Information Flow and Genetics

TWiM #121: A Plague of Pathogens

Podcast and Annotation Information
  • Annotation by Georgia Chaffin, Sophia Misterek, Jack Siemering, Rebecca Seipelt-Thiemann, and Regina McGrane
  • Podcast audio by TWiM: Listen to TWiM #121 Podcast
  • Podcast transcript by Otter.ai and edited by Georgia Chaffin, Sophia Misterek, Jack Siemering, Grace Helle, and Laurel Thompson: Access Podcast Transcripts
  • Papers Discussed:
    • Golnar AJ, Turell MJ, LaBeaud AD, Kading RC, Hamer GL. 2014. Predicting the mosquito species and vertebrate species involved in the theoretical transmission of Rift Valley fever virus in the United States. PLoS Negl Trop Dis. 8(9):e3163. doi: 10.1371/journal.pntd.0003163
    • Zimbler DL, Schroeder JA, Eddy JL, Lathem WW. 2015. Early emergence of yersinia pestis as a severe respiratory pathogen. Nature Communications 6:7487 doi: 10.1038/ncomms8487

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 6:00 minutes

The Most Interesting Things (according to students)

  • There are currently no human vaccines or antivirals against RVFV. With no human-to-human transmission, there has been little urgency in developing treatments for infection (similar to West Nile virus). The fear of increased transmission with climate change and mosquito expansion and adaptation is causing renewed interest in this pathogen.
  • There is a connection between the gut microbiome of the different species of mosquitoes and the development and transmission of RVFV.

“Rift Valley fever virus (RVFV) is a mosquito-borne virus in the family Bunyaviridiae that has spread throughout continental Africa to Madagascar and the Arabian Peninsula. The establishment of RVFV in North America would have serious consequences for human and animal health in addition to a significant economic impact on the livestock industry. Published and unpublished data on RVFV vector competence, vertebrate host competence, and mosquito feeding patterns from the United States were combined to quantitatively implicate mosquito vectors and vertebrate hosts that may be important to RVFV transmission in the United States. A viremia-vector competence relationship based on published mosquito transmission studies was used to calculate a vertebrate host competence index which was then combined with mosquito blood feeding patterns to approximate the vector and vertebrate amplification fraction, defined as the relative contribution of the mosquito or vertebrate host to pathogen transmission. Results implicate several Aedes spp. mosquitoes and vertebrates in the order Artiodactyla as important hosts for RVFV transmission in the U.S. Moreover, this study identifies critical gaps in knowledge which would be necessary to complete a comprehensive analysis identifying the different contributions of mosquitoes and vertebrates to potential RVFV transmission in the U.S. Future research should focus on (1) the dose-dependent relationship between viremic exposure and the subsequent infectiousness of key mosquito species, (2) evaluation of vertebrate host competence for RVFV among North American mammal species, with particular emphasis on the order Artiodactyla, and (3) identification of areas with a high risk for RVFV introduction so data on local vector and host populations can help generate geographically appropriate amplification fraction estimates.” (Golnar et al. 2014).

1.2. Main paper; discussion starts at 31:15 minutes

The Most Interesting Things (according to students)

  • Yersinia pestis evolved from Y. pseudotuberculosis, but Y. pestis causes much more severe disease despite their genetic similarities.  Studying Y. pestis helps us understand the importance of bacterial pathogenesis, immune evasion, and how antibiotic resistance evolves.
  • Y. pseudotuberculosis spreads through the fecal-oral route and often causes mild or no symptoms, but severe cases can be fatal within days.

“Yersinia pestis causes the fatal respiratory disease pneumonic plague. Y. pestis recently evolved from the gastrointestinal pathogen Y. pseudotuberculosis; however, it is not known at what point Y. pestis gained the ability to induce a fulminant pneumonia. Here we show that the acquisition of a single gene encoding the protease Pla was sufficient for the most ancestral, deeply rooted strains of Y. pestis to cause pneumonic plague, indicating that Y. pestis was primed to infect the lungs at a very early stage in its evolution. As Y. pestis further evolved, modern strains acquired a single amino-acid modification within Pla that optimizes protease activity. While this modification is unnecessary to cause pneumonic plague, the substitution is instead needed to efficiently induce the invasive infection associated with bubonic plague. These findings indicate that Y. pestis was capable of causing pneumonic plague before it evolved to optimally cause invasive infections in mammals.” (Zimbler et al. 2015).

