Meet the Microbiologist

Pat Brown founded Impossible Foods with a mission to replace animals as a food production technology. Here, he discusses the ways microbial engineering helps produce the plant hemoglobin that provides the Impossible Burger's meaty qualities.

Links for this episode:

• Take the MTM listener survey(~3 min.)
• The Microbial Reasons Why the Impossible Tastes So Good
• Impossible Foods
• The Conversation: What Makes the Impossible Burger Look and Taste Like Real Beef?
• Wired:The Impossible Burger: Inside the Strange Science of the Fake Meat that 'Bleeds'
• HOM Tidbit: Mendel's letters to von Nägeli
• HOM Tidbit:The Mendel-Nägeli Letters, circa 1866-73 (Scientific American)

CRISPR is a genome-editing tool, but what is its role in microbial biology and evolution? Joe Bondy-Denomy discusses his discovery of the first anti-CRISPR protein and the many unanswered questions surrounding CRISPR biology.

Julie's Biggest Takeaways

CRISPR is a bacterial immune system that identifies and destroys specific nucleotide sequences. These sequences are most commonly associated with foreign DNA from bacteriophage or plasmids.

Bacterial acquisition of new CRISPR spacer sequences is fairly inefficient, and often a bacterium dies before acquiring and fending off a new phage infection. Only about 1 in a million cells emerge from a phage infection with a new spacer sequence, likely driven defective phages that act as a vaccine of sorts to provide spacer sequence material.

40% of bacteria and 85-90% of archaea have had some sort of CRISPR system detected in their genomic sequences.

Most bacteria have Type I CRISPR system. This system includes different proteins that serve unique functions: one holds onto CRISPR RNA, one helps identify complementary sequences, and one cleaves the actual nucleotide sequence. The Type II CRISPR system has a single protein, Cas9, which performs all of these functions by itself. Because of its simplicity, this Type II CRISPR system has become widespread as a DNA manipulation tool.

What are the inputs to CRISPR? How do bacterial cells turn CRISPR genes on and off? Do CRISPR systems serve any other regulatory functions? There are still a number of questions that need to be answered to understand the biological role of CRISPR systems.

• Take the MTM listener survey (~3 min.)
• Joe Bondy-Denomy UCSF Lab Website
• Rauch BJ. Inhibition of CRISPR-Cas9 with Bacteriophage Proteins. Cell 2017.
• Borges AL. Bacteriophage Cooperation Suppresses CRISPR-Cas3 and Cas9 Immunity. Cell 2018.
• Mendoza SD. A Nucleus-Like Compartment Shields Bacteriophage DNA from CRISPR-Cas and Restriction Nucleases. bioRxiv 2018.
• UCSF Sandler Fellows Program
• HOM Tidbit: Coming of Phage Celebrating the Fiftieth Anniversary of the First Phage Course

Julie's Biggest Takeaways:

Parasites are incredibly varied in many characteristics, including their size! Some are microscopic, while others are macroscopic and can be seen with the naked eye. Not just small macroscopic, although some worms at 35 cm can be considered quite large. Some tapeworms can reach 50 feet!

Bobbi Pritt's blog started as an exercise to share the cases she observed while a student at the London School of Tropical Medicine. She wanted to share these cases with students back at the Mayo Clinic, but found the audience grew to include clinical parasitologists, microbiologists, and parasite-interested people worldwide. Part of its success relies on its succinctness: a short, digestible case study with the minimum information needed to make a diagnosis.

Pritt's research focuses on developing molecular tests to detect microorganism RNA or DNA. Molecular tests can be used as a complementary diagnostic test or as the primary test, which can give healthcare workers definitive information to make therapeutic decisions much more quickly than a test that requires culturing the microorganism.

A new bacterium that causes Lyme disease, Borellia mayonii, was found because the molecular tests that detect Borellia burgdorferi are flexible enough to detect multiple species and can differentiate between the different types of organisms. It was an astute technologist working at the bench who recognized the readout was slightly different than

We did a tick drag, taking a white cloth and dragging it through vegetation. The Ixodes ticks that transmit Lyme disease will think the sheet is a host and will grab onto the sheet, allowing easy collection of a large number of ticks to test for bacterial presence.

