Seeking answers in a molecular maze — II

One ongoing project in Dr. Sliwinski’s lab is to continue tracking soil archaea using DNA fingerprinting.

“In addition to my work, other labs around the world have found DNA evidence of archaea in the soil. They are on every continent and in every soil tested,” he says. “Part of what now needs to get done is an accounting of which species of archaea are where, over space and over time.”

He has created a modified version of the DNA fingerprinting technique that he developed in graduate school to separate and differentiate the DNA sequences of different groups of soil archaea.

In testing this new DNA fingerprinting technique with soil samples from an Iowa prairie, one of Dr. Sliwinski’s undergraduate students discovered that a wide range of Thaumarchaeota species inhabit Iowa soil.

For a recent publication in the journal Archaea, Dr. Sliwinski returned to the same temperate forest sites in Wisconsin where he previously studied soil archaea ecology as a graduate student. “I wanted to know whether the community of archaea stays the same or does it change over time,” he explains. “Is it a dynamic community, or is it the same species dominating the area?”

He collected samples in 2001 and 2010–2012 from several plots at two collection sites. In order to compare archaeal communities on a variety of spatial scales, he collected soil samples from spots that were only centimeters apart in the same plots, and collected samples at sites which are kilometers away from each other.  He then isolated DNA from the soil samples and used a molecular biology tool called the polymerase chain reaction (PCR) to make millions of copies of archaeal DNA sequences recovered from the samples.

Dr. Sliwinski then applied his new method of DNA analysis to those sequences. This allowed him to distinguish broad groups of archaea in the soil while simultaneously differentiating similar DNA sequences of more closely related archaea.

With this method he was able to more finely resolve the diversity of archaea soil communities from the Wisconsin study sites over time and on different spatial levels. “What we found was that soil archaea community composition is patchy–the species changed in some patches while in others they remained the same,” Dr. Sliwinski says. “Depending on spatial scale, different species of archaea may be the dominant community members.”

The results also showed that community composition fluctuated over time at each plot.

In the future, Dr. Sliwinski plans to analyze archaeal DNA in soil samples that he has collected from across the country, from Maine to California, and examine whether or not there is a difference in the archaeal communities in soil samples from different states.

“If you just sample enough soil, see what’s there, you might find an outlier — a community where archaea are growing that are different from the archaea that are more cosmopolitan,” he says.

“You might find a little niche of some very interesting species, and those might be the ones that grow easily in the lab. That would be a huge discovery for an undergraduate,” he adds. “If you can get a new species to grow in the lab, you can learn more about them. Then by comparing genomic sequences, you can learn more about the ones that don’t grow in the lab.”

Dr. Sliwinski and his research students have already begun work on culturing soil archaea in the laboratory. This is a project that would typically come first in more traditional microbiology labs. “Historically, new species were discovered by first growing them in the lab, but in the entire history of microbiology, only a tiny fraction of microbes have been successfully cultured,” he says. “Molecular biology has turned it around backwards; you now find environmental DNA that suggests a novel microbe exists in a sample. Then you pick and choose which samples have species you want to study.” 

 Currently, there are only a few types of soil archaea which have been grown successfully in laboratories, says Dr. Sliwinski.

He and his student are currently working to develop an inorganic, silica-based solid matrix with which to fill the petri dishes that they will use in their attempts to cultivate archaea in the lab. This design is distinct from the organic agar gels typically used to grow bacteria and other microorganisms in a laboratory setting.

“Nobody knows why the majority of soil archaea aren’t growing in petri dishes on standard media. One idea is that petri dishes in lab can be a toxic environment. So sugar is great, but if too much sugar is present it might be poisonous to microbes that are adapted to living in a low-sugar environment such as soil,” Dr. Sliwinski explains.

Once the silica matrix petri dishes with growth medium are ready to use, Dr. Sliwinski and his student will then add soil slurries from their samples, allow the microorganisms in the soil slurries to grow on the matrix, and then extract the DNA and test for archaea using PCR. This PCR test will confirm if they are successful in growing soil archaea in the lab.

In addition to his work on archaea and plants, Dr. Sliwinski has found another interesting biological question to investigate using the tools of molecular biology. In the summer of 2016, Dr. Sliwinski and a member of his undergraduate research team joined a project with researchers from the University of Iowa’s Carver College of Medicine which focused on Ebola infections. Infection with Ebola virus causes hemorrhagic fever in humans and has had a high mortality rate during recent epidemics in Africa.

This research was conducted as a part of the FUTURE in Biomedicine Program at UI, which is designed to encourage the growth and success of talented undergraduate researchers and to promote collaborations between faculty at primarily undergraduate colleges and universities in Iowa and researchers at the Carver College of Medicine. The objective of their project was to examine whether the virus could have been transmitted through contact with the skin of infected individuals during the 2013–2015 Ebola epidemic in West Africa.

Dr. Sliwinski and his student approached this question by studying which cells in the skin are actually infected by the virus. In one set of experiments they infected donated human skin samples from surgeries with a genetically-modified vesicular stomatitis virus (foot and mouth disease) and tracked the infection over time. This virus was engineered to express Ebola glycoprotein in order to model with a safer virus to understand how Ebola infects human cells. The glycoprotein is a molecule on the virus which is required for entry into human cells. The modified virus was also designed to express a green fluorescent protein, a molecule used to track the virus during examination of infected skin samples under fluorescent microscopes. These experiments demonstrated that the model virus could infect cells in human skin.

Next, using immunohistochemistry–a method which uses antibodies tagged with fluorescent probes to label specific molecules and cells in tissue samples — and confocal microscopy to examine the samples, Dr. Sliwinski and his student explored further to determine which skin layers and cell types in the samples were actually infected by the modified virus. With these experiments they showed that like Ebola, the model virus expressing Ebola glycoprotein infected both the epidermal and dermal layers of the skin samples. They identified epidermal keratinocytes as one cell type which was infected by the model virus. This work also showed that an important type of immune cell in the skin called Langerhans cells were not infected.

In separate experiments using an in vitro human skin dermis model made from fibroblast cells, keratinocytes, and a dermal equivalent substance, Dr. Sliwinski and his student found that in the dermis only fibroblasts were infected by the model Ebola virus. Together, these findings increase knowledge of which cells in the skin can be infected by Ebola virus and may ultimately help to reveal if Ebola transmission in humans can occur via the skin.

Dr. Sliwinski says some of the interesting questions to ask next include how the virus moves through the cells and layers of the skin. He also says that because his work was done with a model system, further study is necessary to better determine how actual Ebola infection works in the skin of living animals.

Dr. Sliwinski says that what he appreciates most about his position at UNI is this ability to work on the broad range of research questions that he finds interesting. “As a scientist, I’m in a place where I can literally study any molecular biology question,” he adds. “Over my time at UNI, I’ve allowed my research program to expand into three phases: we study archaea, we study plant genes, and we study Ebola.”

Mir Ashfaquzzaman, UNI STEM Graduate Assistant