We’re taking time to get to know the members of the GSA’s Early Career Scientist Committees. Join us to learn more about our early career scientist advocates.

Jessie MacAlpine
Communication and Outreach Subcommittee
University of Toronto

Research Interest

I am passionate about using molecular genetics to understand fundamental biology. During my undergraduate studies at the University of Toronto, I completed a specialist program in the Department of Molecular Genetics and decided to stay for my graduate training. I was fortunate to join the laboratory of Leah Cowen, where I was introduced to the fascinating, complex, and often overlooked world of human fungal pathogens. Throughout my undergraduate and graduate training, I was able to use functional genomics to identify genes important for the virulence of the human fungal pathogen, C. albicans. During my PhD, I dissected the interaction between Lactobacillus bacteria and C. albicans to understand how commensal bacteria can alter fungal virulence and disease. This work identified a small molecule secreted by Lactobacillus that acts against a key C. albicans virulence trait, establishing a novel strategy to thwart fungal disease.

Currently, I am transitioning to a position as a postdoctoral fellow at the National Institute of Allergy and Infectious Disease in the laboratory of Michail Lionakis. At the NIAID, I will extend my studies of human fungal pathogens to gain training in fungal immunology and human genetics.

Overall, my research interests lie in fungal pathogenesis, specifically why certain fungi, like C. albicans, are specialized members of the mucosal microbiota while other ubiquitous environmental fungi cause devastating disease in immunocompromised individuals. With a limited arsenal of antifungal therapeutics and the rising threat of antifungal resistance, I plan to continue to use molecular genetics to understand the interactions between fungi and their human hosts. The goal is to better understand fungal pathogenesis and identify potential therapeutic targets in both fungi and humans to combat fungal disease.  

As a PhD-trained scientist, you have many career options. What interests you the most?

For as long as I can remember, I’ve wanted to be a scientist: to be able to go into the lab, ask tough questions, and use experiments to try to further our understanding of fundamental biology. Throughout my formal education, I focused on pursuing a career as a Principal Investigator at a research-intensive institution. I am passionate about both education and research, so I am very drawn to the fact that PIs can teach classes, mentor trainees, and continue to drive a competitive research program as part of their career. Throughout my scientific training, I’ve been fortunate to work with incredible mentors and supervisors, including Leah Cowen and Teresa O’Meara, who have demonstrated what can be accomplished as a PI.

My mom and sister both recently received advanced degrees in education, so our home is always filled with lively discussions of teaching philosophies and curriculum development. I am very passionate about mentoring the next generation of scientists, and I want to pursue a career where I can continuously mentor, support, and teach students, especially in genetics-related fields. As a PI, I hope to be able to use my future lab to mentor students and continue to ask fundamental biological questions related to pathogenesis and virulence.

In addition to your research, how do you want to advance the scientific enterprise?

Throughout my scientific training, I have been passionate about science communication and outreach. In particular, I am deeply dedicated to ensuring all children can picture themselves as scientists, encouraging youth to pursue science. My passion for youth in STEM originates from my early experiences in the Canadian science fair program. Although neither of my parents are scientists, the science fair exposed me to research at a young age. These formative experiences demonstrated to me what it is like to pursue science as a career much more effectively than my elementary and high school classes.

For the past ten years, I have been a member of the Youth Science Canada Executive Committee, which organizes the annual Canada-Wide Science Fair (CWSF). In this role, I help to organize and run CWSF, which takes place in a new Canadian city each year and sees participation from approximately 500 secondary school students from every corner of Canada. Beyond these organizational efforts, since 2016, I have also helped to write and act in a children’s television show on the Canadian network TVOKids. Targeted to young learners, Blynk and Aazoo features a child asking a common question (e.g., How can I stay up all night? How can I make my vegetables taste better?) and includes an in-depth answer from a scientist. Additionally, I am a freelance journalist with Engineering.com, where I cover news related to computational biology, AI/ML, and cloud computing.

Through all these endeavors, my goal is to continue to engage the public, specifically youth, in the scientific community. I firmly believe that curiosity is a core tenet of being human. Where classical education seems to frequently fail to portray the exciting pursuit of scientific problems, I aim to use my time to ensure everyone knows they can ask complex questions and explore their curiosity. This also relates to my firm commitment to support diverse and inclusive spaces within the scientific community, whether at the science fair, in the lab, or within professional societies like GSA. Because science is a fundamental part of being human, no one should ever be excluded from pursuing it.