One of the outstanding questions in parasitology is the relationship of Blastocystis (formerly known as Blastocystis hominis but may actually be several species) to human health. Blastocystis lives in the intestinal tract and may cause irritable bowel-like syndrome. Definitive evidence on whether Blastocystis causes intestinal disease has yet to be presented, and there is a lot of opportunity for research in this area.

Links for this Episode:

• Take the MTM Listener survey (~3 min.)
• Creepy Dreadful Wonderful Parasites (Bobbi Pritt's blog)
• ParasiteGal: Bobbi Pritt on Twitter
• Pritt B.S. et al. Identification of a Novel Pathogenic Borrelia species causing Lyme borreliosis with unusually high spirochaetaemia: a descriptive study. Lancet Infectious Disease. 2016.
• MTM Episode: Biofilms and Metagenomic Diagnostics in Clinical Infections with Robin Patel
• HOM Tidbit: Patrick Manson. On the Guinea Worm. British Medical Journal.
• Bobbi on This Week in Parasitism (TWiP)

How did discoveries made with bovine papillomavirus help scientists develop the human papillomavirus vaccine? Doug Lowy discusses his journey that began with basic research and led to the production of the HPV vaccine.

In the early 1950s, the U.S. was a high-incidence country for cervical cancer. Through application of screens using the Pap smear, doctors have been able to catch and excise suspicious tissue, leading to a significant drop in incidence. Cervical cancer remains high-incidence in low- and middle-income countries; in high-incidence countries, cervical cancer is the most common form of HPV-associated cancer. In the U.S., cervical cancer represents around 50% of the HPV-associated cancers, with others like penile, anal, and oropharyngeal cancers also represented. Henrietta Lacks, the woman from whom HeLa cells were derived, had a cervical adenocarcenoma caused by HPV-16. The viral DNA had integrated near the myc oncogene to generate high expression of this oncogene. The cell lines have been growing for decades but the epigenetic changes from HPV infection have led to a dependence of the cells on E6 and E7; if they are blocked or removed, the HeLa cells undergo apoptosis. Lowy's work on bovine papilloma virus (BPV) played a key role in development of the HPV vaccine. Other researchers attempting to generate a neutralizing response to the HPV capsid failed, but Lowy and his colleague Reinhard Kirnbauer had successfully achieved neutralization using BPV. By comparing HPV and BPV sequences, Lowy realized there was a single amino acid change in the HPV-16 strain that was being used as a lab standard strain; fixing this restored capsid self-assembly, led to immunogenicity and provided the basis for the HPV vaccine. HPV L1 capsid protein has a repeating structure that induces a very high level of immune protection. Protection is so high that it is sterilizing, meaning that exposed individuals prevent any infection, not just disease. This may serve as the basis for a new strategy, using repeating structures such as ferretin in vaccine development. The incubation between infection and development of cancer can take decades, and the vaccine has not been on the market long enough to assess a difference in cancer incidence. It has resulted in a decrease in cervical dysplasia, the endpoints used in cervical cancer screening via pap smear, but no cancer reduction has been observed yet.

Links for this Episode:

• Harold zur Hausen Nobel Prize for association between HPV and cancer
• Lowy D. HPV Vaccination to Prevent Cervical Cancer and Other HPV-Associated Disease: From Basic Science to Effective Interventions. Journal of Clinical Investigation. Jan 2016.
• Schiller J. and Lowy D. Explanations for the High Potency of HPV Prophylactic Vaccines. Vaccine. August 6 2018.
• VAERS Vaccine Adverse Event Reporting System
• ASM Article: A Brief History of Cancer Virology
• JHU Press: Vaccines Did Not Cause Rachel's Autism

Burkholderia pseudomallei is an endemic soil-dwelling bacterium in southeast Asia, where it causes melioidosis. Direk Limmathurotsakul discusses his work to improve the official reporting numbers and how

Julie's Biggest Takeaways:

Melioidosis can present in a number of ways, such as sepsis, pneumonia, or abscesses. Because the symptoms are not specific, diagnosis requires isolation of the Burkholderia pseudomallei bacterium. Risk factors for disease include diabetes and exposure to the soil and water in which the bacterium lives.