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

Snippet Main
Vision and Change Topics
  • Information Flow and Genetics (V&C_IFG)
  • Microbial Ecology (V&C_ME)
  • Evolution (V&C_E)
  • Information Flow and Genetics (V&C_IFG)
ASM Fundamental Statements
  • Fundamental Statement 19 (ASM_19): Non-cellular infectious agents, such as viruses, prions, viroids, and satellites, are dependent on host cell processes in order to replicate.
  • Fundamental Statement 21 (ASM_21): Microbes and the environment interact with and affect each other.
  • 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 2 (ASM_2): The diversity of microbes has arisen because of processes that include horizontal gene transfer, mutation, reassortment, recombination, and natural selection in varying ecological niches that favor the growth and survival of certain variants.
  • 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 16(ASM_16): Genetic variation can influence microbial structures and their functions.

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • Compare mechanical and biological transmission routes.
  • Recall the evidence that shows a pathogen has disseminated from the insect gut.
 S L
  • Predict how large-scale mosquito extermination efforts might affect RVFV transmission, ecology, and/or public health.
 S H
  • Compare the clinical symptoms and disease progression of Yersinia pseudotuberculosis and Y. pestis.
  • Recall how a critical genetic event drove the evolution of Y. pestis from Y. pseudotuberculosis.
 M L
  • Predict the effect of a hypothetical small molecule inhibitor on Y. pestis infection.
 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

  • in Vivo Infection (16:30–17:45; 21:16–22:00): In vivo infection experiments are those where a pathogen is used to infect an organism in a lab setting.  Here, mosquitoes are experimentally infected with Rift Valley Fever Virus by allowing them to feed on infected mammalian hosts or infected blood. Because the virus needs to move from the midgut into the salivary glands for the mosquito to transmit the virus to a new host, viral load can be detected in the mosquito’s legs to determine if dissemination is possible. The infected mosquito can then be exposed to an uninfected mammalian host to test transmission.

4.2. Main Paper

  • Variant and Ancient Genome Analysis (35:30–37:25; 36:35–38:00): This is a sequencing analysis method where a genome of interest (lab, naturally evolved, or ancient) is compared to a reference genome to identify differences.  For modern strains, Investigating mutations observed for a bacterial protease encoded by different strains of  Y. pestis suggested that point mutations impacted the evolution of Y. pestis and its ability to cause bubonic and pneumonic plague. For the ancient genomes, the Y. pestis genome was found when sequencing tooth DNA taken from Bronze Age grave sites.
  • In Vivo Infection Experiments (39:35–41:50): In vivo infection experiments are those where a pathogen is used to infect an organism in a lab setting.  Here, the researchers used mice as an animal model to study Y. pestis infection, and mutant Y. pestis strains can be used that mimic ancient lineages to understand disease progression. However, mice cannot transmit via the respiratory route like humans.
  • Clinical Strains (44:43–44:50): The podcast talks about a widely used strain of Y. pestis taken from a woman infected with the bacteria which has now become the standard Y. pestis strain used in studies.