As a leader within the Genetics Society of America, what do you hope to accomplish?

I joined GSA’s ECLP program to join a community of like-minded scientists dedicated to creating an inclusive, supportive, and diverse space for researchers to pursue science. As a Co-Chair of the Communication and Outreach Subcommittee, I hope to expand GSA’s commitment to engage broad audiences with the genetics community. I am particularly excited to expand the subcommittee’s social media presence to engage more non-technical audience members with the field of genetics and its impact on our everyday lives. We are in the process of launching a dedicated Instagram presence within the GSA account. In addition to this initiative, I am eager to support the subcommittee’s outreach efforts and encourage our members to develop their communication skills and conduct projects related to their passions.

Beyond my work within the subcommittee, I am also excited to be a part of the ECLP to grow my professional network and use the incredible resources offered by GSA to further my own leadership, communication, and research skills.

Previous leadership experience

Foraging for Fungi Walks, Royal Canadian Institute of Science (2022-Present)

Foundational Genetic Approaches Teaching Assistant, University of Toronto (2022)

Molecular Genetics Teaching Assistant, University of Toronto (2021-2023)

Girls SySTEM Mentor (2021-2022)

Adventures in Science Mentor, University of Toronto (2020-2022)

Math and Science Tutor-Mentor, Tutorbright Toronto (2018-2020)

Board Member, Partners in Research (2016-2019)

Judging Division Head, Thames Valley Science and Engineering Fair (2014-2019)

Executive Committee-Youth Science Canada (2013-Present)

Aspergillus fumigatus isolated from clinical settings is resistant to agricultural fungicides.

Infections have long been a deadly problem for hospital patients. Though modern medicine has an impressive array of antimicrobial drugs at its disposal, pathogens continue to evolve resistance, creating ever more dangerous infections as the microbial “arms race” escalates.

Overprescribing of antibiotics is one source of resistance, but there could be another culprit further afield. In a new publication in G3: Genes|Genomes|Genetics, fungal biologists Michelle Momany and Marin Brewer report their finding that some strains of a pathogenic fungus apparently acquired antifungal resistance in an agricultural setting rather than a clinical one. Genetic analysis revealed that strains of the fungus Aspergillus fumigatus that were resistant to antifungals used in people had also developed resistance to fungicides used only on plants.

Fungal infections endanger both plants and people

Momany and Brewer are part of the Fungal Biology Group at the University of Georgia, one of the largest fungal biology research groups in the world. Fungal diseases are a major problem both clinically and in agriculture, but doctors generally use different compounds to treat people than farmers use to treat crops. A class of antifungals known as azoles, however, is used in both people and plants. Many different azoles are currently available, and though they are considered “moderate” in terms of the risk of fungi developing resistance, azole-resistant strains of the fungus Aspergillus fumigatus have begun turning up more frequently in hospitals.

Unlike drug-resistant bacteria, which can spread from person to person, A. fumigatus is always picked up environmentally. There’s no documented case of anyone getting sick by breathing in Aspergillus exhaled by an infected patient, says Momany. Because of this, fungal researchers suspected A. fumigatus was evolving azole resistance in the field, rather than in patients. Still, there was no way to know for sure that it wasn’t caused by hospital drugs.

From “Big Chicken” to Big Tulip

“The idea came to me when I was reading the book Big Chicken by Maryn McKenna,” says Brewer. In the book, scientists showed that antibiotic-resistant bacteria had moved from chickens to humans – not the other way around – by showing that bacteria isolated from people contained genetic evidence of resistance to compounds used only in chickens. Brewer decided to apply that same logic to Aspergillus. “I thought it would be interesting to look for the signatures for resistance to fungicides only used in agricultural environments,” she says.

The researchers analyzed 700 different isolates of A. fumigatus collected from compost heaps, soil samples, and plant debris from 56 sites around Georgia and Florida. They sequenced and analyzed the genomes of 135 of these isolates. Most of the sites had a history of azole fungicide use, but two were organic sites with no fungicide use for 10 years. They also analyzed publicly available genome data of A. fumigatus samples from the US, the UK, the Netherlands, and India that were resistant to multiple azole compounds. 

