Microbial Ecology

TWiM #186: Crypto-metamorphosis

Podcast and Annotation Information

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 37:37 minutes (yes, it is last)

The Most Interesting Things (according to students)

Hosts can undergo metamorphosis when associated with endosymbionts, and can even show increased size and/or growth rate in certain species and environments.

This article is not licensed for Creative Commons use; see article copyright information. Thus, the abstract and figures cannot be copied here.

1.2. Main paper; discussion starts at 5:30 minutes

The Most Interesting Things (according to students)

Listeria can be modified to train the immune system to recognize and fight tumors in mice.

“Agents that remodel the tumor microenvironment (TME), prime functional tumor-specific T cells, and block inhibitory signaling pathways are essential components of effective immunotherapy. We are evaluating live-attenuated, double-deleted Listeria monocytogenes expressing tumor antigens (LADD-Ag) in the clinic. Here we show in numerous mouse models that while treatment with nonrecombinant LADD induced some changes in the TME, no antitumor efficacy was observed, even when combined with immune checkpoint blockade. In contrast, LADD-Ag promoted tumor rejection by priming tumor-specific KLRG1+PD1loCD62L− CD8+ T cells. These IFNγ-producing effector CD8+ T cells infiltrated the tumor and converted the tumor from an immunosuppressive to an inflamed microenvironment that was characterized by a decrease in regulatory T cells (Treg) levels, a proinflammatory cytokine milieu, and the shift of M2 macrophages to an inducible nitric oxide synthase (iNOS)+CD206− M1 phenotype. Remarkably, these LADD-Ag–induced tumor-specific T cells persisted for more than 2 months after primary tumor challenge and rapidly controlled secondary tumor challenge. Our results indicate that the striking antitumor efficacy observed in mice with LADD-based immunotherapy stems from TME remodeling which is a direct consequence of eliciting potent, systemic tumor-specific CD8+ T cells..” (Deng et al. 2018)

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)
  • Structure and Function (V&C_SF)
  • Impact of Microorganisms (V&C_IM)
ASM Fundamental Statements
  • Fundamental Statement 13 (ASM_13): Intrinsic factors, such as genotype, metabolism, and cell structures, impact the survival and growth of microbes.
  • Fundamental Statement 20 (ASM_20):Microbes are ubiquitous, found in diverse and dynamic ecosystems, where they use available resources and often form complex communities.
  • Fundamental Statement 22 (ASM_22): Most microbes interact with hosts in beneficial or neutral ways, with a minority having a detrimental impact on their host.
  • Fundamental Statement 6 (ASM_6): The distinct structures and processes in microbes can be targets for interspecies competition, antimicrobial treatments, and host immunity.
  • Fundamental Statement 22 (ASM_22): Most microbes interact with hosts in beneficial or neutral ways, with a minority having a detrimental impact on their host.
  • Fundamental Statement 26 (ASM_26): Humans leverage microbes and their products to address problems and improve quality of life.

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • Define trophosomes and explain how they relate to bacteriocytes.
  • Recall how the snail organs changed as the snail became sessile.
 S L
  • Predict what would happen to a sessile snail if it was moved to a low sulfur environment.
 S H
  • Identify common exposures to Listeria.
  • Recall the changes that were made to Listeria so that this normally intracellular pathogen could not exit the host cell.
 M L
  • Predict how prior infections might affect treatment success.
 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

  • Micro Computed Tomograph (CAT) Scans (47:43–50:11): This is a technique that uses X-rays to take images of sections and then use them to create 3-D representations of structures.  It was used to analyze the structures of the digestive systems of snails of different sizes.
  • Electron Microscopy (EM) (50:24–50:33): This is a high resolution microscopy technique.  It was used to see the trophosomes of sessile adult snails were filled with bacteriocytes.

4.2. Main Paper

  • Mutation/Mutants (12:30–13:55): A mutation is a change in the DNA sequence.  Here, the researchers constructed a (mutant) strain of Listeria with a deletion of actA and internalinB to prevent cellular movement of Listeria in host.
  • Mouse Models (14:00–15:15): Model organisms are organisms that are commonly used in experiments because they are usually small, easy to grow, and have well-defined genomes or other data.  Here, the mouse model used for experimental cancer therapy to see if it might be applicable to human tumors.
  • Koch’s Postulates (15:15–18:00): These are a series of “rules” for establishing a pathogen causes a disease.  In this instance, it was applied to the engineered Listeria’s ability to destroy tumor cells.
  • Flow Cytometry (20:09–20:40): This is a cell sorting technique, usually involving fluorescence, to examine cell surface markers on cells.  It was used to look at the surface of CD8 cells because expression of certain markers correlate with specific types and functions of T cells.

