Evolution

TWiM #268: Aspergillus and Aspergillum

Podcast and Annotation Information

  • Annotation by Gabrielle Baca, Benji Torres  Lauren Farrar, Damaris Hernandez  Enrique Rodriguez, Jovani Catalan-Dibene, Rebecca Seipelt-Thiemann, Suparna Chatterjee
  • Podcast audio by TWiM: Listen to TWiM #268 Podcast
  • Podcast transcript by Otter.ai and edited by Isabelle Norris: Access Podcast Transcripts
  • Papers Discussed:
    • Shelton JMG, Collins R, Uzzell CB, Alghamdi A, Dyer PS, Singer AC, Fisher MC. 2022. Citizen Science Surveillance of Triazole-Resistant Aspergillus fumigatus in United Kingdom Residential Garden Soils. Appl Environ Microbiol. 88(4):e0206121. https://doi.org/10.1128/AEM.02061-21
    • Menghani SV, Cutcliffe MP, Sanchez-Rosario Y, Pok C, Watson A, Neubert MJ, Ochoa K, Wu HJ, Johnson MDL. 2022. N,N-Dimethyldithiocarbamate Elicits Pneumococcal Hypersensitivity to Copper and Macrophage-Mediated Clearance. Infect Immun. 90(4):e0059721. https://doi.org/10.1128/iai.00597-21

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 3:44 minutes

The Most Interesting Things (according to students)

The geographical distribution of Aspergillus fumigatus in the UK and the distribution of resistance and susceptible strains.

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

1.2. Main paper; discussion starts at 24:25 minutes

The Most Interesting Things (according to students)

N,N-Dimethyldithiocarbamate increases intracellular copper concentration, which will sensitize S. pneumoniae to killing by macrophages.

“Streptococcus pneumoniae is a Gram-positive, encapsulated bacterium that is a significant cause of disease burden in pediatric and elderly populations. The rise in unencapsulated disease-causing strains and antimicrobial resistance in S. pneumoniae has increased the need for developing new antimicrobial strategies. Recent work by our laboratory has identified N,N-dimethyldithiocarbamate (DMDC) as a copper-dependent antimicrobial against bacterial, fungal, and parasitic pathogens. As a bactericidal antibiotic against S. pneumoniae, DMDC’s ability to work as a copper-dependent antibiotic and its ability to work in vivo warranted further investigation. Here, our group studied the mechanisms of action of DMDC under various medium and excess-metal conditions and investigated DMDC’s interactions with the innate immune system in vitro and in vivo. Of note, we found that DMDC plus copper significantly increased the internal copper concentration, hydrogen peroxide stress, nitric oxide stress, and the in vitro macrophage killing efficiency and decreased capsule. Furthermore, we found that in vivo DMDC treatment increased the quantity of innate immune cells in the lung during infection. Taken together, this study provides mechanistic insights regarding DMDC’s activity as an antibiotic at the host-pathogen interface.” (Menghani et al. 2022)

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

Snippet Main
Vision and Change Topics
  • Evolution (V&C_E)
  • Structure and Function (V&C_SF)
  • Evolution (V&C_E)
  • Microbial Ecology (V&C_ME)
  • Structure and Function (V&C_SF)
ASM Fundamental Statements
  • Fundamental Statement 3 (ASM_3): The evolution of microbes is impacted by their interactions with the environment and a variety of ecological forces, including other microbes, humans, and habitats.
  • 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 8 (ASM_8): Microbes have unique genomes, structures, and/or biochemical characteristics that distinguish them from each other.
  • Fundamental Statement 28 (ASM_28): A minority of microbes are pathogens that can cause diseases and harm host organisms, society, and ecosystems.
  • 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
  • Identify the improvement that Hesse contributed to culturing microbes.
  • Recall the features of Aspergillus fumigatus.
  • Identify the source of fungicidal exposures that may have led to resistance.
 S L
  • Propose how this study is a good example of One Health concepts.
 S H
  • Identify the features/uses of copper in living systems.
  • Evaluate scenarios and pick out an example of nutritional immunity.
 M L
  • Provide experimental conclusions based on alternative, hypothetical results.
 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

  • Soil Sample Collections (12:40–13:30): Soils, which are a natural habitat for microbes, are collected and examined.  Here, they were used to determine the number of triazole-resistant/non-resistant A. fumigatus spores.
  • Survey (13:38–15:00): Surveys are questionnaires used to record environmental and demographic data.  Here, they were used to determine environmental associations from the reported survey taken simultaneously with soil sample collection.