They found that A. fumigatus in the environment that carried resistance to azole compounds had often developed resistance to two other classes of fungicides used only in agriculture, benzimidazoles (MBC) and quinone outside inhibitors (QoI). Even more tellingly, A. fumigatus samples collected from patients carried the genetic markers indicating resistance to MBC and QoI.

“Those signatures showed that they were exposed to fungicides in an agricultural environment, and we’re finding them in strains from patients in hospital environments,” says Brewer.

The first hint that agricultural fungicides might be a problem in hospitals came from the Netherlands, the world’s major supplier of tulips and other ornamental flowers. “It turns out a tulip bulb is a wonderful place for fungi to grow,” Momany says. “Nobody wants decorative flowers with mold spots on them. To prepare these for shipping, they’re treated with azoles.” Discarded plant matter from these industrial tulip farms, she says, can spread azoles into the surrounding environment. Strikingly, Momany points out, A. fumigatus isn’t even dangerous to plants. It’s not the intended target of agricultural azole use, but because it’s so widespread, it gets exposed incidentally. “Once those azoles are in the environment, then the A. fumigatus that’s there develops its own resistance,” she says.

People are likely to encounter azole-resistant A. fumigatus in compost or in flower beds. “Compost is definitely a hot spot for Aspergillus that’s azole resistant,” says Brewer. “People who are immunocompromised should be very careful around compost, flower beds, or dusty areas where there can be a lot of fungi.”

This study is one of the first to characterize a fungal pathogen that has picked up antifungal resistance in an agricultural setting and carried that resistance into the clinic. “There could be a lot of others, and people just haven’t looked,” Brewer says.

CITATION:

Evidence for the agricultural origin of resistance to multiple antimicrobials in Aspergillus fumigatus, a fungal pathogen of humans

S. Earl Kang, Leilani G. Sumabat, Tina Melie, Brandon Mangum, Michelle Momany, and Marin T. Brewer

G3: GENES|GENOMES|GENETICS February 2022, jkab427

https://doi.org/10.1093/g3journal/jkab427

Prize recognizes extraordinary creativity and ingenuity in genetics research.

Joseph Heitman, MD, PhD of Duke University is the recipient of the 2019 Genetics Society of America (GSA) Edward Novitski Prize. Heitman is honored for his work on human fungal pathogens and for identifying the molecular targets of widely-used immunosuppressive drugs. The latter work proved to be a seminal contribution to the discovery of the TOR pathway, which regulates cell growth and activity in response to nutrients.

The Novitski Prize recognizes an extraordinary level of creativity and intellectual ingenuity in the solution of significant problems in genetics research. “Joe is an exceptionally imaginative geneticist, and his work has often been paradigm changing,” says William K. Holloman, a Professor at Cornell University Weill Medical College and one of the scientists who nominated Heitman for the Novitski Prize.

Heitman helped kickstart the study of the previously unknown TOR pathway with a series of inspired experiments during postdoctoral research at the Biozentrum of the University of Basel, under the mentorship of Michael N. Hall and in collaboration with Rao Movva from the pharmaceutical company Sandoz (now Novartis).

The story began with an unmet medical need.  In the 1980s and 1990s, surgeons developing organ transplant procedures were finally starting to see success. Thanks to newly discovered immune-suppressing drugs isolated from soil microbes, the doctors were able to prevent transplanted organs from being rejected by the patient’s immune system.

Yet despite the success of these important drugs, it was not clear how they worked. Although they targeted specific cells of the immune system, the drugs had originally been identified as antifungal chemicals. Heitman and his colleagues reasoned that this ability to kill fungi could be the key to understanding how the drugs worked in humans.

The microbes likely evolved to produce these natural product drugs to fend off fungal competitors in the soil. Perhaps, the team hypothesized, the mechanism by which they inhibited fungal growth would be the same by which they inhibited function of the immune system. After all, despite the obvious differences between fungi and ourselves, we all evolved from a common ancestor and share many components of our basic cellular machinery. That is why Heitman proposed an unconventional strategy for identifying the molecular targets of the new drugs: studying their effects on baker’s yeast.

As one of the best understood fungi, with many genetic research tools already available, yeast supplied a powerful method for honing in on targets of the drugs. The group investigated the effects on yeast of several transplant drugs, including a then-experimental compound named rapamycin.