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

5.1. Snippet Paper

  • Epidemics (6:51–9:01; 10:42–11:10): Rift Valley Fever Virus (RVFV) has caused epidemics in Africa and has spread into the Middle East, specifically Saudi Arabia.
  • Viral Emergence and Transmission (7:00–9:00): Emergence of RVFV and its spread to livestock and people in Africa.
  • Case Fatality (10:00–11:00): The case fatality rate is the percentage of people with symptoms of infection who die as a result of the infection. The case fatality rate of RVF is low.
  • Zoonoses (11:15–13:30): Zoonotic diseases are transmitted from animal to human. RVF is both zoonotic and vector-borne.
  • Transmission and Vector Competence (13:30–16:00): RVFV infects mosquitoes and is transmitted to offspring (i.e., vertical transmission). Mosquitoes are infected by RVFV by feeding on Cows, sheep, goats, or humans who have viremia. Vector competence refers to the ability of RVFV to infect mosquitoes and move from the midgut to the salivary glands, where it can be transmitted to an uninfected host.
  • Bioterrorism/Select Agent (22:00–23:00): The USDA maintains surveillance on specific pathogens and toxins that have the potential to be weaponized. These are called select agents. RVFV is a select agent that has no readily available vaccines or treatments, and the pathology due to infection can be severe.

5.2. Main Paper

  • Biofilms (34:15–34:45): Yersinia pestis, the bacterium that causes plague, has evolved the ability to form biofilms, which are sticky communities of bacteria that attach to surfaces. In this case, Y. pestis forms a biofilm in the flea’s digestive tract, specifically between the esophagus and midgut. This biofilm blocks the passage of blood into the flea’s gut. This causes the flea to become increasingly hungry, biting more frequently in an attempt to feed. Since the blood is unable to pass through the gut, the flea regurgitates blood mixed with bacteria back into the bite of a new host. This is how Y. pestis is transmitted from flea to host.
  • Plasmids (35:10–36:10): Plasmids are small circular pieces of DNA that exist separately from a bacterium’s main chromosome and can carry genes that give the bacterium new traits. Y. pestis carries a plasmid known as Pla, containing a protease (an enzyme that breaks down proteins). This protease plays a significant role in the type of infection Y. pestis causes. A single change in one amino acid of this protease shifts how the bacterium behaves in the body, making it more likely the infection remains in the lungs (pneumonic plague) rather than moving to the lymph nodes (bubonic plague). This changes the severity and contagiousness of the disease.
  • Biosafety Levels (41:45–42:30): Biosafety levels (BSL) refer to a set of safety protocols that are used when working with biological agents in laboratories. These levels range from BSL-1 (least restrictive) to BSL-4 (most restrictive). Y. pestis can be spread through the air and cause deadly infections, requiring it to be studied in BSL-3 labs. These labs have strict safety measures, including special ventilation systems and protective clothing to prevent pathogens from escaping or infecting lab workers.
  • Yersinia pseudotuberculosis (42:30–44:15): Y. pseudotuberculosis is a close relative of Y. pestis. Unlike Y. pestis, it is typically spread via the fecal-oral route, meaning people get infected by ingesting food and water contaminated with feces from other infected animals. It causes mild, self-limiting intestinal illness, usually diarrhea, and is found mainly in Europe and Asia. It does not form biofilms in fleas or cause the more severe infections normally associated with plague.
  • Bioterrorism/Select Agent (45:45–47:00): Some pathogens are considered especially dangerous because they can be used deliberately to cause harm in acts of bioterrorism. These pathogens are classified as “select agents” by government agencies such as the USDA and CDC. Y. pestis is on this list due to its high fatality rate, ability to spread through the air (pneumonic plague), and its historical use as a biological weapon. It reportedly was used by ancient Chinese forces, during WWII by the Japanese army, and was studied for further weaponization during the Cold War by the Soviet Union.
  • Y. pestis Evolution (49:20–54:30): Scientific evidence from genomics (DNA studies) and archaeology shows that Y. pestis evolved from Y. pseudotuberculosis. This transformation occurred after Y. pseudotuberculosis acquired new genetic material, including the pPCP1 plasmid. This newly acquired plasmid allowed the bacterium to produce the protease, which helps it remain in the lungs to cause more severe disease. This evolutionary step marked the emergence of a more deadly form of the bacterium, capable of causing not just intestinal illness, but life-threatening bubonic and pneumonic plagues.