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

5.1. Snippet Paper

  • Metamorphosis (40:17–44:45; 47:20–47:40; 50:40–52:20): Snail goes from grazing animal to 100% dependent on bacterial endosymbionts. Crypto metamorphosis  is a metamorphosis that can’t be seen from the outside.
  • Differentiation (45:30–47:40; 50:24–50:40): Trophosomes are differentiated esophageal glands that work as a bacteria housing organ. Bacteriocytes are host cells containing a symbiotic bacterium.
  • Chemosymbiotic Holobionts (53:18–54:54): Chemosymbiotic bacteria produce things which are needed by the snail, and holobiont because there are two distinct organisms involved.

5.2. Main Paper

  • Epidemiology and Public Health (6:40–7:30): Listeria is a food-borne pathogen that can cross placental barrier and cause meningitis in neonates.
  • Tolerance and Cell Mediated Immunity (7:30–11:00): A variety of cell-mediated immune cells, their roles, and activation were discussed.
  • Engineered Bacteria (11:00–24:50): A Listeria double mutant could be used to design cell mediated immunity against tumors.
  • Mechanisms of Pathogenesis (12:30–13:55): Actin tails mediate movement of Listeria inside host cell and from cell to cell.
  • Microbe/Pathogen Associated Molecular Patterns (MAMPs/PAMPs) (23:50–24:50): Microbe/Pathogen Associated Molecular Patterns and Toll-Like receptors are molecules from (internally) bacteria that signal the presence of invaders to the host immune system.

6. Podcast Questions

  1. What is the relationship of the trophosome and bacteriocyte in the snails?
    1. Trophosomes are snail organs that are filled with bacteriocytes.
    2. Bacteriocytes are the small, immature versions of trophosomes.
    3. Bacteriocytes are found in gills, trophosomes are found internally.
    4. Trophosomes are external versions; bacteriocytes are internal.
  2. What structural changes did the researchers identify as the snail changed from free-moving to sessile?
    1. The snail formed disc-like structures for attachment.
    2. The outer structures curved in and became a pocket.
    3. The esophageal gland enlarged and filled with bacteria.
    4. The outer cells exuded polysaccharides for attachment.
  3. What would likely happen to a sessile snail if it was moved to a low sulfur environment?
    1. The snail would grow very large.
    2. The snail would remain very small.
    3. The snail would return to free-living.
    4. The snail would float to the surface.
  4. How are people commonly infected with Listeria? [pick all that apply]
    1. Eating contaminated meat
    2. Inhaling contaminated droplets
    3. Drinking contaminated milk
    4. Touching infected surfaces
  5. Which two genes did the researchers mutate in their Listeria strain so that it would not exit a cell once it was phagocytosed?
    1. internalin B
    2. toll receptor
    3. cyt B
    4. actA
  6. The researchers note that we have no vaccine to Listeria, an intracellular pathogen, because immunity to this pathogen is long-lived, unlike for flu or tetanus.  The Listeria involved in these anti-tumor experiments differed from environmental Listeria in several ways, including expressing common tumor antigens and having dysfunctional ability to exit the cell.  If you were testing this agent in a person, how do you think the outcome would be affected by a recent food poisoning event?
    1. It would be more effective than without the food poisoning.
    2. It would be less effective than without the food poisoning.
    3. It would be equally effective with or without food poisoning.
    4. It would be completely ineffective with the food poisoning.

7. Figure Reading Exercises

The following are two figure reading exercises, one from the snippet paper (Figure 1) and one from the main paper (Figure 5).

7.1. First Figure Reading Exercise  

7.1.1. Learning Objectives

Students will be able to: 

  • Identify key features in the 3-dimensional renderings.
  • Analyze the data to make conclusions about snail changes over its metamorphosis.
  • Predict what would happen to a sessile snail if it was moved to a low sulfur environment.
  • Predict the likelihood of classical endosymbiotic relationship features.
Experimental Background (Chen et al., Figure 1)

When most people consider metamorphosis, they usually remember metamorphosis for insects such as the butterfly, where there are egg, larva, pupa, and adult butterfly stages.  There are also other metamorphoses that are less visible, but no less elegant.  One such metamorphosis is described and investigated by Chen et al. (2019).  This group of researchers studied the large marine snail Gigantopelta chessoia that lives part of its life as small free-moving individuals, then becomes sessile and grows very large.  In this process, the giant snail has an internal metamorphosis that mirrors its lifestyle change.  The small free-living snail captures most of its nutrient and other needs on its own, but as it becomes sessile it enters into an endosymbiotic relationship as it forms new internal structures to accommodate the endosymbiont.  The endosymbionts provide considerable energy to the snail via chemotrophy involving sulfur compounds.  To characterize the internal structure changes, Chen et al (2019) performed micro computational tomography (micro CAT scans) and generated 3-dimensional renderings (panels a-f).