Rapamycin turned out to have a similar effect on yeast cells as it did on human immune cells—blocking progress of the cell cycle. This not only supported their hypothesis, it made it possible for them to screen for the chemical’s molecular targets by looking for the yeast genes that made the cells vulnerable to rapamycin treatment.

Their strategy paid off quickly. Heitman isolated rapamycin-resistant yeast mutants, and identified three genes needed to make the yeast sensitive to the drug. Among them were two previously uncharacterized genes that they named TOR1 and TOR2, for “Target of Rapamycin” and tor, the German word for gateway (in honor of Basel’s medieval city gate Spalentor). The third gene encodes FKBP12, the cellular receptor that binds rapamycin to form a complex that inhibits TOR.

Spurred by the discovery of the fungal targets of rapamycin, other research groups later confirmed that the human version of these yeast genes, mTOR, was also the target of the drug’s immunosuppressive activity.

“Joe exploited yeast genetics in a creative, clever, and unique way, which ultimately led to the appreciation that immunosuppressive drugs work through a pathway that is conserved from yeast to humans. His work was instrumental in understanding this important network,” says colleague Sue Jinks-Robertson, a Professor at Duke University who nominated Heitman for the Novitski Prize.

Thanks to the subsequent work of many researchers, including Heitman, Tor proteins are now understood to be at the center of a critical pathway for both biology and medicine. Animals, plants, and fungi all use Tor proteins to sense nutrient levels and set in motion appropriate physiological responses. Without Tor, cell growth and division become uncoupled from nutrient status, which is why dysfunction of the pathway is implicated in many human disorders, including cancer, cardiovascular disease, and diabetes, as well as the aging process.

As Heitman established his own research group, linking fundamental insights in fungal biology to medically important problems became a recurring theme in his work. He has made many contributions to our understanding of fungal species that cause diseases, including findings on how such fungi sense their environment, what factors affect their virulence, and how antifungal drugs work. His studies have defined fungal mating type loci, including their evolution and links to virulence, and illustrated convergent transitions from outcrossing to inbreeding in fungal pathogens of plants and animals.  He has led efforts to establish new genetic and genomic methods for studying pathogenesis in Cryptococcus species, which cause a range of life-threatening infections—and up to 15% of all AIDS-related deaths—but are of a quite different type than most other fungal pathogens of humans.

Studying Cryptococcus has also produced broader insights into how many other fungal diseases evolve and cause outbreaks. For example, Heitman’s work on Cryptococcus has contributed to a growing consensus that, contrary to previous assumptions, fungal pathogens have a robust sex life. His group even discovered an entirely new mode of fungal reproduction.

Like many fungi, Cryptococcus occurs as two different mating types. In the lab, one cell of each type can pair up to mate and sexually reproduce. But one of these two mating types is extremely rare in the wild, so Cryptococcus was thought to mostly go without sex—if it ever occurred at all. The vast majority of cells were thought to reproduce by splitting into genetic clones.

However, Heitman and colleagues found that Cryptococcus cells can mate with themselves by fusing with others of the same mating type or undergoing endoreplication to produce new offspring. This “unisexual reproduction” involves many of the same intricate series of steps as sexual reproduction, including the transition from haploid to diploid and the dividing up of chromosomes by meiosis to ensure offspring receive the correct final number. The main difference? Unisexual reproduction can occur between two genetically identical cells or genomes, such as “mother” and “daughter” cells that have just split by normal cell division.

But why go to all this trouble? Why not just keep multiplying by asexual division? The main evolutionary benefit of sex is to shuffle genetic variants in a population into new combinations. In unisexual reproduction, mixing around the genomes of two parents with already matching variants would make no difference—the offspring would have the same combinations as their parents.

Heitman’s research suggests an answer: their evidence shows unisexual reproduction can generate new genetic diversity by triggering new mutations. This allows fungal species that rarely engage in bisexual outcrossing an escape mechanism from the evolutionary dead end of endless clonal division.

Heitman and others are now studying the roles of sexual reproduction in the evolutionary dynamics of fungal pathogens to better understand how they spread, switch hosts, become more virulent, and a host of other important clinical questions, such as how the emerging pathogen Cryptococcus gattii expanded its geographic range to cause an ongoing outbreak in North America.

