Wisconsin Public Television University Place Wednesday Nite at the Lab  Nancy Keller Aspergillus Can Kill Us  January 25, 2012 UP672   cc >> Tom Zinnen: Welcome, everyone, to Wednesday Nite at the Lab. I'm Tom Zinnen. I work here at the UW Madison Biotechnology Center. I also work for UW Extension Cooperative Extension, and on behalf of those organizations and our other sponsors, Wisconsin Public Television, the Wisconsin Alumni Association, and the Division of Continuing Studies, thanks for coming to Wednesday Nite at the Lab. We do this every Wednesday night, 50 times a year. Tonight, it's my pleasure to introduce to you Nancy Keller. She's a professor in the Department of Medical Microbiology and Immunology and in the Department of Bacteriology. She received her doctoral training in the Department of Plant Pathology at Cornell University. Her postdoctoral training with the US Department of Agriculture in New Orleans first introduced her to the genus Aspergillus where she worked on the identification of enzymes required to produce the mycotoxin aflatoxin. Since that period of time, Dr. Keller has not only retained her interest in understanding how Aspergillus fungi produce toxin metabolites and using this knowledge to design controlled strategies to help combat diseases caused by pathogenic Aspergilli, I would just say Aspergilluses but that's all right. 

[LAUGHTER] She has also expanded her research to work in the field of drug discovery utilizing knowledge gained from the natural product formation by fungi. This is one of the funnest titles we've ever had: "Aspergillus Can Kill Us and Thrill Us." Please join me in welcoming Professor Keller to Wednesday Nite at the Lab. [APPLAUSE]  >> Nancy Keller: Well, hopefully we'll have a little fun tonight. And can everyone hear me okay? >> Yes. >> Okay. Wave if you can't or otherwise. Okay. So, what I thought I would do is introduce you, first, to Aspergillus. I think many of you know Aspergillus but perhaps some don't. For those of you who were sitting here earlier and you saw the beautiful waving of these objects on the YouTube, that was a video, an artist's rendering, of Aspergillus. 

So, this Aspergillus is a genus of fungus that was first described in 1729, I'll give you a little history here, by a priest scientist. I guess we call, what do you call a priest? Reverend? Micheli or Priest Micheli. And I was trying really hard to see if I could find some of the pictures that he drew of Aspergillus from his very early microscope. I couldn't find his but I did find this drawing from Louis Pasteur. Actually, maybe I can do what is asked and use this little hoo-dicky here. So this is Louis Pasteur's drawing of Aspergillus glaucus. And I part that really somewhat resembles the Aspergillus is this particular figure right here. And let me just show you what he was trying to draw at the time. Aspergillus is a genus of fungus that produces its sexual spores, and, yes, fungi have both sexual and asexual spores. Pardon me, these are the asexual spores, on top of a stalk that has a vesicle on the top and cells that produce these spores that fly into the air. 

And I'm quite sure we're probably breathing in some of these spores right now. If you're a healthy person, it doesn't make any difference because your immune system will kill the spores. Now, where did this name come from? So here's the really interesting part with this fungus. So Reverend Micheli was a scientist. He was also a Catholic priest. >> Monsignor. >> Monsignor. And what he did notice right away is that the Aspergillus stalk actually looked quite a bit like an instrument that's used by the priests in the Catholic church. This instrument is known as an aspergillum, and hence he called the fungus Aspergillus. And this gives you an idea of how similar the likeness is between this fungus and the Aspergillus. 

You can see here the priest sprinkling the holy water, and it does look very much like the spore is coming off of the Aspergillus. And this, for those of you who are interested and mesmerized by that YouTube I showed earlier, this is the address. So, now that I've introduced you to Aspergillus, I'll take a moment to tell you how I got interested in working with this. Tom did give a bit of my background, but why, you might ask, did I start working with fungi and Aspergillus. And I will tell you that I actually did have an earlier episode that brought me to fungi. I was a Peace Corps volunteer before I went to graduate school, and I was a volunteer in Africa in a country called Lesotho. And where I worked, what I noticed that oftentimes the food was really in very poor condition. We usually had enough food, but it was often tainted with mold. And a lot of my students got sick over the course of the many years I taught there. So I became very interested in agriculture and quality of food, and when I was in the Peace Corps, one of the advisers in Lesotho was actually an alumni of Cornell University where he'd taken international agriculture courses, and he's the one that got me interested in Cornell and plant pathology. 