In 2012, only 4 people were officially reported to have died of melioidosis in Thailand, but microbiological records suggest the real number was closer to 696. Scientists like Direk worked with the government to improve reporting requirements and the numbers now reflect a more accurate assessment of the disease burden. More accurate official reporting can lead to more public health campaigns, resources, and support for both scientists and patients.

Social media campaigns and a YouTube competition help to raise local awareness of melioidosis. The YouTube competition engages the community by allowing them to enter videos in their own dialect, which then inform others about how to minimize risk factors for melioidosis.

The AMR Dictionary gives simple definitions to jargon surrounding the problem of antimicrobial resistance. The definitions are translated into multiple languages in ways that make sense with colloquialisms. For example, in Thai, many people refer to antibiotics as antiseptics or anti-inflammatory drugs, and the dictionary takes local use into consideration in its definitions.

Links for this Episode:

MTM Listener Survey

Limmathurotsakul website at MORU Tropical Health Network

Melioidosis.info

Melioidosis: the Most Neglected Tropical Disease

Antibiotic Footprint

AMR Dictionary

When will the next disease outbreak occur? Why are some pests better at spreading disease than others? Disease Ecologist Barbara Han talks about her research that addresses these questions with computer modeling, as well as how modeling predictions can inform field and bench research.

Take the listener survey: asm.org/mtmpoll

Visit asm.org/mtm for all links and notes.

Why have scientists struggled to generate a protective HIV vaccine? Dan Barouch lays out the unique challenges and discusses the ongoing clinical trial with an adenovirus-based vaccine developed in his lab.

Julie's Biggest Takeaways

HIV poses unique and unprecedented challenges for vaccine development including:

• Viral diversity: extremely wide range of viral diversity.
• No natural precedent: No human has cleared HIV based on their immune responses.
• Unknown correlates of protection: scientists are unsure what immune responses are important to induce.

Barouch's group uses a vaccine strategy comprised of computationally optimized mosaic HIV Env proteins, which represent pieces of the outermost glycoprotein, Env, that have been tied together in a way expected to generate protective immunity. Early data from animal and human trials suggests these mosaic antigens generate an immune response to a wider array of HIV types than previous vaccines. Clinical trials are ongoing to see if a strategy of mosaic antigen vaccination, followed by a boost with Env protein, is protective in people.

Attenuated HIV hasn't been used as a vaccine strategy because of fears it could revert to a disease-causing form; similar fears have prevented a whole-killed virus platform for vaccine development.

A clinical trial testing safety in 3 locations around the world demonstrated that this vaccine strategy in people elicited immune responses shown to be protective in animals. An efficacy trial is ongoing in sub-Saharan Africa, with results expected in 2021. The trial is double blinded: neither the doctor nor the patient know who was administered the candidate vaccine or who was administered the placebo.

HIV latent infection causes complications in vaccine development because

• HIV latency is seeded early, possibly in the first few days of infection.
• Once latency is established, the individual is infected for life. Any low level of HIV infection in vaccinated people could potentially seed this latent infection.
• Quickly-seeded latency means immune responses must react extremely quickly.

Featured Quotes

"The challenges in the development of a prophylactic HIV vaccine are among the toughest challenges in biomedical and scientific research."

"HIV poses unique challenges for vaccine development and truly unprecedented challenges that have never been posed before by vaccination. One such challenge is the viral diversity: HIV exists not as a single sequence, but as numerous different viral sequences — not only throughout the world, but also throughout regions, communities, and even within the same individual. So to create a vaccine against HIV, the immune responses have to be relevant for a vast diversity of viral sequences."

"At what efficacy level would an HIV vaccine be licenced by both the industry partners as well as the government regulators in a particular country, and at what level of efficacy would it actually have a major public health impact? It's a moving target over time; it really depends on what the current state of the epidemic is at the time the vaccine is ready to be licensed."

"It's critical to have high-quality research part of the clinical efficacy trials so that success or failure or something in between, that the HIV research field learns from it, and learns what worked well and what didn't work well, and how to make better vaccines moving forward."

"I always encourage young scientists to pursue their dreams and to tackle hard problems. There's a lot of easy problems to solve but some of the hardest problems are the most impactful in the end."

