Impact of Microorganisms

TWiM #204: Programmable Bacteria for Antitumor Immunity

Podcast and Annotation Information
  • Annotation by Tony Uriostegui, Aidan Shoemaker, Jeremy Ritzert, and Rebecca Seipelt-Thiemann
  • Podcast audio by TWiM: Listen to TWiM #204 Podcast
  • Podcast transcript by Otter.ai and edited by Grace Helle, Harshita Sharma, Tony Uriostegui, Aidan Shoemaker, and Jeremy Ritzert: Access Podcast Transcripts
  • Papers Discussed:
    • Din MO, Danino T, Prindle A, Skalak M, Selimkhanov J, Allen K, Julio E, Atolia E, Tsimring LS, Bhatia SN, Hasty J. 2016. Synchronized cycles of bacterial lysis for in vivo delivery. Nature. 536(7614):81-85. doi: 10.1038/nature18930.
    • Chowdhury S, Castro S, Coker C, Hinchliffe TE, Arpaia N, Danino T. 2019. Programmable bacteria induce durable tumor regression and systemic antitumor immunity. Nat Med. 25(7):1057-1063. doi: 10.1038/s41591-019-0498-z.

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 12:10 minutes

The Most Interesting Things (according to students)

Using circuits in biology to create a plasmid for lysis to potential lead to targeted therapeutics was interesting.

This article is not licensed for Creative Commons use; see article copyright information. Thus, the abstract cannot be copied here.  A version of this paper is available on PubMed at: https://pmc.ncbi.nlm.nih.gov/articles/PMC5048415/

1.2. Main paper; discussion starts at 20:02 minutes

The Most Interesting Things (according to students)

Using bacteria to only seek out the tumor cells and colonize the tumors was interesting.  The nanobody with the bacteria lysis restores immune visibility because tumors selectively turn it off by expressing CD47.

This article is not licensed for Creative Commons use; see article copyright information. Thus, the abstract cannot be copied here. A version of this paper is available on PubMed at: https://pubmed.ncbi.nlm.nih.gov/31270504/

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

Snippet Main
Vision and Change Topics
  • Structure and Function (V&C_SF)
  • Impact of Microorganisms (V&C_IM)
  • Microbial Ecology (V&C_ME)
  • Structure and Function (V&C_SF)
  • Impact of Microorganisms (V&C_IM)
ASM Fundamental Statements
  • Fundamental Statement 26 (ASM_26): Humans leverage microbes and their products to address problems and improve quality of life.
  • Fundamental Statement 18 (ASM_18): The regulation of gene expression is influenced by external and internal molecular cues and signals.
  • 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 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 quorum sensing.
  • Identify the biological circuit components.
 S L
  • Predict phage lysis protein expression and tumor killing based on the circuit described and specific conditions.
 S H
  • Identify the biological circuit components.
  • Define nanobody and its advantages in the treatment.
 M L
  • Predict tumor outcomes if the anti-tumor activity had been solely microbe based.
 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

  • Genetic Engineering (12:10–13:35): These are techniques to modify bacteria using recombinant DNA techniques, such as polymerase chain reaction and restriction enzymes.  This was used in constructing the circuits used in the snippet study and also the main paper.
  • Synchronized Lysis Circuit (13:00–13:35): This is the engineered lysis circuit constructed using genetic engineering.  When the quorum sensing molecule AHL accumulates enough due to a localized number of bacteria, the quorum sensing induces gene expression of a phage lysis protein which kills tumor cells in the vicinity.
  • Genetic Engineering (13:37–14:50): These are techniques to modify bacteria using recombinant DNA techniques, such as polymerase chain reaction and restriction enzymes.  This was used in constructing the circuits used in the snippet study and also the main paper.
  • in vivo Bacterial Growth (17:26–17:42): These are techniques to quantify how many bacteria grew at the tumor site.   They measured this using the fluorescent molecule expressed in the bacteria and also colony forming units from homogenized tissues.

