Meet the Microbiologist

How do arboviruses evolve as they pass between different hosts? Greg Ebel discusses his research on West Nile virus evolution and what it means for viral diversity. He also talks about using mosquitos' most recent blood meal to survey human health in a process called xenosurveillance. Julie's Biggest Takeaways: Mosquitoes and other arthropods have limited means of immune defense against infection. One major defense mechanism is RNA interference (RNAi). RNAi uses pieces of the West Nile viral genome to select against the viral genome, which helps select for broadly diverse viral sequences. The more rare a viral genotype, the more likely it is to escape negative selection inside the mosquito host, allowing this viral sequence to increase in frequency. West Nile virus passes largely between birds and mosquitos. Culex mosquitos tend to prefer birds, and this leads to an enzootic cycle for the virus passing between birds and mosquitos. The viral life cycle inside the mosquito has several important steps: The virus first enters as part of the mosquito blood meal. The virus infects epithelial cells of the mosquito midgut. After 3-5 days, the virus leaves the midgut (midgut escape) to enter the mosquito hemolymph. In the next mosquito blood meal, virus is expelled with saliva, which has anticoagulant activity. West Nile virus selection undergoes cycles of selection as it passes from vertebrates (mostly birds) to invertebrates (mosquitos): In vertebrates, the virus must escape to cause viremia in a short period of time for replication to occur before the immune system recognizes and eliminates the virus. This leads to purifying selection, or elimination of amino acid variation that decreases viral protein function. In mosquitos, the virus spends several days in the midgut epithelial cells and then hemolymph, leading to a longer selection time. This leads to more viral diversity in the mosquito host. RNAi further drives population diversity. Through stochasticity, a single viral population will often come to dominate a single infected mosquito. How do scientists know which virus replicates best? Competitive fitness tests measure which virus grows to a higher population in a given environment. A manipulated virus (one passaged in a mosquito or selectively mutated at distinct sequences) and its non-manipulated parent sequence are inoculated at known proportions, and given a certain amount of time to replicate. By measuring the final proportions, Greg and his team can determine which sequence was more fit in that given environment. Xenosurveillance uses mosquitoes to detect a wide array of pathogens at clinically relevant levels. Testing began with in vitro blood-bag feeding, and was validated with studies in Liberia and Senegal. The microorganism sequences are so diverse that the information was used to identify novel human viruses. These studies also provide insight into mosquito feeding habits, which helps in disease modeling. Links for this Episode: Greg Ebel Lab Website Rückert C. et al. Small RNA Responses of Culex Mosquitoes and Cell Lines during Acute and Persistent Virus Infection. Insect Biochemistry and Molecular Biology. 2019. Grubaugh N.D. et al. Mosquitoes Transmit Unique West Nile Virus Populations during Each Feeding Episode. Cell Reports. 2017. Grubaugh N.D. and Ebel G.D. Dynamics of West Nile Virus Evolution in Mosquito Vectors. Current Opinion in Virology. 2016. Fauver J.R. et al. Xenosurveillance Reflects Traditional Sampling Techniques for the Identification of Human Pathogens: A Comparative Study in West Africa. PLoS Neglected Tropical Diseases. 2018. Fauver J.R. The Use of Xenosurveillance to Detect Human Bacteria, Parasites, and Viruses in Mosquito Bloodmeals. American Journal of Tropical Medicine and Hygiene. 2017. Tracey McNamera: Canaries in the Coal Mine TEDxUCLA New York Times: Encephalitis Outbreak Teaches an Old Lesson. 1999. ASM Article: The One Health of Animals, Humans, and Our Planet: It's All Microbially Connected