Heitman has been a GSA member since 1998, served as an editor for the GSA journal GENETICS 2011–2017, and has served on the GSA Conferences Committee since 2017. He has also served as a member of the Fungal Genetics Policy Committee from 2013–2019 including as chair of the Committee 2017–2019. The Prize will be presented at the 30th Fungal Genetics Conference, which will be held March 12–17, 2019, at Asilomar Conference Grounds in Pacific Grove, CA. This will be the tenth consecutive Fungal Genetics Conference that Heitman has attended.

Heitman is the James B. Duke Professor and Chair of the Department of Molecular Genetics and Microbiology at Duke University and director of the Tri-Institutional Molecular Mycology and Pathogenesis Training Program.

The Novitski Prize recognizes a single experimental accomplishment or a body of work in which an exceptional level of creativity and intellectual ingenuity has been used to design and execute scientific experiments to solve a difficult problem in genetics. It recognizes the beautiful and ingenious experimental design and execution involved in genetics research. The Prize, established by the Novitski family and GSA, honors the memory of Edward Novitski (1918–2006), a Drosophila geneticist and lifelong GSA member who specialized in chromosome mechanics and meiosis through the construction of modified chromosomes.

Sexual reproduction is scarce in skin infection culprit.

While some people love to feel the burn during a workout, we generally seek that sensation in our muscles—not our feet. Treading barefoot in damp, communal environments like gym showers and the perimeters of pools can expose us to the fungus Trichophyton rubrum, the most common cause of athlete’s foot. Despite its name, athlete’s foot isn’t found exclusively in fitness fanatics—it affects around 15% of people worldwide. New work published in GENETICS shows that across this global range, the T. rubrum genome varies surprisingly little.

T. rubrum is widespread and comes in many varieties called morphotypes that differ in characteristics such as which parts of the body they can infect and the appearance of their colonies. In this study, the researchers found that T. rubrum samples from around the world were remarkably genetically similar to one another despite representing many different morphotypes. The data also suggest T. rubrum rarely, if ever, sexually reproduces. Mating in many fungi occurs between cells of different mating types, but of the 135 samples tested, the mating types of all but a single Mediterranean strain were identical.

The researchers found no evidence of mating when they paired the Mediterranean strain with strains of the opposite mating type, which supports the idea that the fungi reproduce clonally. This result comes with some caveats, though: the lab conditions may not have favored mating, and it’s possible that mating does occur when the Mediterranean strain comes in contact with some of the other strains in the wild. Overall, the results are consistent with one hypothesis that has been put forth about the fungi, which that is the species recently experienced a sharp decrease in sexual reproduction. The authors suggest this might have occured when T. rubrum began specializing for growth on humans.

Given that pathogens must dodge the defenses of their constantly adapting hosts, it may seem strange that T. rubrum exhibits such low genetic diversity, but it’s not alone in this trait. For reasons that haven’t been fully established, bacteria that cause tuberculosis and Hansen’s disease (leprosy) also come in a variety of types despite being highly clonal.

Although T. rubrum infections are treatable and rarely progress to serious disease, they’re common and often extremely uncomfortable. Those of us who wear sandals in the gym shower would certainly agree it’s well worth it to learn more about how this pesky fungus operates.

CITATION:

Whole-Genome Analysis Illustrates Global Clonal Population Structure of the Ubiquitous Dermatophyte Pathogen Trichophyton rubrum
Gabriela F. Persinoti, Diego A. Martinez, Wenjun Li, Aylin Döğen, R. Blake Billmyre, Anna Averette, Jonathan M. Goldberg, Terrance Shea, Sarah Young, Qiandong Zeng, Brian G. Oliver, Richard Barton, Banu Metin, Süleyha Hilmioğlu-Polat, Macit Ilkit, Yvonne Gräser, Nilce M. Martinez-Rossi, Theodore C. White, Joseph Heitman, Christina A. Cuomo
GENETICS 2018 208: 1657-1669; https://doi.org/10.1534/genetics.117.300573
http://www.genetics.org/content/208/4/1657

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Rapid genomic changes observed in Candida albicans soon after exposure to the oral cavity.