6. Podcast Questions

  1. Biting insects such as mosquitos have the potential to transmit pathogens mechanically and/or biologically. What is the difference between these two routes?
    1. Mechanical transmission is when the pathogen is transferred by contaminated surfaces and biological transmission is when the pathogen is absorbed through the skin barrier.
    2. Mechanical transmission is when the pathogen is transferred by airborne droplets and biological transmission is when the pathogen is inhaled through the host respiratory system.
    3.  Mechanical transmission is when the pathogen is transferred by touching insect feces and biological transmission is when the pathogen is ingested during host feeding behavior.
    4. Mechanical transmission is when the pathogen is transferred by the biting structures and biological transmission is when the pathogen is injected with a biological fluid such as saliva.
  2. The podcasters discussed how the researchers identify when River Valley Fever Virus (RVFT) has disseminated from the insect gut.  What did the researchers use to determine dissemination has occurred?
    1. They look for tagged bacteria in nearby tissues.
    2. They look for bacteria in the legs of the insect.
    3. They look for the ability to infect other insects.
    4. They look for the ability to deposit bacteria in eggs.
  3. If mosquitoes were completely eradicated from a region, how might Rift Valley Fever Virus (RVFV) persist despite the loss of its primary vector?
    1. The virus could evolve into a free-living, pathogenic microbe.
    2. The virus could infect birds and be transmitted through contact.
    3. RVFT could infect another insect vector to support transmission.
    4. RVFT could be eradicated from the region after a few months.
  4. Which of the following best differentiates the disease caused by Yersinia pseudotuberculosis from that caused by Yersinia pestis?
    1. Y. pseudotuberculosis causes only systemic infections, while Y. pestis causes mild gastrointestinal disease.
    2. Y. pseudotuberculosis has the ability to infect humans, while Y. pestis infects both humans and animals.
    3. Y. pestis and Y. pseudotuberculosis cause the same disease, but Y. pseudotuberculosis is a milder disease.
    4. Y. pseudotuberculosis causes enteric illness, while Y. pestis has systemic, bubonic, and pneumonic forms.
  5. What role did pla play in Yersinia evolution?
    1. The loss of the pla gene in Y. pestis led to reduced virulence and limited its ability to adapt to new environments.
    2. The acquisition of pla enabled Y. pestis to express a protease that enhanced its ability to cause pneumonic plague.
    3. Mutations in the pla gene rendered it non-functional, preventing Y. pestis from adapting to respiratory transmission.
    4. The expression of the pla gene in Y. pestis was solely due to quorum sensing pathway regulatory mechanisms.
  6. If Y. pestis researchers identified a small molecule treatment that inactivated the Pla protease, what would you predict for treatment effectiveness?
    1. This would be an effective treatment for pneumonic plague.
    2. This would be an effective treatment for bubonic plague.
    3. This would be an effective treatment for systemic plague.
    4. This would be an effective treatment for all plague.

7. Figure Reading Exercises

The following are two figure reading exercises, one from the snippet paper (Figure 2) and one from the main paper (Figure 1).

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to: 

  • Identify key features of a horizontal bar chart.
  • Identify parameters that contribute to the vertebrate host competence index calculation.
  • Analyze the data presented to make a conclusion about the most competent host for RVFV.
  • Predict how changes to key features would likely affect vertebrate host competence.
Experimental Background (Golnar et al., Figure 2)

Rift Valley fever virus (RVFV) is a pathogen carried by mosquitos that has spread from continental Africa. An outbreak in Saudi Arabia in 2000 showed it can spread from the continent.  This pathogen causes three main disease forms that are found in the eye, brain, or systemic as a hemorrhagic fever.  While death is rare overall, it may be as high as 50% for the hemorrhagic form.  Since its ability to spread across continents was identified, there has been an interest in identifying competent transmission vectors on other continents.  In this study, Golnar et al. (2017) use laboratory and mathematical modeling data to predict the competence of different insect vectors and vertebrate hosts to carry and transmit RVFV.  Their modeling predictions are reported as a competence index and their calculation method and parameters are noted in the figure legend.

Horizontal bar chart showing vertebrate competence index scores with different species. Callithrix jacchus is at the top, with a steep dropoff following.