How can the intricate relationship between soil microbiota and plants be managed for improved plant health? Linda Kinkel discusses new insights into the plant rhizosphere and the ways that some Streptomyces isolates can protect agricultural crops against bacterial, fungal, oomycete, and nematode infections. Julie's Biggest Takeaways: The soil microbiome is extremely dynamic, with boom-and-bust cycles driven by nutrient fluxes, microbial interactions, plant-driven microbial interactions, and signaling interactions. Finding the source of these boom-and-bust cycles can help people to manage the microbiome communities and produce plant-beneficial communities for agricultural purposes. Rhizosphere soil is soil closely associated with the root and is distinct from rhizoplane soil that directly touches the root. The endophytic rhizosphere are those microbes that get inside the root. Many scientists view these communities as a continuum rather than sharply delineated. Plants provide necessary carbon for the largely heterotrophic soil microbiota, and these microorganisms help the plants in several ways too: Microbes mediate plant growth by production of plant growth hormones. Microbes provide nutrients through mechanisms like nitrogen fixation or phosphorus solubilization. Microbes protect the plant from stress or drought conditions. Through a University of Minnesota plant pathology program, potatos were passaged in a field for over 2 decades to study potato diseases. Over time, researchers found fewer diseases in test crops, which led the plot to be abandoned in the late 1970s. In the 1980s, Dr. Neil Anderson planted potatoes to see if they would develop disease, but neither Verticillium wilt nor potato scab developed among the plants. Soil from the field (and on the potatoes) contained Streptomyces isolates that showed antimicrobial activity against bacteria, fungi, nematodes, and oomycetes. This discovery led Neil, new University of Minnesota professor Linda, and their collaborators to study the antimicrobial activity of natural Streptomyces isolates from around the world. Inoculation quickly adds specific microbial lineages to soil microbiome communities. Alternatively, land can be managed by providing nutrients to encourage the growth of specific species, like Streptomyces, within a given plot, but this takes longer to develop. How are soil microbiomes inoculated? Microbes can be: Added to the seed coating before planting. Placed in the furrow when the seed is planted. Distributed into the irrigation system. Links for this Episode: Linda Kinkel website at University of Minnesota Essarioui A. et al. Inhibitory and Nutrient Use Phenotypes Among Coexisting Fusarium and Streptomyces Populations Suggest Local Coevolutionary Interactions in Soil. Environmental Microbiology. 2020. Schlatter D.C. et al. Inhibitory Interaction Networks Among Coevolved Streptomyces Populations from Prairie Soils. PLoS One. 2019. Schlatter D.C. et al. Resource Use of Soilborne Streptomyces Varies with Location, Phylogeny, and Nitrogen Amendment. Microbial Ecology. 2013. Small Things Considered blog: Are Oomycetes Fungi or What? International Year of Plant Health HOM Tidbit: Austin-Bourke P.M. Emergence of Potato Blight, 1843-1846. Nature. 1965.

Pathogenic E. coli are different than lab-grown or commensal E. coli found in the gut microbiome. Alfredo Torres describes the difference between these, the method his lab is using the develop vaccines against pathogenic E. coli, and how this same method can be used to develop vaccines against Burkholderia infections. Julie's Biggest Takeaways: coli plays many roles inside and outside the scientific laboratory: Laboratory E. coli strains used by scientists to study molecular biology. Commensal E. coli strains contribute to digestion and health as part of the intestinal microbiome. Pathogenic E. coli strains have acquired factors that allow them to cause disease in people The pathogenic E. coli associated with diarrheal disease are the ones named for their O-antigen and flagellar H-antigen, such as O157:H7. There