Whether or not you treat your body like a temple, it presents a hostile and rapidly-changing environment for the many microorganisms that call you home. In contrast to the microbes that hang out inside humans, those that are cultured in the lab can expect predictable conditions designed to help them thrive. But could the real-world stresses of the host environment contribute to a pathogen’s ability to adapt? A new report in GENETICS sheds some light on this question, showing the surprisingly immediate genomic effects of exposing fungal populations to the inside of a mouth.

Forche et al. studied the effects of infection on population variability in the fungus Candida albicans, which is normally a harmless commensal bystander but, under the right conditions, can cause opportunistic infections. One of the places this can happen is in the mouth, which is one of the few locations where C. albicans can grow as either a commensal or a pathogen. The authors took C. albicans that had been cultured in vitro and briefly infected the oral cavity of mice, then collected the fungi at intervals over a few days and analyzed them for genotypic and phenotypic changes.

The authors found that as few as 24 hours in the mouths of mice was enough to increase the diversity of colony appearance. Genotypic differences, including aneuploidy and loss of heterozygosity, became more common, too. Although the authors note that the transition from in vitro to in vivo systems—and back again—may be a contributing factor, this observation nonetheless demonstrates the astonishingly rapid diversification of C. albicans during infection. Among the genotypic differences identified, changes in chromosome number were relatively common; in particular, trisomy 6 was identified in C. albicans isolates from multiple mice. The authors suggest that this variation might confer advantages during infection, which could be a promising direction for future study.

They also analyzed the number of genetic events (e.g. aneuploidy, recombination) in different isolates, and strikingly, they found that isolates with more than five such events were more common than would be expected by chance. This was not true of C. albicans cultured in vitro, suggesting that a subpopulation of highly variable C. albicans were overrepresented after exposure to the oral niche. Whether the dramatic genetic rearrangements in these rare individuals are beneficial in the long-run—and how the host responds to such variation—remain questions to be explored.

CITATION:

Rapid phenotypic and genotypic diversification after exposure to the oral host niche in Candida albicans

Anja Forche, Gareth Cromie, Aleeza C. Gerstein, Norma V. Solis, Tippapha Pisithkul, Waracharee Srifa, Eric Jeffery, Darren Abbey, Scott G. Filler, Aimée M. Dudley, Judith Berman

Genetics July 2018 209: 725-741; https://doi.org/10.1534/genetics.118.301019

http://www.genetics.org/content/209/3/725

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Expression analysis provides clues about what makes some peanut strains more susceptible to fungal toxin contamination.

In 1960, 100,000 turkeys across hundreds of English poultry farms died from aflatoxin contamination in the peanut meal in their feed. Aflatoxin is a potent carcinogen produced by fungi of the genus Aspergillus, which can grow on peanuts. Although the peanut butter in your supermarket is tested for aflatoxin before it reaches you, fungal toxin contamination remains a major problem for peanut producers. Some peanut strains are more likely to become contaminated than others, but the reasons for this difference aren’t clear.

In a report in GENETICS, Korani et al. examined the transcriptomes of peanuts and fungi to gain a better understanding of what makes some peanuts resistant to contamination with aflatoxin. The authors chose two strains of peanut—one that was resistant to aflatoxin contamination and one that was susceptible. They infected some seeds with the aflatoxin-producing fungus Aspergillus flavus, leaving some seeds uninfected as controls. They used RNA-seq to examine differential gene expression in both the peanuts and the fungus.

By comparing gene expression between uninfected and infected seeds and between resistant and susceptible strains, the authors were able to identify consequential pathways. They found that resistant plants had higher expression of genes involved in the synthesis of jasmonates, which are plant hormones known to be involved in plants’ response to biotic stresses, such as insects. This suggests that jasmonates might inhibit fungal production of aflatoxin.

The authors also examined the transcriptome of A. flavus grown on resistant or susceptible peanut seeds. Interestingly, they found significant differences in the expression of a number of metabolic genes, particularly those involved in carbohydrate processing. These results suggest that different peanut strains may alter the metabolism of fungi growing on them, leading to decreased aflatoxin production.

This study provides clues as to potential mechanisms of aflatoxin contamination-resistance in peanuts. The authors note that the study was not designed to identify individual genes important in resistance phenotypes; they analyzed whole pathways instead. Further studies will be needed to demonstrate causality, but a better understanding of these resistance mechanisms will help farmers ensure that our beloved peanut butter remains safe to consume.