4.2. Main Paper

  • Vaccines (28:00–29:33): Vaccines are used to induce immunity by exposure to less virulent, dead, or pathogen components.  Here, the podcasters discussed how the Pneumococcal vaccine uses an antigen derived from the polysaccharide capsule of Pneumococcus.
  • RPMI Medium (33:04–33:30; 50:10–51:00): This is a liquid medium typically used for culture of mammalian cells.  The authors used this when they tested the retention of biocidal activity of dimethyl dithiol carbonate (DMDC) in the host environment.
  • Ice Bath (33:38–34:20): This is a technique used to stop active transport.  It was used to find whether DMDC based effects on Pneumococcus were dependent on a mechanism involving active transport.
  • Spectroscopy (33:45–36:31): There are many types of spectroscopy, including atomic absorption spectroscopy and inductively coupled plasma optical emission spectroscopy which are used to identify and quantify compounds in a mixture.  Here the authors used these methods to quantify copper.
  • Fluorescent Activated Cell Sorting (FACS) (44:20–45:01): This is a method for quantifying molecules on the surface of cells or inside cells, often using either antibodies coupled to a fluorescent tag, such as for cell surface markers, or a directly fluorescent molecule or stain, such as DAPI for DNA.  Fluorescence is quantified for each cell, usually displayed as a scatterplot. The researchers used FACS to characterize the white blood cells recruited to the lung after different treatments.

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

5.1. Snippet Paper

  • Azole Class of Drugs (10:17–11:30): These are fungicidal and inhibit the fungal cytochrome p450 dependent enzyme. lanosterol 14ɑ-demethylase prevents the conversion of lanosterol into ergosterol, inhibiting fungal membrane synthesis
  • One Health (17:22–24:00): The concept that recognizes the health of the individual is connected to the health of animals and our shared environment.

5.2. Main Paper

  • Nutritional Immunity (25:19–27:44): The human body’s enzymes rely on transition metals, like copper, with unstable electrons, that are highly reactive. This makes it a great cofactor in many biological processes. Due to their importance, the human body tries to sequester precious metals, thereby depriving pathogens of access to them. This is a way to defend us against infection, called nutritional immunity.
  • Mechanisms of Antibacterial Metals (29:42–31:54): Macrophages use transition metals, also known as bactericidal metals, to defend the human body against infection. Some of these metals include copper and zinc. Aluminum is also a bactericidal metal, but due to the oxygen in our environment, the aluminum passivates very quickly, making it no longer antimicrobial. The human body also sequesters metals that are used as cofactors, which inhibits pathogen growth due to the lack of cofactors.

6. Podcast Questions

  1. Fanny Hesse began the use of  _____ in microbiology media plates rather than gelatin.  This improved microbe growth because the plates _______.
    1. Ethylene blue; could be used to distinguish aerobes
    2. Agar; remained solid so you could isolate single colonies
    3. Yeast extract; to enhance growth of chemoheterotrophs
    4. Glucose; to enable more ATP to be produced per volume
  2. Which are features of Aspergillus fumigatus that the podcasters mentioned? [Pick all that apply]
    1. Is a fungus
    2. Conidia survive 70C
    3. Is a scavenger
    4. Spores are inhaled
    5. Grows at 37C to 45C
    6. Has a role in global nutrient cycling
  3. How did the fungus likely come into contact with the fungicide that many strains are shown to be resistant to?
    1. It was used in meat processing.
    2. It was discarded into landfills.
    3. It was used agriculturally.
    4. Soil microbes produce it.
  4. How does this study show the principles of One Health?
    1. This study shows microbes (Pneumococcus) are evolving faster than multicellular eukaryotes.
    2. This study underscores the importance of antimicrobial stewardship in garden settings.
    3. This study spotlights the use of citizen survey programs in helping move science forward.
    4. This study highlights the interconnectedness of human health and the local environment.
  5. How is copper used in living systems? [Pick all that apply]
    1. As a cofactor in enzymes.
    2. As a chaperone in protein folding
    3. As a nucleation site for crystals.
    4. As an energy source in respiration.
    5. Is sequestered as a mechanism of defense.
    6. Is bactericidal in macrophages.
  6. What is an example of nutritional immunity?
    1. Our bodies utilize pathogen associated particles to harvest ATP from bacterial systems.
    2. Our bodies sequester copper so it is unavailable for pathogens to use in their own enzymes.
    3. Our bodies use nutrients scavenged from pathogens to fuel cells in the immune system.
    4. Our bodies use cofactors to speed up glucose metabolism in neutrophils and macrophages.
  7. The podcasters discussed an experiment where Pneumococcus were exposed to the antimicrobial compound in an ice bath and the cells were not killed.  What would the results have suggested if the cells had been killed by the antimicrobial in the ice bath?
    1. The antimicrobial mechanism does not involve transporters.
    2. The antimicrobial mechanism does not involve respiration.
    3. The antimicrobial mechanism does not involve fermentation.
    4. The antimicrobial mechanism does not involve lipid synthesis.