Links for This Episode

• MTM Listener Survey
• Barouch lab at the Center for Virology and Vaccine Research.
• MTM: Mark Connors.
• The Lancet: Evaluation of a Mosaic HIV-1 Vaccine in a Multicentre, Randomised, Double-Blide, Placebo-Controlled, Phase 1/2 a Clinical Trial (APPROACH) and in Rhesus Monkeys.
• The Lancet: A Step Forward for HIV Vaccines.
• Journal of Virology: Similar Epitope Specificities of IgG and IgA Antibodies Elicited by Ad26 Vector Prime, Env Protein Boost Immunizations in Rhesus Monkeys.
• PLoS One: First-in-Human Randomized, Controlled Trial of an oral, replicating adenovirus 26 vector vaccine for HIV-1.
• HOM Tidbit: I am the Berlin Patient: A Personal Reflection.
• HOM Tidbit: Doctor who cured Berlin Patient of HIV: 'We knew we were doing something very special'.

Erica Ollmann Saphire discusses her research on Ebola virus glycoprotein and the changing nature of structural biology.

The Ebola virus glycoprotein sequence can vary up to 50% between Ebola virus species, presenting a challenge to develop pan-Ebola therapeutics or vaccines. Erica Ollmann Saphire discusses her work on antibodies that neutralize all Ebola virus species and the changing nature of the structural biology toolkit used to study them.

Check out all our great podcasts at asm.org/podcast

• MTM Listener Survey: asm.org/mtmpoll
• Ollmann-Saphire Lab Site
• Protein Database
• Isolation of Potent Neutralizing Antibodies from a Survivor of the 2014 Ebola Virus Outbreak. Science 2016.
• Systemic Analysis of Monoclonal Antibodies against Ebola Virus GP Defines Features that Contribute to Proteciton. Cell 2018.
• Structural Basis of Pan-Ebolavirus Neutralization by a Human Antibody against a Conserved, yet Cryptic Epitope. mBio 2018.
• Tenacious Researchers Identify a Weakness in All Ebolaviruses. mBio 2018.
• HOM Tidbit: How "Lassa," a small Nigerian Town, was Stigmatized by having a Killer Virus Named after it.

We pull back the curtain as former show hosts Merry Buckley and Carl Zimmer talk Meet the Scientist origins, favorite interviews and microbial topics.

Julie's Biggest Takeaways:

Though the show started before podcasts were as popular as they are now, this didn't pose a problem for Merry or Carl when soliciting guests - scientists were happy to have their work featured and to discuss their research.

Inviting guests may involve bringing in a mix of experienced and early-career researchers, but both Merry and Carl agreed that the science is the major deciding factor when selecting guests.

The ability to steer away from technical jargon and to use accessible, everyday analogies is one of the features shared by favorite guests. Carl uses the example of Bonnie Bassler, who explains bacterial quorum sensing as a communication mechanism.

Delving into the personal motivations and experiences of guests can be tough, even when these experiences relate to science. Merry uses Abigail Salyers' claim of the English teacher who supported her through her high-school pregnancy and Julie uses Ilaria Capua's experience when falsely accused of trafficking viruses for money.

Scientists can make themselves more visible to scientists and nonscientists by promoting their research on social media, particularly on Twitter.

Links for this Episode:

MTM Listener Survery

Merry Buckley on twitter

Carl Zimmer website

Carl Zimmer on TWiV

She has her Mother's Laugh

How does an engineer approach microbial genetics? cworks with microbes of all kinds to optimize metabolic and agricultural systems. Here he discusses his work with Rhodobacter to make biofuels and for membrane protein expression, with Agrobacterium and plant pathogenic viruses to make drought-resistant plants, and with Clostridium and yeast cocultures for lignocellulose digestion.

Take the listener survey at asm.org/mtmpoll

Full shownotes at asm.org/mtm

Links for this Episode:

• Wayne Curtis Lab site at Penn State University
• PLoS One: Molecular Cloning, Overexpression, and Characerization of a Novel Water Channel protein from Rhodobacter sphaeroides
• Protein Expression and Purification: Advancing Rhodobacter sphaeroides as a Platform for Expression of Functional Membrane Proteins
• Biotechnology for Biofuels: Consortia-Mediated Bioprocessing of Cellulose to Ethanol with a symbiotic Clostridium phytofermentans/Yeast Co-Culture
• HOM Tidbit: Genentech "Cloning Insulin" blog
• HOM Tidbit: Genentech press release announcing insulin cloning