And ever since I got my degree, I have always worked with fungi. I really enjoy fungi very much. They're fascinating organisms. And I've spent most of my career with fungi with Aspergillus. So this title we had that Aspergilli can kill you or thrill you, well, it is true. And I want to introduce you to some of the big players in Aspergillus. There's probably about 180 species to 200 hundred species, and some of them are very well-known. There's a fungus called Aspergillus fumigatus which is the most common cause of the medical diseases posed by Aspergillus. And then the Aspergillus that I first started working with was Aspergillus flavus. Aspergillus flavus and its cousin, Aspergillus parasiticus, are the very common food contaminating fungi which produce the mycotoxin called aflatoxin in our food crops. 

And we'll talk a little bit more about aflatoxin later, but it is the most potent natural carcinogen known to humankind. I'll also point out that Aspergillus flavus is the second most common Aspergillus to actually invade the body and cause various diseases. A disease of the mammal caused by Aspergillus is known as an Aspergillosis. Now, we do have Aspergilli that we like that are on the positive side, and why are they useful? Well, Aspergilli, and other fungi, are one of our most common organisms we need for composting and destroying, recycling, carbon and nitrogen. There's a particular Aspergillus called Aspergillus nidulans, which is one of the best established model organisms for genetics. We actually work with this one quite a bit. The history of this particular fungus is very interesting too and would take us into World War II, but maybe after if you're interested. Then we have the food production Aspergilli. And Aspergillus niger is a fungus where we get a lot of enzymes that we use both for food, citric acid. Also this is the fungus where we get a lot of the enzymes that we use to make what's, I think it's called stone-washed jeans. 

>> Can I interrupt you? >> Yes. >> What do you mean by fungus? >> By fungus? A fungus is an organism that has its own kingdom, and, let's see, I'll give you an example of one you probably know well: mushrooms. Mushrooms are fungi. A fungus is one; fungi are many. And they're just a kingdom of life. >> Well, why is the mushroom a fungus? >> Why is it a fungus? Well, the fungi are put into this kingdom based on their DNA sequences and the enzymes and proteins they make, and this one is classified, mushrooms belong to this kingdom based on their evolution and the sequences of their DNA and their proteins. >> And they reproduce by spores. >> Most of them do. Okay? I'm sorry, I was there making an assumption that everyone knew what a fungus was. Yes, they reproduce by spores, most of them. 

I think you probably have seen Aspergillus, or a related fungus called Penicillium, in your refrigerator on food that you don't eat readily. Or on your shower curtains. I'm quite sure you have all seen these fungi. Now, the Aspergilli we like, for those of who like Asian food especially, are Aspergillus oryzae and Aspergillus sojae. I'll show you some pictures of these. These are the fungi that are used in making, oh, soy sauce and sake and various fermented foods. And then we have a series of Aspergilli where we actually get a lot of the pharmaceuticals we use to combat drugs, I mean, combat other organisms. For example, these echinocandins right here. These are actually compounds made by Aspergillus nidulans and Aspergillus sydowii, and they kill other fungi. So fungi are always competing with each other. 

and they sometimes like to kill the other fungus so they can get their own turf. Lovastatin, maybe some of you are familiar with statins or even use these statins. These are compounds that help people with high cholesterol, and there are many others too. So now I'm going to give you an example of some of these particular good and bad Aspergilli. Aspergillosis, as I told you earlier, is just the generic name for diseases caused by Aspergillus. And this is becoming a very serious problem in the world because we're now able to keep people alive where normally we wouldn't, and we keep them alive often by using, let's say, steroids which lower the immune system. So what we're finding now is a lot of people that have diseases which lower their immune system are getting Aspergillus diseases. For example, people that have AIDS often get Aspergillus diseases, diabetics, people with leukemia. You're particularly susceptible if you've had some organ transplant. The range of diseases are from fairly mild, a lot of people have allergies, that can be mild or serious. 