Figure 2. “Rift Valley fever virus host competence index values for 20 vertebrate hosts based on experimental infection studies characterizing viremia profiles in PFU/ml or TCID50.The vertebrate host competence index value depends on the viral titer circulating in the blood and the duration of the infectious viremia [38]. Each value represents the sum of daily probabilities that an infected vertebrate host will transmit RVFV to a biting mosquito. This value was obtained by inserting the recorded daily viremia of experimentally infected hosts into the viremia-vector competence equation [% infectious = 0.062 (Log10 viremia)−0.276 (R2 = 0.27; p<0.001; N = 27)] (Figure S1, C). When a vertebrate host’s viremia was calculated to be negative the daily infectiousness was set to zero. Conversion from TCID50 to PFU/ml was obtained by the equation: PFU/ml = TCID50/ml×0.69 [39], [40]. *Denotes a vertebrate species found in the U.S.” (Golnar et al. 2014)

7.1.2. Questions

  1. What does the asterisk notation indicate?
    1. The species is competent to carry RVFV.
    2. The species is found in the United States.
    3. The species is found internationally.
    4. The species is competent to transmit RVFV.
  2. Which of the following are components of the competence calculation? [pick all that apply]
    1. Number of infected hosts present in the local environment
    2. number of viral particles circulating in the host’s blood
    3. how long the virus remains infectious when in the host
    4. probability that an infected host will transmit to an insect
  3. According to these data, which species is most competent to carry and transmit RVFV?
    1. Callithrix jacchus
    2. Ovis aries
    3. Saccostomus campestris
    4. Bos taurus
  4. Capra aegagrus hircus is the domestic goat, which has many different breeds that are genetically different due to selective breeding for meat, milk, and fiber.  If the angora goat was found to inactivate the virus early in the infection, how would this affect the competence index for this breed?
    1. It would not affect the competence calculation.
    2. It would make the prediction of competence difficult.
    3. It would increase competence to be above other goat breeds.
    4. It would decrease competence to be below other goat breeds.

7.2. Second Figure Reading Exercise 

7.2.1. Learning Objectives

  • Identify key features of phylogenetic trees, strip plots, and western blots.
  • Identify key features of experimental design including controls.
  • Evaluate the data to make conclusion regarding the impact of pPCP1 acquisition and pla mutation to Y. pestis evolution.
  • Analyze bacterial burden data to make conclusions about how pPCP1 affects organ colonization.
  • Evaluate the data to make conclusions about how pla variants impact Pla protein.
Experimental Background (Zimbler et al., Figure 1)

Yersinia pestis is the bacterium responsible for causing Bubonic Plague, which caused at least three pandemics, one in the 500 AD, one in the mid-1300s, and one in the late 1800s. The most deadly to date was the one in the 1300s which killed 1-2 million people worldwide. Y. pestis is a recent relative of another bacterium, Y. pseudotuberculosis, which is a mild pathogen. A visual comparison of the relatedness of sixteen modern and historic Y. pestis strains and Y. pseudotuberculosis is shown as a phylogenetic tree (panel a). Included in this visualization are events related to acquiring plasmids pPCP1 or pMT1, as well as a mutation in the pPCP1 plasmid-encoded gene called pla (pla I259T; former isoleucine at codon 259 is now threonine).  To investigate how the modern, highly virulent pandemic strains evolved from its much less virulent ancestor, Zimbler et al. (2015) infected mice intranasally with different historic strains (Angola, Pestioides A, Pestioides E, and Pestoides F) or modern pandemic strains (CO92 and KIM) and quantified bacterial burden in the lungs (panel b; top) and spleen (panel b; bottom) as colony formin units (c.f.u). Whether the strains harbor the pPCP1 plasmid are noted below the graphs.  To further determine whether pPCP1-encoded pla was expressed in each strain, they performed western blot analysis on bacterial cell lysates for the Pla protein, a protease that can cleave itself (autoprocessed), as well as a loading control protein, RpoA, which is a component of RNA polymerase (panel c).