CITATION:

Insight into Genes Regulating Post-harvest Aflatoxin Contamination of Tetraploid Peanut from Transcriptional Profiling

Walid Korani, Ye Chu, C. Corley Holbrook, Peggy Ozias-Akins

GENETICS May 1, 2018 vol. 209 no. 1 143-156; DOI: 10.1534/genetics.118.300478

Biolistic genetic transformation in C. neoformans produces few off-target side effects.

While genome editing is a staple of genetics research, there remains anxiety about unintended side effects of genetic transformation, one of the most common longstanding genome-editing techniques. Some researchers fear that the process of introducing exogenous DNA into a cell may cause unwanted mutations, adding confounding variables to their experiments—but others aren’t content to accept this lore.

In G3, Friedman et al. report their study of the off-target effects of transformation in the common fungal pathogen Cryptococcus neoformans. They created 23 new strains using biolistic transformation, a standard procedure for this organism that involves shooting gold beads coated with DNA into cells, to add a marker to a neutral site in the strains’ genomes. They then sequenced the genomes of these new strains. Across all 23 strains, they found only four point mutations; of these, just one changed an amino acid in the encoded protein. They also found one case of insertion of a second, partial copy of the drug resistance marker.

They used the same transformation method to create more than 100 strains, each with a single gene replaced by a marker gene. By carrying out RNA-Seq on this group, they identified six strains that expressed the marker at unusually high or low levels. On average, these outlier strains had 1.67 off-target point mutations, and three of them (50%) carried multiple copies of the marker. The greater number of mutations in these six strains compared to the first set of 23 likely reflects selection for mutations that compensate for the genes the researchers replaced. Nonetheless, the overall number of off-target effects was still low, and the authors write that the mutations would be unlikely to have consequences as drastic as deleting the intended gene would. Therefore, they argue, effects observed when a gene is deleted using this protocol are likely most often due to the deletion and not to off-target effects, although additional confirmation of any deletion’s effects is still prudent.

The study illustrates the importance of testing conventional wisdom, and it will be important to investigate whether these findings apply to other species used in research and other transformation techniques. In the process of conducting this study, the researchers also sequenced a frequently used C. neoformans laboratory strain’s genome—a vital resource because this fungus is estimated to kill hundreds of thousands of people each year.

CITATION:

Unintended Side Effects of Transformation Are Very Rare in Cryptococcus neoformans
Ryan Z. Friedman, Stacey R. Gish, Holly Brown, Lindsey Brier, Nicole Howard, Tamara L. Doering and Michael R. Brent
G3: GENES|GENOMES|GENETICS 2018 8: 815-822; https://doi.org/10.1534/g3.117.300357
http://www.g3journal.org/content/8/3/815

Rhinocladiella mackenziei is a fungus that infects the human brain. It is the most common cause of neurological fungal infections in arid regions of the Middle East, and it is fatal in 70% of cases. However, little is understood about this lethal pathogen—not even its natural habitat.

To learn more about the biology of R. mackenziei, Moreno et al. turned to its genome. They resequenced the genome of two strains isolated from patients and compared them to known sequences from R. mackenziei, as well as other related fungi.

These comparisons gave clues about the natural lifestyle of the fungus. For example, R. mackenziei carries genes similar to fungi from habitats polluted by aromatic hydrocarbons, such as those found in gasoline. This suggests that R. mackenziei might flourish in oil-contaminated desert soil, where these genes would give it a competitive advantage over organisms that are unable to thrive in such a harsh environment.

The authors also identified a number of secreted virulence factors which could permit R. mackenziei to more easily establish itself in the brains of infected humans. The genomes harbor a wide array of genes involved in metabolism of diverse substrates, as well as nitrogen and iron uptake. This metabolic adaptability means that R. mackenziei probably isn’t a true pathogen; a pathogen would have lost some of these pathways because it could rely on its host for nutrients. Rather, this desert fungus is equipped to survive a number of harsh conditions, so its ability to infect human brains is most likely opportunistic.

CITATION

Genomic Understanding of an Infectious Brain Disease from the Desert

The yeast Candida albicans lives on and even inside many of us. Most of the time, its silent presence goes unnoticed, but this fungus can turn on its host, causing infections ranging in severity from annoying to life-threatening. For the yeast to become pathogenic, some of the C. albicans must transform from small, round cells into long, thread-like filaments, a process that can be triggered by environmental cues. To learn more about how these yeast morph, Azadmanesh et al. examined C. albicans filamentation under ten different conditions—and their results may have implications for the ways we study and treat infections.