Often you can get a ball in your lung. That sounds horrible but it's really not the worst thing in the world. We're pretty good at removing those aspergillomas, that's what the fungal balls are called. It's when you start getting invasive growth that the fungus is growing through your body that they're very hard to control. Now, I told you there's about 180 species of Aspergillus, but there's not that many that cause disease. One criterion is that you have to grow at 37 degrees because that's the human body. So these are some of the ones that are more frequently found in humans. The genetic model Aspergillus nidulans. Amphotericin B is an antifungal. We're having a big problem now with a lot of these Aspergilli being resistant to antifungal drugs. 

Aspergillus niger is a dark Aspergillus. Aspergillus terreus is particularly difficult to treat. It is more resistant than the other Aspergilli to antifungals. Aspergillus flavus, and the most common species, as I told you, Aspergillus fumigatus. Here's an SEM, a scanning electron micrograph, of the conidiophore, the asexual spores, here's one right here, of this fungus. You're going to see a couple gross pictures right now. These are just some examples of what can happen if you get these infections. I will tell you by the time you see a lesion on your skin, that's not a very good sign. I don't know what tissue this is, but you can see a sporulating colony right there. This is fingernails that have been infected with an Aspergillus. 

The very common disease is this aspergilloma. You can see here is lungs and here is a ball. You really can get rid of these balls pretty well. And this is a case where they didn't get rid of the ball very well though. And this is a lung from someone who died, and here is one of the, this is a huge aspergilloma. And if you do sections and you stain the lung or any other part of the body which has Aspergillus in it, you'll see the fungus, and here you will recognize that Aspergillus or the aspergillum. So it's a dead give away that's the fungus you have. Now, the other player which can kill you is the Aspergillus flavus that produces toxins in your food, and when you eat the food, you get sick. The toxins that fungi produce in food and feed are called myco. Myco is from the Greek name for fungus. 

So mycotoxins. And really the history of mycotoxins is quite fascinating. It is felt by historians, although we cannot prove this, that the 10th plague of Egypt is most likely caused by mycotoxins. If you read the documentation, it really sounds exactly like a mycotoxin outbreak. And I've given some of the history over the ages when we go back where we can figure out that certain diseases were associated with mycotoxins. I'm not going to go through all of this now but bring you to what I call modern mycotoxicology. And this is what actually made us understand that fungi can produce toxins that are very harmful. This happened in 1961 when there was a shipment of peanuts from Brazil to England. The peanuts were used as feed for turkey and chickens, and it resulted in over hundreds of thousands of poultry dying from England. This is a massive outbreak, of course, and this really set the stage for the scientists to try to figure out what happened. 

They called it turkey X disease. And ultimately what they found, it was a compound produced by the fungus Aspergillus flavus, that's where we got the name aflatoxin, that caused this disease. And what happens here are four turkey poults. This one was fed aflatoxin. You can see it's stunted, and the liver has a fatty discolored tissue look. This turkey is not going to make it. Since then, aflatoxin periodically comes into the news. One of the more interesting news items, I guess, about, geez, when was the Desert Storm war? That was about... >> '91. >> '91. 

Wow, okay. So it was weaponized and put in scud missiles. Probably it was all destroyed before it could do anything as the bomb exploded, but nevertheless. And all of us who were working with aflatoxin got phone calls from the federal government to ask if we had sent any strains of Aspergillus to anyone we shouldn't have. That was a funny question to ask because Aspergillus flavus is all over the world, but I did get a phone call. This is what it's going to look like most commonly. You'll recognize this corn ear, and this fuzzy green stuff is Aspergillus flavus, and there's the molecule of aflatoxin. I know only certain people enjoy to look at chemical structures. I'm quite found of them so I won't give you too many. You see that, you don't want to ingest it. 

Aflatoxin, we're still working on it. I do a lot of work with this. We still have not been able to control it. The fungus is ubiquitous and when the environmental conditions are right, it's going to grow in our corn and our peanuts and our tree nuts. What we do do in this country, though, is we analyze these particular crops to look at the level of aflatoxin. And the food in our country will not have high levels of aflatoxin. However, as you can see here, we do not look at our pet foods so carefully. Since I've been working with Aspergillus there's been three dog poisonings. I'll just go through this. The first one was in Texas where I actually was working at that time at Texas A&M. 