are about 30 E. coli strains with various combinations of O-H factors known to cause diarrheal disease in people. The E. coli Shiga toxin (though not the bacterium itself) can pass through the epithelial cell layer to become systemic, and eventually the toxin will accumulate in the kidneys. This can lead to patients experiencing hemolytic uremic syndrome (HUS) and kidney failure, leading to lifelong dialysis or need for a transplant. An immune response that prevents the E. coli from attaching will prevent the bacterium from secreting toxin in close proximity to the epithelial cells and decrease likelihood of HUS development. Burkholderia is a bacterial genus whose member species have been weaponized in the past, and which remain potent disease-causing agents around the world. B. mallei causes glanders, a disease mostly of horses and their handlers. It is a respiratory infection that can become systemic if not treated. B. pseudomallei causes melioidosis, a disease that can manifest in many ways. It is endemic in many tropical regions around the world, found in over 79 countries so far. Coating gold nanoparticles with antigens against which the immune response will be protective is a method Alfredo has used for a number of candidate vaccines, including one against E. coli and one against B. pseudomallei. The nanoparticles can have the gold cleaved off to provide different functional variants of the same vaccine. Links for this Episode: Alfredo Torres webpage at University of Texas Medical Branch McWilliams BD and Torres AG. Enterohemorrhagic Escherichia coli Adhesins. Microbiology Spectrum. 2013. Sanchez-Villamil JI et al. Development of a Gold Nanoparticle Vaccine against Enterohemorrhagic Escherichia coli O157:H7. mBio. 2019. Wiersinga WJ et al. Melioidosis. Nature Reviews Disease Primers. 2018. Khakhum N. et al. Evaluation of Burkholderia mallei ΔtonB Δhcp1 (CLH001) as a live attenuated vaccine in murine models of glanders and melioidosis. PLOS Neglected Tropical Diseases. 2019. Torres AG. Common Sense Can Keep You Safe in E. coli Outbreak. Galveston County Daily News. 2020. ABRCMS: Annual Biomedical Research Conference for Minority Students MTM: Burkholderia pseudomallei & the neglected tropical disease melioidosis with Direk Limmathurotsakul HOM Tidbit: Kiyoshi Shiga Biography in Clinical Infectious Diseases

Does the fetus have a microbiome? How does the placenta prevent infection? Carolyn Coyne talks about placental structure and biology, and why studying the maternal-fetal interface remains a critical area of research. Julie's Biggest Takeaways: The placenta forms within 3-5 days post conception as a single layer of cells surrounding the fertilized embryo. These cells differentiate and develop into more complex structures. Very few microbes cause fetal disease. Of those that do, the disease-causing microorganisms are diverse and can lead to serious congenital defects or even death of a developing fetus. These microbes are largely grouped into the TORCH (now TORCH-Z) microorganisms: Toxoplasma gondii Other (a variety of different bacteria and viruses) Rubella Cytomegalovirus Herpesviruses Zika virus The fetus is immunologically immature and unable to protect itself. Some of the maternal immunological molecules (such as maternal antibodies) cross the placenta to protect the fetus, but that only happens during later stages of fetal development. Between the first and second trimesters, the maternal vasculature reorganizes and maternal antibodies can begin to reach the fetus. This increases over time, until the end of the third trimester, when there is a higher concentration of maternal antibodies in fetal blood than in maternal blood. In the later stages of development, the placenta is coated in a layer of fused cells, leading to a shared cytoplasm that covers the entire surface area of the placenta. This fused-cell layer is formed from syncytiotrophoblasts, and the fusion is facilitated by the activity of an endogenous retrovirus fusion protein. Syncytiotrophoblasts are extremely