norspermidine; high spermidine
  4. Based on the model of biofilm and dispersal regulation by norspermidine and spermidine, what effect would you predict if you exposed Vibrio cholerae biofilm to a small molecule inhibitor of norspermidine?
    1. The biofilm will remain intact due to the high cyclic-di-GMP levels.
    2. The biofilm will begin dispersal due to the high cyclic-di-GMP levels.
    3. The biofilm will remain intact due to the low cyclic-di-GMP levels.
    4. The biofilm will begin dispersal due to the high cyclic-di-GMP levels.
  5. What is a microbiome?
    1. A genomic comparison of all strains of a particular microbe species.
    2. A small lab environment used to mimic the natural environment.
    3. A complex community of microbes present in an environment.
    4. A group of structurally attached species existing in an environment.
  6. What effects on mouse characteristics were observed when mice were virally infected compared to non-infected or pre-infected mice?
    1. Appetite loss, weight loss, Firmicutes bacteria decrease
    2. Appetite increase, weight gain; Firmicutes bacteria decrease
    3. Appetite loss, weight loss, Bacteroidetes bacteria decrease
    4. Appetite increase, weight gain; Bacteroidetes bacteria decrease
  7. Gut metabolites were altered in virally infected mice compared with non-infected mice. The researchers identified the biological pathways of those with altered levels.  What were the significant biological pathways that were affected?
    1. Gene expression
    2. Signal transduction
    3. Quorum sensing
    4. Lipid metabolism
  8. Which experiment could be used to test whether the host response or the viral infection induces the changes to the Firmicutes and Bacteroidetes in the gut microbiome?
    1. Infect mice with a bacterium or with a virus and compare the gut microbiome composition.
    2. Inoculate mice with a heat-killed virus or no virus; compare the gut microbiome composition.
    3. Infect mice with a very different virus or no virus; compare the gut microbiome composition.
    4. Inoculate mice with a mixture of Firmicutes and Bacteroidetes and test for viral immunity.

7. Figure Reading Exercises

The following are two figure reading exercises, both from the snippet paper (Figures 1 and 2).

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to: 

  • Identify key features and interactions in a model schematic.
  • Predict biofilm/dispersal effects when key players in the process/model are changed.
  • Defend a strategy to control Vibrio cholerae spread.
Experimental Background (Bridges and Bassler, Figure 1)

Vibrio cholerae is the causative agent for cholera, which has resulted in seven major outbreaks in recorded history.  In the absence of treatment, which occurs often in undeveloped areas of the world, it has a 50-60% mortality rate.  Clean water, proper hygiene, and access to medical care are important factors in its control, so undeveloped world areas are particularly at risk.  Understanding how this organism regulates the change between its sessile (biofilm) and motile forms will also inform making better containment protocols.  Cells cycle between attachment to a surface, where cells are non-motile, and dispersal, where they become swimmers and leave the biofilm community to colonize elsewhere. While the processes controlling biofilm formation are well characterized, the signals triggering dispersal are not. In this study, Bridges and Bassler (2021) identified the key signals and cellular factors involved in Vibrio dispersal from biofilms.  Their model is shown in the schematic.

schematic/model
Figure 1. “Polyamine sensing in V. cholerae. Schematic showing the previously proposed polyamine detection and import mechanisms in V. cholerae. Norspermidine (Nspd, triangles) promotes biofilm formation and spermidine (Spd, circles) represses biofilm formation. The primarily eukaryotic polyamine spermine (not pictured) also signals through the NspS-MbaA pathway. See text for details. OM: outer membrane; IM: inner membrane.” (Bridges and Bassler, 2021, no changes).