4.2. Main Paper

  • Nanobody (30:15–32:02): This is a small antibody-like protein engineered based on llama and alpaca antibodies that have only the heavy chain.  These are used to target the CD47 molecule rather than a much bigger antibody molecule.
  • Intertumoral Delivery (32:38–32:55): This is an injection into a specific site, here into the tumor. This would cause it to be localized to the tumor.
  • Intravenous Delivery  (36:07–36:32): This is an injection into the blood.  This would cause it to be systemic, not just localized to the tumor.
  • T Cell Response (41:52–43:35): T cells are a specific type of white blood cells.  There are a variety of kinds, such as CD8+ and CD4+.  The researchers talked about how T cells were mobilized in the experiment and which kinds.
  • Bacterial Localization (43:38–44:12):  These are techniques to localize bacteria to see if they disperse from the injection site.  The researchers used a luminescent tag to track where bacteria were located within the mice.

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

5.1. Snippet Paper

  • Quorum Sensing (14:22–14:48; 16:16–16:55): A quorum sensor system was engineered to trigger lysis or fluorescence once the bacteria reach a threshold number of bacteria.
  • Lysis of Bacteria (14:43–15:09; 15:51–16:15) Bacteria lysis releases the therapeutic phage lysis protein.
  • Evolution of Bacteria (17:02–18:26): The ability of bacteria to evolve was noted as a possible concern for therapeutic use.  In this system, the bacteria still lyse when re-isolated and tested, so the researchers did not see in mouse evolution of the engineered bacteria.

5.2. Main Paper

  • Bacteria Growth at Tumor Sites (19:10–19:56; 20:43–20:47; 36:16–36:53): The podcasters discussed why bacteria grow at tumor sites.
  • Phagocytosis and Antigen Presentation (21:55–23:32; 31:34–32:01; 41:30–41:44): In the absence of the programmed bacteria, when CD47 is present on the cell, it is not destroyed by phagocytosis.  When CD47 is not present, the cell is phagocytosed, degraded, and used to produce peptides for presentation to immune cells. When the programmed bacteria are present and reach the quorum level, the CD47 nanobody is released and triggers phagocytosis and destruction of the tumor cells.
  • Immune System Response to Tumor (23:34–25:06; 34:08–35:45; 40:24–46:13): The podcasters discussed how the nanobodies can trigger the adaptive & innate immune system.
  • Host Receptors (25:33–26:00; 26:46–27:42): CD47 is a molecule on healthy red blood cells.  When they get old, they have less and are then targeted for destruction.
  • Nanobody (27:44–30:13; 32:58–33:05): This is a small antibody-like protein engineered based on llama and alpaca antibodies that have only the heavy chain.  These are used to target the CD47 molecule rather than a much bigger antibody molecule.

6. Podcast Questions

  1. What is quorum sensing?
    1. It is bacterial cell communication using small molecules.
    2. It is a receptor-mediated way for bacteria to take up metals.
    3. It is a sensor system for detecting toxins from other bacteria.
    4. It is a method for using microbes to detect contaminants.
  2. What is the role of quorum sensing in the biological circuit described in the snippet discussion?
    1. It is used as a pore-forming complex to enhance destruction of specific tumor cells.
    2. It is used as a method of inducing gene expression to detect fluorescent molecules.
    3. It is used as a sensor for detecting threshold numbers of bacteria at the tumor site.
    4. It is used as a delivery system for anti-tumor antibody molecules as given in serum.
  3. In the circuit system, the AHL molecule is the sensor which then drives expression of the phage lysis proteins which kill tumor cells. If a molecule was produced near the tumor site that bound and sequestered all the AHL molecule, what would you expect to happen? [Pick all that apply]
    1. The AHL molecule would be produced.
    2. The AHL molecule would not be produced.
    3. Phage lysis proteins would be produced.
    4. Phage lysis proteins would not be produced.
    5. Tumor cells would be destroyed.
    6. Tumor cells would not be destroyed.
  4. What is the molecule that is “targeted” on the tumor in the main paper’s circuit, and where is it normally found?
    1. Interferon; phagocytic cells
    2. CD47; healthy red blood cells
    3. CD8; cytotoxic T cells
    4. CD4; helper T cells
  5. What is a nanobody and what advantage does this molecule provide the circuit?
    1. A small antibody-like protein; size and specificity
    2. A small molecular complex; creates pores in cells
    3. A small molecule inhibitor; toxifies tumor cells
    4. A small surface marker protein; targets tumor cells
  6. In the podcast the researchers describe an experiment where they intoduced tumor cells into each flank of a mouse’s leg, allowed tumors to form, then injected one of the flanks with their engineered bacteria (bacterial injection). They found that eventually both tumors were decreased, the bacterial injected flank and the non-bacterial injected flank.  If they had only seen the bacterial injected flank tumor decrease, what would have been their conclusion?
    1. Killing is mediated by circulating T cells.
    2. Killing is localized to the engineered bacteria.
    3. Killing is influenced by engineered T cells.
    4. Killing is mediated by cytotoxic phagocytes.