resistant to infection with a number of different pathogens, and pathogen types. In initial tests experiments, Carolyn and her research team discovered that these cells releasing certain antimicrobial molecules to share protective properties. Syncytiotrophoblasts secrete type III interferons, which play a big role at barrier surfaces such as the airway and the gut—but unlike these barriers, the syncytiotrophoblast cells secrete type III interferons constitutively. Links for this Episode: Carolyn Coyne Website on the University of Pittsburgh School of Medicine Arora N. et al. Microbial Vertical Transmission during Human Pregnancy. Cell Host & Microbe. May 2017. Coyne C.B. The Tree(s) of Life: The Human Placenta and My Journey to Learn More About It. PLoS Pathogens. April 2016. Ander S.E. et al. Human Placental Syncytiotrophoblasts Restrict Toxoplasma gondii Attachment and Replication and Respond to Infection by Producing Immunomodulatory Chemokines. mBio. January 2018. Wells A.I. and Coyne C.B. Type III Interferons in Antiviral Defenses at Barrier Surfaces. Trends in Immunology. October 2018. Ander S.E. Diamond M.S. and Coyne C.B. Immune Responses at the Materna-Fetal Interface. Science Immunology. January 2019. HOM Tidbit: Women in Microbiology HOM Tidbit: Small Things Considered blog post: Retroviruses, the Placenta, and the Genomic Junk Drawer

Are there drugs that can treat coronaviruses? Timothy Sheahan talks about his drug discovery work on a compound that can inhibit all coronaviruses tested so far, and tells how his career path took him to pharmaceutical antiviral research and then back to academia. Julie's Biggest Takeaways: Even though the MERS-CoV was discovered as a human pathogen in 2012, it was likely percolating as a disease agent for a long time before that. Banked camel serum provides evidence that the virus had been circulating in camels for several decades prior. Differentiated ex vivo lung cultures allow study of virus infection in a 3D model representation for studying viral infection, including target cell types of both MERS-CoV and SARS-CoV. SARS-CoV prefers ciliated epithelial cells Ace2 MERS-CoV prefers nonciliated epithelial cells DPP4 Coronavirus disease in people takes place over a course of about 2 weeks. In mice, the disease is similar, but progression is faster, taking about 1 week. The drug remdesivir (RDV) is a nucleoside analog that inhibits the coronavirus RNA-dependent RNA polymerase (RDRP). Remdesivir activity has not been tested against nCoV2019, but similarity to other viruses is promising. Bioinformatic approaches show that the nCoV2019 RDRP is 99% similar and 96% identical to SARS-CoV RDRP. Remdesivir works against every coronavirus tested so far, including viruses with highly divergent RDRP sequences, so remdesivir is likely to be effective again nCoV2019. Experiments must still be performed before reaching this conclusion, of course. Tim also hopes to discover the genetic determinants that will allow a chronic hepatitis C virus (HCV) infection in mice, but not standard inbred mice. He uses outbred mice meant to mimic the diversity of the human population, and strengthen the results. Understanding these determinants would inform human studies to better understand chronic HCV infection. Links for this Episode: MTM Listener Survey, only takes 3 minutes. Thanks! TWiV 584: Year of the Coronavirus Timothy Sheahan website at University of North Carolina Sheahan T.P. et al. Broad-Spectrum Antiviral GS-5734 Inhibits both Epidemic and Zoonotic Coronaviruses. Science Tranlational Medicine. 2017. Sheahan T.P. et al. Comparative Therapeutic Efficacy of Remdesivir and Combination Lopinavir, Ritonavir, and Interferon Beta against MERS-CoV. Nature Communications. 2020. Agostini M.L. et al. Coronavirus Susceptibility to the Antiviral Remdesivir (GS-5734) is Mediated by the Viral Polymerase and the Proofreading Exoribonuclease. mBio. 2018. ASM Coronavirus Resource Page HOM Tidbit: Baltimore D. In Vitro Synthesis of Viral RNA by the Poliovirus RNA Polymerase. PNAS. 1964.