7.1.2. Questions

  1. What features are indicated by labels IM and OM in the model diagram, and what is the name for the space between them?
    1. Outer membrane; inner membrane; periplasm
    2. Orthologous and internal measures; cytoplasm
    3. Osmotic and ionic media components; nucleus
    4. Organism and intramolecular stages; biofilm
  2. The mechanism by which norspermidine and spermidine interact at the cell surface, in the periplasm and enter the cell to regulate biofilm and dispersal is displayed as a model schematic. Using this model diagram, you can deduce that PotD1 ________ . [Pick all that apply]
    1. Binds MbaA
    2. Binds norspermidine
    3. Binds spermidine
    4. Binds NspS
    5. Is found in the periplasm
  3. A mutation that affects entry of norspermidine and spermidine into the periplasm will likely result in which of the following?
    1. Biofilm formation due to degradation of c-di-GMP
    2. Motility induction due to degradation of c-di-GMP
    3. Motility induction due to no degradation of c-di-GMP
    4. Biofilm formation due to an accumulation of pGpG
  4. Deletion of PotD1 results in enhanced biofilm formation.  Based on the schematic, what is the reason  this happens?
    1. The transport of spermidine into the cell is blocked allowing it to bind NspS and inactivate MbaA, keeping cyclic-di-GMP low.
    2. The transport of norspermidine into the cell is activated allowing it to bind NspS and internalize MbaA, keeping cyclic-di-GMP low.
    3. The transport of spermidine into the cell is blocked allowing it to bind NspS and inactivate MbaA, keeping cyclic-di-GMP high.
    4. The transport of norspermidine into the cell is blocked allowing it to bind NspS and inactivate MbaA, keeping cyclic-di-GMP high.
  5. Inhibition of MbaA might be a good strategy to control the spread of cholera infection.  Pick the statement that justifies this as a good strategy.
    1. Inhibition of MbaA would stop production of c-di-GMP, maintaining high intracellular levels of pGpG which kills them.
    2. Inhibition of MbaA would enhance production of pGpG, maintaining low intracellular levels of c-di-GMP and thus attachment.
    3. Inhibition of MbaA would stop production of pGpG, maintaining high intracellular levels of c-di-GMP and thus attachment.
    4. Inhibition of MbaA would enhance production of c-di-GMP, maintaining low intracellular levels of c-di-GMP which kills them.

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Identify key features in microscopy images, line graphs, and box plots.
  • Analyze biofilm and other related data from strains lacking different genes and draw conclusions regarding the role of the genes in cholera biofilm formation.
  • Make a recommendation for a gene to target for cholera control procedures.
Experimental Background (Bridges and Bassler, Figure 2)

Vibrio cholerae is the causative agent for cholera, which has resulted in seven major outbreaks in recorded history.  In the absence of treatment, which occurs often in undeveloped areas of the world, it has a 50-60% mortality rate.  Clean water, proper hygiene, and access to medical care are important factors in its control, so undeveloped world areas are particularly at risk.  Understanding how this organism regulates the change between its sessile (biofilm) and motile forms will also inform making better containment protocols.  Cells cycle between attachment to a surface, where cells are non-motile, and dispersal, where they become swimmers and leave the biofilm community to colonize elsewhere. While the processes controlling biofilm formation are well characterized, the signals triggering dispersal are not. In this study, Bridges and Bassler (2021) mutagenized Vibrio cholerae and identified strains that are unable to disperse. Two of the genes identified as variant in these mis-regulated strains were mbaA, which encodes an inner membrane protein, and potD1, which encodes a periplasmic protein.  To investigate confirm these genes are in fact responsible for the dispersal defect, they engineered strains that were deleted in each gene and examined a variety of dispersal metrics, including microscopy (panel A), biofilm biomass accumulation (panel B), difference in abundance of a known biofilm regulator molecule (cyclic-di-GMP) using a reporter gene (panel C), and activation of a biofilm-related operon as cellular density increased, also using a reporter gene (panel D).