7. Figure Reading Exercises

The following are two figure reading exercises, one from the snippet paper (Figure 1abc) and one from the main paper (Figure 4a-g).

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to: 

  • Identify key features in a schematic, microscopy image, and/or line graph.
  • Identify key molecules in the biological circuit and their role in the circuit.
  • Analyze the data to make conclusions about the expression of the different circuit genes.
  • Predict under which conditions key molecules in the biological circuit should be expressed.
  • Analyze the data and make conclusions about the biological circuit’s success.
Experimental Background (Din et al., Figure 1abc)

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, Din et al. (2016) cleverly engineer a bacterium with the ability to target and deliver therapeutics in the body.  This has treatment potential for a number of diseases, but particularly cancers, which affect 1 in 5 people over their lifetime.  The bacteria (non-pathogenic E. coli) were engineered with two plasmids to drive expression of four genes: an activator gene [luxI], a reporter gene [sfGFP], a therapeutic gene [hlyE], and a lysis gene [ɸX174E] (panel a).  Expression of the genes, and thus fluorescence (due to gfp), lysis (due to ɸX174E), and therapeutic delivery of HylE protein should only occur when the quorum sensing molecule AHL reaches a threshold level, that is when the local E. coli population is high.  To test their system in vitro, the researchers populated a growth chamber and examined bacterial fluorescence, growth, and lysis over time using fluorescent microscopy (panel b).  These measures were also quantified over time as fluorescence and bacterial number (panel c).

7.1.2. Questions

  1. Which gene in the schematic encodes the fluorescence reporter and which color shows its coding region (panel a)?
    1. hylE; orange
    2. sfGFP; green
    3. luxI; blue
    4. ɸX174E; red
  2. What molecule in the schematic is the quorum sensor molecule and what shape and color shows its location in the circuit (panel a)?
    1. HylE; orange rectangle
    2. GFP; green rectangle
    3. LuxR; dark blue ovals
    4. AHL; sky blue circle
  3. What conditions are required for luxI to be produced? [pick all that apply]
    1. When AHL is at high levels
    2. When LuxR is present
    3. Following when lysis occurs
    4. When HylE is at high levels
  4. What conditions are required for lysis to occur? [pick all that apply]
    1. When HylE is at present
    2. When AHL is at high levels
    3. When ɸX174E is produced
    4. When LuxR is present
  5. Which microscopy image(s) in panel b show(s) successful production of the green fluorescent protein reporter?
    1. Left image
    2. Middle image
    3. Right image
    4. None
  6. According to the circuit description, which of the circuit genes should be expressed in the center microscopy image of panel b?
    1. hylE
    2. sfGFP
    3. luxI
    4. ɸX174E
  7. Why are there fewer cells in the right microscopy image than the center microscopy image in panel b?
    1. Cells are being lysed.
    2. Cells are losing GFP.
    3. Cells die due to aging.
    4. Cells stop dividing.
  8. Which of the culture conditions shown in the microscopy images (panel b) would have the greatest amount of the quorum sensor molecule AHL?
    1. Left image
    2. Middle image
    3. Right image
    4. All are equal
  9. Fluorescence and cell number are both quantified in data panel c, which has two y-axes.  Match the measure with its y-axis and the color line that represents the data for that measure? [pick all that are correct]

L = left y-axis; R = Right-y axis; U = Blue line; A = Black line

    1. _______ Fluorescence
    2. _______ Cell number
  1. Based on the data presented for culture appearance and culture dynamics, what level of success did the researchers achieve?
    1. Excellent; the researchers show us convincing evidence for production of all four of the gene products.
    2. Very good; the researchers show us convincing evidence for production of three of four gene products.
    3. Good; the researchers show us convincing evidence for production of two of the four gene products.
    4. Ok; the researchers show us convincing evidence for production of only one of four gene products.