C. albicans filamentation can be triggered by a variety of stimuli, from the surface the yeast are growing on to chemicals floating around them. Acidic environments, for example, make filamentation less likely, which is thought to be one reason maintaining a healthy balance of lactic acid-generating bacteria helps prevent vaginal yeast infections. Azadmanesh et al. determined which genes are required for filamentation under ten different environmental conditions, and for each condition, they also examined how gene expression changes during filamentation.

The researchers identified several genes needed for filamentation in all the conditions tested. In most cases, the reasons these genes are needed isn’t clear, but some have roles that make sense given what we know about how filamentation works. A few of the genes, for example, are involved in regulating the actin cytoskeleton, and modifications to the actin cytoskeleton are required for filamentation.

Surprisingly, though, the core genes required for filamentation under all conditions are the exceptions. Mostly, they found that both the genetic requirements for filamentation and the gene expression changes vary significantly in different conditions. This means that when researchers are comparing previous studies of C. albicans filamentation, they may not be comparing two like things: the programs of filamentation may be different in each case. The group’s work also has medical implications. The genes required for filamentation are different in solid and liquid media, suggesting that C. albicans infections in the gastrointestinal and genitourinary tracts are likely different from those found in bodily fluids like blood—which may be an important factor to consider when studying these infections and designing treatments.

CITATION:

Azadmanesh, J.; Gowen, A.; Creger, P.; Schafer, N.; Blankenship, J. Filamentation Involves Two Overlapping, but Distinct, Programs of Filamentation in the Pathogenic Fungus Candida albicans.
G3, 7(11), 3797-3808.
DOI: 10.1534/g3.117.300224
http://www.g3journal.org/content/7/11/3797

At first glance, the yeast Candida krusei seems as innocuous as microbes come: it’s used for fermenting cocoa beans and gives chocolate its pleasant aroma. But it’s increasingly being found as a pathogen in immunocompromised patients—and C. krusei infections aren’t always easy to cure. This yeast is naturally resistant to fluconazole, a first-line antifungal that’s vital not just for treating many fungal infections but also for preventing them in susceptible populations. In the September issue of G3, Cuomo et al. unveil the first whole-genome sequence of a clinical sample of C. krusei, providing leads on genes that may be important for the species’ fluconazole resistance.

Some draft assemblies of the C. krusei genome had been produced prior to these researchers’ work, but they were fragmented, a problem caused by a large amount of heterozygosity. The key to their success was to generate long sequencing reads, producing an assembly with not many more scaffolds than C. krusei has chromosomes.

With their new draft in hand, Brand et al. searched the C. krusei genome for genes that are associated with pathogenesis in the more familiar Candida albicans—the cause of thrush, vaginal yeast infections, and sometimes potentially lethal systemic infections. C. krusei, they found, has few copies of gene families associated with pathogenesis in C. albicans, including oligopeptide transporters, aspartyl proteases, and phospholipase B genes. Candida species that often cause disease usually have expansions in these gene families, so given that C. krusei is rarely pathogenic, this finding makes sense.

When they focused on genes involved in drug resistance, the group found that C. krusei differs from C. albicans; many of the sites that are often mutated in the target of azole drugs in resistant C. albicans are not mutated in C. krusei. This implies the cocoa-fermenting yeast’s natural resistance to fluconazole arises from a different mechanism than the one that commonly allows C. albicans to resist azole drugs.

The researchers also didn’t find the gene MDR1, which encodes a drug transporter often responsible for resistance to multiple drugs. They did, however, find copies of genes related to CDR1, CDR2, and few others that encode other transporters associated with drug resistance—although the way they function in C. krusei is unknown. These genes could be a starting point for discovering the mechanisms behind antifungal resistance in C. krusei, potentially leading to better treatments and preventive measures for highly vulnerable populations.

CITATION:

Cuomo, C.; Shea, T.; Yang, B.; Rao, R.; Forche, A. Whole Genome Sequence of the Heterozygous Clinical Isolate Candida krusei 81-B-5.
G3, 7(9), 2883-2889.
DOI:10.1534/g3.117.043547
http://www.g3journal.org/content/7/9/2883