  • This article is not licensed for Creative Commons use; see copyright information. Thus, the abstract and figures cannot be copied here.

7.1.2. Questions

  1. What color denotes the trophosome, where the endosymbionts live?
    1. Blue
    2. Green
    3. Yellow
    4. Gray
  2. What is the change in size for the snail over the 3-dimensional renderings?
    1. Doubles (2x)
    2. Triples (3x)
    3. Five times (5x)
    4. Ten times (10x)
  3. How would you describe the changes in the trophosome as the metamorphosis occurs (panels a-f)? [pick all that apply]
    1. The trophosome gets larger as the changes occur.
    2. The snail gets larger as the metamorphosis proceeds.
    3. The trophosome makes up more of the snail body.
    4. The metamorphosis changes the external snail structures.
  4. What would likely happen if a sessile snail at the 2.2mm stage was moved to a low sulfur environment?
    1. The snail would grow very large.
    2. The snail would remain very small.
    3. The snail would return to free-living.
    4. The snail would float to the surface.
  5. Many times endosymbionts and their hosts are involved in horizontal gene transfer (HGT).  What would you predict for the amount of horizontal gene transfer between these species compared to permanent, inherited endosymbiont-host relationship? Why?
    1. This relationship would be less likely to involve HGT because the endosymbiont lives free at least some of the time.
    2. This relationship would be more likely to involve HGT because the endosymbiont increases in mass as the snail ages.
    3. This relationship would be less likely to involve HGT because the endosymbiont lives in the sulfur-rich thermal vents.
    4. This relationship would be more likely to involve HGT because the endosymbiont has a smaller surface to volume ratio.

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Identify key features in a bar graph, line graph, and cell sorting style scatterplot.
  • Identify controls in these experiments and their purpose.
  • Analyze the data and make conclusions about the types of cells involved in the response.
  • Analyze the data and make conclusions about the engineered bacterial delivery system’s long term efficacy as tumor therapy.
Experimental Background (Deng et al. 2018, Figure 5)

Microbes account for almost 99% of the world’s species, an estimated 1 trillion species, and make up 13-15% of the biomass.  While we often see reports of pathogenic microbes, there are also naturally neutral and beneficial microbes.  Here, Deng et al. (2018) engineer a common, food-borne, intracellular pathogen bacterium (Listeria monocytogenes) in an effort to reshape the immune response towards killing tumor cells.  This has treatment potential for a number of diseases, but particularly cancers, which affect 1 in 5 people over their lifetime.  In their study, they found that injecting bacteria that were engineered to constitutively express tumor antigen (strain LADD-AH1) reduced mouse tumor size compared to injection with the strain that does not express the tumor antigen (strain LADD).  To test the long term efficacy of this treatment, the researchers first quantified several types of immune cells in survivors and naive mice, including CD8+ T cells primed to tumor antigen (panel A).  They next used a cell sorting technique* to identify other very specific groups of immune cells.   The cell surface markers/molecules they quantified were KLRG1 (a marker for memory killer T cell; panel B), Ki67 (a proliferation marker; panel C), T-bet (a marker for memory T cells; panel D), all along with an AH1-specific receptor (AH-1; x -axis for panels B-D).  In their final experiment for this study, the researchers were interested to determine the long term efficacy on recurrent tumors, that is whether having undergone a first successful tumor treatment could affect new tumor formation.  To do this, the researchers injected naive and survivor mice with tumor cells.  Survivors were also treated with a control immunoglobulin (IgG) or an antibody to the CD8 molecule.  All were monitored for tumor growth (panel E).