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Identify key features in a schematic, line graph, bar-type graph, and scatterplot.
  • Identify key molecules in the engineered bacterial delivery system and their role in the system.
  • Predict under which conditions the therapeutic molecule will be released into the environment.
  • Analyze the data and make conclusions about the researcher’s competing hypotheses regarding systemic activation of the immune system and localized bacterial effects.
  • Analyze the data and make conclusions about the engineered bacterial delivery system as a tumor therapy.
  • Predict when this treatment would and would not be effective.
Experimental Background (Chowdhury  et al., Figure 4a-g)

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, Chowdhury et al. (2016) cleverly engineer a bacterium with the ability to target and deliver therapeutics in the body.  This has treatment potential for a number of diseases, but particularly cancers, which affect 1 in 5 people over their lifetime.  The bacteria (non-pathogenic E. coli) were engineered with a plasmid to drive expression of two genes: an activator gene [luxI] and a lysis gene [ɸX174E] when the bacterial population reached a threshold level.  This was done using a quorum sensor molecule, AHL.  Bacteria also were engineered to constitutively express a therapeutic gene encoding a nanobody of 15 kiloDaltons (eSLC-CD47nb) or not (eSLC).  Expression of the activator and lysis genes, as wells as the therapeutic delivery of nanobody protein (if encoded), should only occur when the quorum sensing molecule AHL reaches a threshold level, that is when the local E. coli population is high.  To test their system in vivo, in particular whether decreases in tumor mass were due to system-wide activation of the immune system or due to localized lysis related to bacterial lytic factors, the researchers introduced tumor cells into each flank of mice and allowed tumors to form.  They then used intratumoral injection at days 0, 4, 7, and 11 to deliver their engineered bacteria (eSCL or eSCL-CD47) or phosphate buffered saline into one tumor (one flank) of each mouse (panel a).  They then quantified tumor mass in the injected tumors (panel b) and in the non-injected corresponding tumors (panel c).  They compared the growth dynamics of the pair of tumors using a scatterplot with injected tumor mass on the x-axis and non-injected tumor mass on the y-axis (panel d).  They next turned to investigating whether immune cells had infiltrated the tumor.  They excised the non-injected tumors and performed a type of cell sorting analysis to quantify the number of Ki-67+CD8+ T cells, which are known to be involved in tumor killing (panel e).  They also quantified whether these immune cells were activated by measuring gamma-interferon produced when they were incubated with a tumor peptide peptide (panel f). In line with their original purpose for this set of experiments, to determine whether bacteria were directly responsible or the tumor decrease was due to their activation of the immune system, they next investigated where the bacteria could be found in the mice, with a specific focus in each tumor (injected and non-injected), as well as spleen (a known immune tissue), and liver (panel g).   Bacteria growing in the tissue’s homogenate were quantified as colony forming units (CFU).