microscopy, line, and box plots
Figure 2. “Polyamine signaling regulates V. cholerae biofilm dispersal. (A) Representative images of the designated V. cholerae strains at 16 hr. (B) Quantitation of biofilm biomass over time measured by time-lapse microscopy for WT V. cholerae and the designated mutants. In all cases, N = 3 biological and N = 3 technical replicates, ± SD (shaded). a.u.: arbitrary unit. (C) Relative c-di-GMP reporter signals for the indicated strains. Values are expressed as the percentage difference relative to the WT strain. N = 4 biological replicates. Each black bar shows the sample mean. Unpaired t-tests were performed for statistical analysis, with p values denoted as *p<0.05; ***p<0.001. (D) The corresponding PvpsL-lux outputs for the strains and growth conditions in (B). For vpsL-lux measurements, N = 3 biological replicates, ± SD (shaded). RLU: relative light units.” (Bridges and Bassler 2021, no changes)

7.2.2. Questions

  1. The Vibrio cells are different in the three microscopy images in panel A.  What makes them different?
    1. They are mutant in different genes in biofilm formation.
    2. They are different Vibrio species that all cause cholera.
    3. They have been treated with three different antibiotics.
    4. They are strains isolated from different environments.
  2. Match the strain to the symbol used to denote its data for panels B and D.
Strain Symbols
a. _____ ΔmbaA 1.Blue circle
b. _____ ΔpotD1 2. Black circle
c. _____WT 3. Blue square
  1. Biofilm biomass was quantified for each strain (panel B). Which strain shows the greatest difference compared to wild-type?  What is the difference?
    1. mbaA deletion; forms more extensive and lasting biofilms
    2. mbaA deletion; forms loose biofilms that disperse quickly
    3. potD1 deletion; forms loose biofilms that disperse quickly
    4. potD1 deletion; forms more extensive and lasting biofilms
  2. The intracellular level of the biofilm regulator molecule cyclic-di-GMP was quantified in each strain using a reporter signal (panel C).  Wild-type is motile and not biofilm-forming under these growth conditions.  Which strain(s) has/have a significant defect in regulation of biofilm formation based on these data?  What is your evidence? [pick all that apply]
    1. potD1 deletion; the internal concentration of c-di-GMP is significantly lower than wild-type
    2. mbaA deletion; the internal concentration of c-di-GMP is significantly lower than wild-type
    3. potD1 deletion; the internal concentration of c-di-GMP is significantly higher than wild-type
    4. mbaA deletion; the internal concentration of c-di-GMP is significantly higher than wild-type
  3. The activation of the biofilm-regulating operon that includes the gene vpsL was quantified in each strain using a reporter signal (panel D).  Which strain(s) has/have a significant defect in gene expression of the representative of biofilm operon based on these data?  What is your evidence? [pick all that apply]
    1. mbaA deletion; expression of the vpsL reporter is significantly lower than wild-type
    2. potD1 deletion; expression of the vpsL reporter is significantly higher than wild-type
    3. mbaA deletion; expression of the vpsL reporter is significantly higher than wild-type
    4. potD1 deletion; expression of the vpsL reporter is significantly lower than wild-type
  4. Inhibition of one of these genes/proteins might be a good strategy to control cholera infection.  Which gene? What is your reasoning?
    1. potD1; the strain that lacks this gene forms more extensive biofilms making spread less likely.
    2. mbaA; the strain that lacks this gene forms looser biofilms making treatment more likely.
    3. potD1; the strain that lacks this gene forms looser biofilms making treatment more likely.
    4. mbaA; the strain that lacks this gene forms more extensive biofilms making spread less likely.

8. Paper Information and Licensing

8.1. Snippet paper

8.2. Main paper

License

Icon for the Creative Commons Attribution 4.0 International License

Podcast Annotation and Resources in Microbiology Copyright © 2025 by Rebecca Seipelt-Thiemann; Nancy Boury; Gwendowlyn S. Knapp; Amaya Garcia Costas; and Patrick Armstrong is licensed under a Creative Commons Attribution 4.0 International License, except where otherwise noted.

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