  • This technique quantifies how much of a specific molecule is present on the cell surface by displaying the data as a scatterplot and marking it into quadrants. Cells that are low for both surface molecules will be found in the lower left quadrant, cells with high levels for both surface molecules will be found in the upper right quadrant.  The other patterns, high/low or low/high will be found in the lower right and upper left quadrants.
bar graph, FACS, line graph
Figure 5. “LADD-AH1 induces memory effector CD8+ T cells that protect survivors from CT26 rechallenge. Survivors and naive mice were reinoculated with CT26 in their left thoracic flanks. (A–D) Before reinoculation, (A) frequency of peripheral AH1 tetramer+ of CD8+ T cells, (B–D) Representative staining of AH1 tetramer and (B) KLRG1, (C) Ki67, or (D) T-bet of peripheral CD8+ T cells in survivors. (E) S.c. growth of 5 × 105 CT26 in naive mice (n = 5) and survivors treated with control IgG or α-CD8. Tumor volumes ±SEM are shown. In scatter plots, each circle represents one mouse. FACS plots show representative analysis for one mouse per group. A was analyzed by Mann–Whitney U tests. E was analyzed by two-way ANOVA. *P < 0.05 and ****P < 0.0001. Results are representative of at least two independent experiments.” (Deng et al. 2018)

7.2.2. Questions

  1. Match the important features/parts of the data displayed in panel A to their names.
Notation Treatment
a._____ circle 1. Statistical significance
b. _____ bar 2. A survivor mouse data point
c. _____ asterisk 3. A naive mouse data point
d. _____ square 4. Mean
  1. What can you conclude about the presence of CD8+ T cells that also have the AH1-specific receptor in naive and survivor mice (panel A)? [pick all that apply]
    1. Only the survivor mice have these cells in high abundance.
    2. These cells are equally abundant in naive and survivor mice.
    3. Only the naive mice have these cells in high abundance.
    4. These cells are not abundant in naive or survivor mice.
  2. In which quadrant of panel B would you find the datapoint for a cell that has high surface expression of KLRG1 and low surface expression of the AH1-specific receptor?
    1. Upper right
    2. Upper left
    3. Lower right
    4. Lower left
  3. Which of the following statements are true about the CD8+ T cells in panels B-D?
    1. Most of the CD8+ T cells that are T-bethi are also AH1hi
    2. Most of the CD8+ T cells are Ki67hi regardless of AH1 level.
    3. Most of the T cells are either AH1lo KLRG1lo or AH1hi KLRG1hi
    4. Most of the CD8+ T cells that are T-bethi are also KLRG1lo
  4. Knowing that T cell markers are markers for specific functional classes of T cells, as noted in the figure background, what can you conclude about the CD8+ T cells that are present in survivor mice (panels B-D)?
    1. All of the peripheral CD8+ T cells are non-proliferating memory T cells specific for AH1.
    2. Most of the peripheral CD8+ T cells are non-proliferating memory T cells specific for AH1.
    3. Some the peripheral CD8+  T cells are non-proliferating memory T cells specific for AH1.
    4. There are no peripheral CD8+ non-proliferating memory T cells that are specific for AH1.
  5. In their final experiment for this study, the researchers were interested to determine the long term efficacy on recurrent tumors, that is whether having undergone a first successful tumor treatment could affect new tumor formation.  The data for this experiment is shown in panel E.  Match the treatment group to what it will tell us about the survivors.
condition control/explanation
a. ____ Naive mice 1. will show us the role of T cells in tumor recurrence
b. ____ Survivor mice treated with anti-CD8 antibody 2.  will show us whether tumor cells can form tumors in mice
c. ____ Survivor mice treated with immunoglobulin G (IgG) 3. will show us whether the treatment is effective for recurrent tumors
  1. What can you conclude about the treatment’s ability to affect tumor recurrence?
    1. The treatment was successful. No tumors formed in survival mice treated with IgG.
    2. The treatment was partially successful. Some tumors formed in survivors with anti-CD8.
    3. The treatment was not successful. Some tumors formed in both survivor mice groups.
    4. The treatment was partially successful. Fewer tumors formed in survivors than naive.

8. Paper Information and Licensing

8.1. Snippet paper

8.2. Main paper

  • Deng W, Lira V, Hudson TE, Lemmens EE, Hanson WG, Flores R, Barajas G, Katibah GE, Desbien AL, Lauer P, Leong ML, Portnoy DA, Dubensky TW Jr. 2018. Recombinant Listeria promotes tumor rejection by CD8+ T cell-dependent remodeling of the tumor microenvironment. Proc Natl Acad Sci U S A. 115(32):8179-8184. https://doi.org/10.1073/pnas.1801910115
  • This article is licensed for Creative Commons use using CC BY-NC-ND 4.0, which allows non-commercial re-use with no adaptation with proper attribution. See https://www.pnas.org/doi/full/10.1073/pnas.1801910115

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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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