7.2.2. Questions

  1. Which statement(s) describe(s) the tumor injection protocol (panel a and figure background)? [pick all that apply]
    1. Injection of the therapeutic bacteria is done four times after tumors are formed.
    2. Injection of the tumor cells is done a total of four times after bacteria are injected.
    3. Bacteria are injected into both flanks of a mouse; tumor cells are injected into one flank.
    4. Tumors are formed in both flanks of a mouse; bacteria are injected into one tumor.
  2. What conditions are required by the bacteria for lysis to occur? [pick all that apply]
    1. When CD8+ T cells are present
    2. When AHL is at high levels
    3. When ɸX174E is produced
    4. When the nanobody is produced
  3. Match the treatment with the color and name that shows its data across all experiments.
Notation Treatment
a. _____ blue (PBS) 1. Engineered bacteria with the lysis circuit and expressing the CD47 nanobody
b. _____ green (eSLC) 2. Engineered bacteria with the lysis circuit
c. _____ red (eSLC-CD47nb) 3. Engineered bacteria expressing the CD47 nanobody
d. _____ brown (eCD47nb) 4. No bacteria
  1. What can you conclude about the engineered bacteria’s ability to affect tumor volume for the treated and untreated tumors (panels b and c)? [pick all that apply]
    1. Only the bacteria with the lysis circuit and the nanobody reduce both tumors.
    2. The bacteria with the lysis circuit with or without nanobody reduce both tumors.
    3. The bacteria with the lysis circuit and the nanobody reduce only treated tumors.
    4. The bacteria with the lysis circuit with or without nanobody reduce treated tumors.
  2. The tumor volume of the untreated versus the treated is displayed as a scatterplot in panel d.  If the tumor volumes were equal for a particular mouse, where would you find the data point for that mouse?  Why would you find it there?
    1. Above the dotted line; the axes aren’t the same scale
    2. Below the dotted line; tumor mass for treated would be less.
    3. On the dotted line; the coordinates for x and y would be equal.
    4. In the lower left quadrant; the growth is reduced for both.
  3. The tumor volume of the untreated versus the treated is displayed as a scatterplot in panel d. All data points are above the dotted line, which has a slope of 1.  What does this allow you to conclude?
    1. Only treated tumors are responding to the treatment.
    2. Untreated tumor mass is more than treated tumor mass
    3. Only untreated tumors are responding to the treatment.
    4. Treated tumor mass is more than untreated tumor mass
  4. Data are displayed as strip plots in panels e and f.  Match each strip plot feature to its description/name.
Notation Feature Name
a. _____ dot 1. Statistical significance
b. _____ horizontal line 2. Standard error
c. _____ asterisk 3. Individual mouse data point
d. _____ error bar/whisker 4. Mean
  1. What can you conclude about the engineered bacteria’s ability to attract the mouse’s immune system for the treated and untreated tumors (panel e)?
    1. The bacteria with the lysis circuit with or without nanobody increase CD8+ T cells entering the tumor regardless of the tumor treatment.
    2. Only the bacteria with the lysis circuit and the nanobody increase CD8+ T cells entering the tumor, but it only happens for treated tumors.
    3. Only the bacteria with the lysis circuit and the nanobody increase CD8+ T cells entering the tumor in both treated and untreated tumors.
    4. The bacteria with the lysis circuit with or without nanobody increase CD8+ T cell infiltration into the tumor, but only for treated tumors.
  2. What do the levels of IFNγ produced by CD8+ T cells isolated from the tumors indicate (panel f)?
    1. Direct bacterial colonization and bacterial-induced lysis of all tumors.
    2. Inhibition of T cell infiltration and activity by eSLC–CD47nb bacteria.
    3. Systemic activation of adaptive immunity in all test conditions.
    4. Systemic activation of adaptive immunity by eSLC–CD47nb bacteria.
  3. Based on the bacterial location experiments (panel g), are the bacteria found systemically or are they localized to the treatment injection site? What is your evidence?
    1. Systemically; bacteria disseminated to the liver and spleen after injection.
    2. Localized; bacteria remained confined to treated tumors over time.
    3. Systemic; bacteria were able to disseminate to the untreated tumor.
    4. Localized, then systemic; bacteria are found in the spleen after day 6.
  4. Metastasis is when tumor cells migrate to a new location in the body.  They often acquire new mutations in doing this. If the original tumor was CD47-positive and metastasis A was CD47-positive, but metastasis B lost expression of CD47, what would you predict for the success of using this treatment?
    1. Not effective for any of the three tumor sites because the tumors have changed.
    2. Effective for all three tumor sites because this treatment was shown to be effective.
    3. Effective for the original tumor and metastasis A because they still express CD47.
    4. Not effective for the metastases because the metastases are different tumors.

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.

Share This Book