Viral gastroenteritis around the world causes 200,000 deaths globally each year. Mary Estes talks about her work on 2 gastroenteritis-causing viruses, rotavirus and norovirus, and tells the story of her discovery of the first viral enterotoxin. She also describes how noroviruses have changed from human volunteer studies to studies using "miniguts," a system now used with many enteropathogenic microorganisms. Julie's Biggest Takeaways: Rotaviruses and noroviruses kill 200,000 people annually, despite an available rotavirus vaccine and current anti-infective measures. Rotavirus is generally associated with gastrointestinal disease in the very young and the very old, while norovirus infects people at all life stages. Rotavirus is so stable that even when viral samples are extremely dessicated by lyophilization, the samples remain perfectly infectious. Rotavirus stability is largely due to 3 concentric capsid cells. NSP4 is a rotavirus enterotoxin, and the first viral enterotoxin to be discovered. It affects the concentration of the intracellular calcium pools. By activating the calcium chloride channel, NSP4 forces chloride and water to be excreted, directly leading to diarrhea. NSP4 is secreted from infected cells and can also disrupt calcium concentrations of neighboring cells, amplifying the effect of a single infected cell. Rotarix® and RotaTeq® are 2 different attenuated rotavirus vaccines. One contains a single attenuated viral strain while the other contains 5 attenuated viral strains; both vaccines have high efficacy in developed countries and slightly lower efficacy in developing countries. Why vaccine efficacy is lower in developing countries is uncertain, with many hypotheses including microbiome-based effects under study now. Human enteroids, or "miniguts," offer insight into complex virus-cell interactions. These stem-cell derived miniguts can be generated from different types of animal stem cells, and the enteroids they become reflect the same host-barrier restriction as the animal of origin. The miniguts can be used to culture many sorts of viruses and other microorganisms, such as bacteria and protozoa. Links for this Episode: Mary Estes Website at Baylor College of Medicine Hyser J.M. et al. Rotavirus Disrupts Calcium Homeostasis by NSP4 Viroporin Activity. mBio. 2010. Crawford S.E. et al. COPII Vesicle Transport is Required for Rotavirus NSP4 Interaction with the Autophagy Protein LC3 II and Trafficking to Viroplasms. J Virology. 2019. Ettayebi K. et al. Replication of Human Noroviruses in Stem Cell-Derived Human Enteroids. Science. 2016. In J.G. et al. Human Mini-Guts: New Insignts into Intestinal Physiology and Host-Pathogen Interactions. Nat Rev Gastroenterol Hepatol. 2016. Finkbeiner S.R. et al. Stem Cell-Derived Human Intestinal Organoids as an Infection Model for Rotaviruses. mBio. 2012. Henning S.J. and Estes M.K. Women in Science: Hints for Success. Gastroenterology. 2015. Kapikian A.Z. et al. Visualization of a 27-nm Particle Associated with Acute Infectious Nonbacterial Gastroenteritis. Journal of Virology. 1972. HOM Tidbit: Smith K.N. The Iron Long was just an Engineer's Side Project. Forbes. 2019. HOM Tidbit: Ramirez M. Living Inside a Canister: Dallas Polio Survivor is One of Few People Left in U.S. Using Iron Lung. Dallas Morning Star. 2018.

The most abundant organism on Earth lives in its seas: the marine bacterium SAR11. Steve Giovannoni describes how the origins of SAR11 provided its name, and the ways that studying SAR11 have taught scientists about ocean ecology. He also discusses how the different depths of the ocean vary in their microbial compositions and what his big questions are in marine microbiology. Different depths of the ocean have different habitats, but the microbes vary continuously, based in part on light availability: Surface light facilitates photosynthesis by algal cells. These primary producers fix carbon for the entire ecosystem! Because nutrients are readily available, the cell concentration in surface waters can reach nearly 1,000,000 cells/ml. The twilight zone offers dim light. Microbes in this area mainly use carbon sources generated by the surface-dwelling microbes. Below a few hundred meters, cell concentrations drop to 10,000-100,000 cells/ml. The deep ocean has no light and the microbes that live here have significantly different biochemistries and metabolisms. SAR11 is small in both physical size and genome size (0.37–0.89 µm and 1.3 million base pairs, respectively). It is nevertheless the most abundant organism on the planet, with more than 1028 cells estimated to exist worldwide. These cells convert between 6-37% of the carbon fixed in the oceans daily. SAR11 in different niches have ecotypes