Much more severe is the constant human poisoning in Kenya. Now, I have to back up a little bit. I had told you aflatoxin was a carcinogen and it is. And you can die from liver cancer from aflatoxin, but when you have it in high amounts, it is toxic and can kill people and animals in a week to a few weeks. When you see a poisoning case, you know there has been massive amounts. And then in 2006, '05 and '06, there was another big dog poisoning case in the United States. There's been a whole lot of breakouts just recently. There's frequently aflatoxin contamination in items from China, in milk and cooking oil recently, and just again in December there was aflatoxin found in dog food from three different companies. And this is really becoming a very serious problem with the dog food. So there's a little interesting article if you guys might want to read it. 

Let's switch, though. I've given you a lot of bad news about the Aspergilli. There's a lot of good news too. And so here is, and this is quite an interesting juxtaposition, Aspergillus oryzae. Why do I say it's interesting? Well, we have now actually sequenced the genome of many Aspergilli. I think all of you probably are familiar that the human genome has been sequenced which has given us a lot of very interesting information. Well, so have many fungi. And what we found out is when Aspergillus oryzae genome was sequenced, I am going backwards, they said, huh, it's Aspergillus flavus. What's going on here? Well, it turns out that Aspergillus oryzae, which we make our food with, actually does seem to be a tamed Aspergillus flavus. There's a lot of speculation as to what happened over the years, how we got this safe Aspergillus flavus. 

That is something we could talk about after the lecture if you're interested. But in any case, Aspergillus oryzae is the fungus that is used to prepare a lot of very yummy condiments, foods and liquids. So it's really interesting. There's a YouTube, and I'm not going to do this because this would take too much time, but a very interesting YouTube which shows how you make soy sauce and how they put the fungus into the soy beans. So soy sauce comes from soy beans that's been fermented with Aspergillus. This is a little recipe here. You have soy bean cake, they do have wheat and down. Here we add Aspergillus oryzae. You put it in these different vats, and you get a mash called koji. >> Is that in China? >> That was in Japan. 

You can buy koji. And now what I have to show you, I have a little surprise here, koji and soy sauce and Aspergillus are so important in Japan that the national fungus is Aspergillus oryzae, and they've made a toy Aspergillus oryzae. My one graduate student is from Japan, and when she went back home to visit... [LAUGHTER] She brought me one of these. [LAUGHTER] We actually hang this in our lab. So this is Aspergillus oryzae, the good Aspergillus. What else do we get from Aspergillus? Thanks to penicillin, he will come home. Maybe some of you have seen this. This is from World War II. So what's this all about? Well, I think some of you might know about this finding, this accidental finding, by Sir Alexander Fleming in 1929. 

He noticed he had a petri dish of bacteria, and what he saw is that a fungus had landed into this petri dish and it was killing the bacteria. There was an inhibition zone that would not let the bacteria grow. What he and others subsequently found is that the fungus produced a compound called penicillin. Now he actually identified this from a fungus called Penicillium, but good old Aspergillus nidulans also makes penicillin, and much of the genetics in biochemistry was worked out with this model system. It turns out that we get a lot of useful pharmaceuticals from Aspergilli. Now, we had mentioned lovastatin. I'm just guessing some of you might actually have this prescription. And here is the chemical structure. Aspergillus terreus is the fungus of choice to make lovastatin. Now, I've given you a sort of brief introduction to the different Aspergilli, and this is a nice little segue right now where I'm just going to tell you a little bit about what we do in our laboratory. 

As I said earlier, I know chemical structures aren't everybody's cup of tea, but you have to admire the diversity here, I'm sure. And when you look at these, what I want you to think is that, oh, wow, fungi make these. Not all these are from Aspergillus. Actually, I couldn't even possibly put in all the compounds from Aspergillus, but this is just to show you that the compounds are very diverse. And these compounds are very bioactive. And that's the main reason why Aspergilli can sometimes be a problem for us or a benefit. They make compounds that are destructive to the animal body, though in small amounts it might be good. Here's, I'm sure I have it, yeah, here's penicillin here. Here's aflatoxin. Here's the statin, lovastatin. 

Some of these are good. This is another antibiotic. This is bad. That's bad. Bad. Bad. Really bad. Bad. Good. Neutral. We have one somewhat neutral one. 