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 2).

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to:

  • Identify important features of geographical data representations.
  • Interpret visually-presented, geographic strain feature data.
  • Analyze the results to make a conclusion about geographic distribution of fungi types.
  • Construct a recommendation to protect the health of gardeners based on these data.

Experimental Background (Shelton et al., Figure 1)

Aspergillus fumigatus is a health hazard for immunocompromised people, causing a variety of illnesses from allergic reactions to chronic lung conditions.  This fungal species is ubiquitous in soils and its spores are small enough to remain aloft in the air column.  To determine the degree to which A. fumigatus is present in a commonly used garden item, compost, Shelton et al. (2022) spearheaded a citizen science project to collect and characterize compost samples across the United Kingdom.  The geographic location of compost samples without A. fumigatus, with A. fumigatus, and with A. fumigatus that is resistant to the antifungal agent tebuconazole are shown in the panels, as noted in the figure legend.

7.1.2. Questions

  1. What do the green dots indicate about the fungi isolated from the compost sample sent from that location?
    1. No Aspergillus fumigatus was isolated
    2. Resistant A. fumigatus was isolated.
    3. Aspergillus fumigatus was isolated.
    4. Many fungi and spores were isolated.
  2. If a person were to become infected with the Aspergillus fumigatus indicated by the red dots, would it be reasonable to treat them with tebuconazole? What is your reasoning?
    1. No, because these fungi are resistant to tebuconazole.
    2. Yes, because these fungi are not resistant to tebuconazole.
    3. No, because these fungi were not identified as A. fumigatus.
    4. Yes, because these fungi are sensitive to tebuconazole.
  3. What can you conclude about the distribution of tebuconazole-sensitive and tebuconazole-resistant strains of Aspergillus fumigatus in compost from these geographic areas?
    1. Compost from the coastal areas is mostly sensitive Aspergillus fumigatus.
    2. Compost from the eastern areas is mostly free of Aspergillus fumigatus.
    3. Compost from the southern areas have more resistant Aspergillus fumigatus.
    4. Compost from all areas of the UK have both types of Aspergillus fumigatus.
  4. Based on these data, what would be a good recommendation for gardeners using compost?
    1. If you live in any of the southern or coastal areas, you should wear a mask when working with compost.
    2. You should wear a mask when working with compost, particularly if you have a weak immune system.
    3. If you work with compost, you should wear a mask and take prophylactic doses of tebuconazole.
    4. If you live in the eastern or northern areas, you should wear a mask when working with compost.

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Identify key features of bar charts related to this study.
  • Identify key experimental design features, including variables and controls.
  • Evaluate the data to make conclusions about the relationship between intracellular copper and N,N-dimethyldithiocarbamate (DMDC).
  • Analyze the data to predict which treatment would be most toxic to bacteria.