phylogenetic tree, bar style charts, and western blot
Figure 1. “pPCP1 is required by ancestral Y. pestis to cause primary pneumonic plague. (a) Genomic maximum parsimony tree and divergence based on 16 Y. pestis genomes. The division between modern, pandemic strains and ancestral isolates is indicated. Tree was adapted from Morelli et al.5 (b) Bacterial burden within the lungs and spleens of mice (n=10) infected i.n. with the indicated Y. pestis strains. Each point represents the number of bacteria recovered from a single mouse at 48 h post inoculation. The limit of detection is indicated by the dashed line, and symbols in the dotted line indicate c.f.u. below the limit of detection. Symbols below the limit of detection represent mice that did not have detectable numbers of bacteria. A solid line indicates the median of c.f.u. recovered. The presence or absence of pPCP1 in each strain is indicated below. (c) Immunoblot analysis of whole-cell lysates of the indicated Y. pestis strains with antibodies to Pla and RpoA (as a loading control). The lower band represents the autoprocessed form of Pla (see Supplementary Fig. 5a). Full blots are shown in Supplementary Fig. 6. Panel is representative of three independent replicates. Data are combined from two independent experiments, and error bars represent the s.e.m. (*P≤0.05, **P≤0.01, ***P≤0.001, NS, not significant by Mann–Whitney U-test).” (Zimbler et al. 2015)

7.2.2. Questions

  1. What event do the researchers highlight as the difference between ancestral and modern Y. pestis (panel a, dashed line separator)?
    1. Acquisition of the pMT1 plasmid
    2. Acquisition of the pPCP1 plasmid
    3. Mutation of the pPCP1 pla gene
    4. Branching of the Pestoides F to AEF
  2. Based on the phylogenetic tree, which ancestral bacterium is most similar to modern strains?
    1. Y. pestis Pestoides F
    2. Y. pestis Angola
    3. Y. pseudotuberculosis
    4. Y. pestis Pestoides E
  3. What feature in the lung colonization data panel (panel b; top) shows the median of each strain’s ability to colonize the lung? Which feature indicates whether the median is statistically significantly different to the other strains?
    1. Distribution of the shapes; asterisks
    2. Horizontal lines in the graph; asterisks
    3. Horizontal lines above and in the graph
    4. Asterisks; distribution of the shapes
  4. What can you conclude about the ability of modern and ancestral Y. pestis strains to colonize the lung?
    1. Both can colonize lungs equally, regardless of genetic differences.
    2. Only modern strains possess Pla and can colonize lungs.
    3. Strains with pPCP1 can grow in lungs; those without cannot.
    4. All ancestral strains grow better in lungs than modern strains.
  5. What can you conclude about the ability of modern and ancestral Y. pestis strains to colonize the spleen?
    1. Both can colonize spleen equally, regardless of genetic differences.
    2. Only modern strains possess Pla and can colonize spleen.
    3. Strains with pPCP1 can grow in spleen; those without cannot.
    4. Modern strains grow better in spleen than ancestral strains.
  6. To determine the relationship of pla gene expression and presence of the pPCP1 plasmid, the researchers used western blot analysis on the historic and modern strains (panel c).  What is the relationship of  pPCP1 plasmid and expression of Pla?
    1. Only strains with a pPCP1 plasmid also express Pla.
    2. All of the strains express Pla regardless of the pPCP1 plasmid.
    3. None of strains with the plasmid express it in these conditions.
    4. Some of the strains without the plasmid also express it.
  7. To identify how the virulence-related mutation affected the Pla protein, the researchers used western blot analysis on the historic and modern strains (panel c).  Based on these data, how does the mutation of codon 259 from isoleucine to threonine (I259T) affect the Pla protein?
    1. It reduces the protein’s stability so we see less protein present.
    2. It truncates translation of the protein so we see a smaller protein.
    3. It causes the protease to cleave itself so we see some smaller protein.
    4. It introduces a stop codon so there is less RNA and also less protein.

8. Paper Information and Licensing

8.1. Snippet paper

8.2. Main paper

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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