with different specialties but look physically similar and have very similar genome sequences. Naturally, the most abundant cells in the ocean have the most abundant parasites: bacteriophages called pelagiphages infect SAR11 all over the world. SAR11 and pelagiphages are under constant evolution, though there doesn't seem to be a CRISPR system in the Pelagibacter genome; these bacteria largely use other mechanisms to evade phage infection. SAR11 is like a house with the lights on all the time, in that the cells constitutively express most metabolic genes. For example, SAR11 metabolizes dimethylsulfoniopropionate (DMSP) into dimethyl sulfide (DMS) and methanethiol (MeSH), which can be produced as soon as the cells are exposed to DMSP. While this may seem energetically expensive, the cells must capitalize on their encounters with this transient resource, often found only at low concentrations, and this capitalization requires the investment of protein production. The cost of metabolic gene regulation outweighs the benefits in this particular case. SAR11 and SAR202 are the poles on the spectrum of heterotrophic marine bacteria. SAR11 is very efficient at accessing and using the organic compounds that come from the phytoplankton (also called the labile organic matter). SAR202, found in the deeper part of the ocean, specializes in hard-to-access carbon compounds that other bacteria can't access. Links for This Episode MTM Listener Survey, only takes 3 minutes. Thanks! Stephen Giovannoni website at Oregon State University OSU High Throughput Microbial Cultivation Lab Carini P. et al. Discovery of SAR11 Growth Requirement for Thiamin's Pyrimidine Precursor and its Distribution in the Sargasso Sea. ISME J. 2014. Sun J. et al. The Abundant Marine Bacterium Pelagibacter Simultaneously Catabolizes Dimethylsulfoniopropionate to the Gases Dimethyl Sulfide and Methanethiol. Nature Microbiology. 2016. Moore E.R. et al. Pelagibacter Metabolism of Diatom-Derived Volatile Organic Compounds Imposes an Energetic Tax on Photosynthetic Carbon Fixation. Environmental Microbiology. 2019. HOM Tidbit: Sagan L. On the Origin of Mitosing Cells. 1967. HOM Tidbit: Cellmates (Radiolab podcast episode) ASM Article: The Origin of Eukaryotes: Where Science and Pop Culture Collide

Can a protein be contagious? Jason Bartz discusses his work on prion proteins, which cause spongiform encephalopathy and can be transmitted by ingestion or inhalation among some animals. He further discusses how prions can exist as different strains, and what techniques may help improve diagnosis of subclinical infections. Links for this Episode: Jason Bartz Creighton University website Holec SAM, Yuan Q, and Bartz JC. Alteration of Prion Strain Emergence by Nonhost Factors. mSphere. 2019. Yuan Q et al. Dehydration of Prions on Environmentally Relevant Surfaces Protects Them from Inactivation by Freezing and Thawing. Journal of Virology. 2018. Bartz JC. Prion Strain Diversity. Cold Spring Harbor Perspectives in Medicine. 2016. Bartz JC. From Slow Viruses to Prions PLoS Pathogens. 2016. Deleault NR, Harris BT, Rees JR, Supattapone S. Formation of native prions from minimal components in vitro. Proceedings of the National Academy of Sciences. 2007. Planet Money Episode 952: Sperm Banks

Microbial interactions drive microbial evolution, and in a polymicrobial infection, these interactions can determine patient outcome. Deb Hogan talks about her research on interkingdom interactions between the bacterium Pseudomonas and the fungus Candida, 2 organisms that can cause serious illness in cystic fibrosis patients' lung infections. Her research aims to better characterize these interactions and to develop better diagnostic tools for assessing disease progression and treatment. Links for this Episode: Deb Hogan Lab Website Demers EG et al. Evolution of Drug Resistance in an Antifungal-Naive Chronic Candida lusitaniae Infection. PNAS. 2018. Lewis KA et al. Ethanol Decreases Pseudomonas aeruginosa Flagella Motility through the Regulation of Flagellar Stators. Journal of Bacteriology. 2019. Gifford AH et al. Use of a Multiplex Transcript Method for Analysis of Pseudomonas aeruginosa Gene Expression Profiles in the Cystic Fibrosis Lung. Infection and Immunity. 2016. Grahl N et al. Profiling of Bacterial and Fungal Microbial Communities in Cystic Fibrosis Sputum Using RNA. mSphere. 2018. Microbiology Resource of the Month: The Aeminium ludgeri Genome Sequence HOM Tidbit: https://www.sciencedirect.com/science/article/pii/S0065216408705628 HOM Tidbit: The Frozen Potential of Microbial Collections

Why is C. diff such a serious disease and what are clinical microbiologists doing to improve patient outcomes with better diagnostic tools?