Experimental Background (Menghani et al., Figure 2)

Streptococcus pneumoniae is an encapsulated bacterium that causes bacterial pneumonia and significant numbers of deaths in both the elderly and the very young. Bacterial strains resist treatment by acquiring antibiotic resistance and altering its capsule.  Developing new antibiotics to treat S. pneumoniae infection is therefore a major need.  In a prior study, this group had identified a new antimicrobial called  N,N-dimethyldithiocarbamate (DMDC) that is dependent on copper.  In this follow-up study, Menghani et al. (2022) investigate the mechanism DMDC uses to kill pathogens.  To determine whether DMDC treatment affected copper uptake in S. pneumoniae cells, they incubated bacteria in plain medium (untreated control) or with copper and/or two different concentrations of  DMDC.  They quantified copper uptake into the bacteria using graphite furnace atomic absorption spectroscopy (GFAAS).

Bar chart showing increase in intracellular copper from several treatments. Described in caption.
 Figure 2.

“DMDC and copper treatment leads to a significant increase in intracellular copper. GFAAS analysis of bacterial pellets showed a marked statistically significant increase in the copper content within the bacteria treated with 250 mM Cu21 plus 16 mM DMDC and within the bacteria treated with 250 mM Cu21 plus 32 mM DMDC in comparison to the untreated control. Experiments were performed in triplicate, with statistical significances of differences determined by an unequal-variance t test (ns, not significant; *, P , 0.05; **, P , 0.01; ***, P , 0.001; ****, P , 0.0001).” (Menghani et al. 2022, no changes)

7.2.2. Questions

  1. What bacterial treatment is noted by the orange bar?
    1. Untreated
    2. 250 μM Cu2+
    3. 16 μM DMDC
    4. 32 μM DMDC
  2. What do the asterisks at the top of the graph indicate and why are they important for interpreting the results?
    1. Statistical significance at different P levels; indicates sample measures are truly different.
    2. Statistical significance at biologically-relevant P levels; indicates samples were quantifiable.
    3. Statistical significance; indicates the null hypothesis was not rejected for these samples.
    4. Statistical significance; indicates sample quantifications were successful for spectroscopy.
  3. In this experiment, the ______ treatment is the negative control and the _____ treatment is the positive control.
    1. 250 μM Cu2+; 16 μM DMDC
    2. No negative control; 250 μM Cu2+
    3. Untreated; no positive control
    4. 16 μM DMDC; untreated
  4. Which treatment yields the highest concentration of intracellular copper in bacteria?
    1. Untreated
    2. 32 μM DMDC and 250 μM Cu2+
    3. 16 μM DMDC and 250 μM Cu2+
    4. 250 μM Cu2+
  5. What evidence is there that copper uptake is DMDC dose-dependent?
    1. The higher the DMDC concentration, the greater the copper uptake inhibition.
    2. Divalent copper and DMDC are both required for consistent and stable copper uptake.
    3. The higher the DMDC in copper-containing medium, the more copper is taken up.
    4. Copper is taken up and found intracellularly whether DMDC is present in the medium or not.
  6. Intracellular copper is toxic. Based on this fact and the results of this experiment, which treatment is most toxic to bacteria?
    1. 16 μM DMDC alone
    2. 16 μM DMDC and 250 μM Cu2+
    3. 32 μM DMDC alone
    4. 32 μM DMDC and 250 μM Cu2+

8. Paper Information and Licensing

8.1. Snippet paper

  • Shelton JMG, Collins R, Uzzell CB, Alghamdi A, Dyer PS, Singer AC, Fisher MC.2022. Citizen Science Surveillance of Triazole-Resistant Aspergillus fumigatus in United Kingdom Residential Garden Soils. Appl Environ Microbiol. 88(4):e0206121. doi: 10.1128/AEM.02061-21
  • This article is not licensed for Creative Commons use; see the article’s copyright information. Thus, the abstract and figures cannot be copied here.

8.2. Main paper

  • Menghani SV, Cutcliffe MP, Sanchez-Rosario Y, Pok C, Watson A, Neubert MJ, Ochoa K, Wu HJ, Johnson MDL. 2022. N,N-Dimethyldithiocarbamate Elicits Pneumococcal Hypersensitivity to Copper and Macrophage-Mediated Clearance. Infect Immun. 90(4):e0059721. doi: 10.1128/iai.00597-2
  • This article is licensed for Creative Commons use using CC BY 4.0, which allows re-use and adaptation with proper attribution and notation of any changes.

License

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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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