So what are we doing? I forgot to, I wanted to put in this slide, too, to tell you that the production of these compounds by the fungus of course has nothing to do with humankind, but they're a benefit to the fungus. And it depends in the lifestyle and the life stage of the fungus when it's going to make all of these different compounds. So right here is our conidiophore again from the aspergillum. All Aspergilli will produce one of these. And each one of these little spores will fly in the air, and when they land on a substrate that they can obtain nutrition, they're going to germinate. Right there it's germinating. Now that substrate could be, oh, a seed. Or it could be decayed vegetative matter. Or it could be your lungs. After it has germinated, it will form mycelium. 

Under the right conditions that mycelium could form the asexual stage again, and you have this circle. Under some conditions, mycelium can produce the sexual stage, and it makes these organelles, which are called sclerotia or cleistothecia, where we get a sexual spore. Now, I've colored some of these different parts of the fungus different colors because they are different colors and a lot of this has to do with some of the chemical structure. And in these different tissues you get different compounds. So, the fungus is very fluid and it will produce different structures and different secondary metabolites depending on the environment. One interesting thing is Aspergillus produces its asexual spores in the light and will only have sex when it's dark. [LAUGHTER] There are other things that the fungus responds to, and, in particular, I'm going to focus right here, is a lot of these chemicals that the fungus is producing are in response to stresses, particularly when the fungus meets other microbes. And that would be, where's that little arrow? Here it is. And by other microbes it could be another fungus or it could be a bacterium. So let me show you how we have taken advantage of this information to understand how the fungus makes these chemicals and hopefully ultimately find some therapies to stop the fungi that we don't want from invading us but also maybe find new drugs. 

We made a fantastic discovery, really. We were very fortunate to find a gene called laeA. This is now in the fungal world very famous. I don't think anybody here has heard of it, but anyone who works with fungus now knows about it because it turns out to be a global regulator of all those compounds I showed you earlier. This square right here is what we call a thin layer chromatography plate. And if you put extracts of any organism on the plate and put it in a solvent, the compounds will move up the plate. And what you can see here, this is the wild type fungus, is that as you put an extract of the fungus on the plate, you can see many different compounds separating out. And some of these compounds are the structures I showed you earlier. What we found is this particular protein, laeA, when you remove it, this delta means it's removed, is you lose production of many of these chemical structures. We'll just ignore this right here. 

I think the main point I want to show you is that this particular protein is required for the expression of the genes that make these chemical, these great variety of chemicals in Aspergillus and now, it turns out, other fungi. Now, this little plate right here gives you an example of just how important that laeA is. I want you to look a little closely at this plate. This is a petri dish, and what we have here is, can you see these little white objects? >> Yeah. >> Okay. These are larvae of a fungivore. A fungivore is an insect that eats fungi, and what we did is we gave it a choice. We put all of the larvae in the middle of the plate, and in this section we had the wild type Aspergillus, you can actually see wild type there, and in this section we had the Aspergillus where we had removed that laeA protein so it no longer produced many chemicals, many toxic. And when we found is that all of the larvae, except for, let's see, we got a little goofy one that's sort of out here by itself, but all of the larvae made the choice to go to the fungal strain that produced less toxins. So we know a lot of these toxins are, indeed, a protection for the fungus. 

What we then asked is if this protein is important for making toxins, maybe it's important in Aspergillus fumigatus, the fungus that causes disease in humans. So what we did is we removed that protein from the human pathogen Aspergillus fumigatus. We found something very interesting. It does appear to be a virulence factor. This particular slide here is we used the Muri model, that is the mouse model, to test for pathogenicity. And what you can see is when you remove laeA, there is a greater percent survival of the mice. We have a clue as to how this happens. Here is the spore of wild type Aspergillus fumigatus, and here is the spore of the mutant where we removed laeA. You can see it looks a little bit different. The more important slide are these red ones. 

What we did is we labeled the fungal spore with a dye that makes it appear green. And right here is a macrophage. So in our lungs, we have macrophage that one of their big jobs is to swallow up fungal spores and kill the spores before they germinate and go into the rest of our body. And as you can see, the macrophage are so-so at gobbling up wild type Aspergillus, but when we remove the laeA it just sucked them all in, and it kills them much more readily. Part of this is due to the loss of toxins in this fungus. So, we next asked, okay, laeA regulates a lot of the toxins in Aspergillus fumigatus, but which one of these are important for pathogenicity? So what you're looking at now is a schematic of the eight chromosomes of Aspergillus. So every fungus has a certain number of chromosomes depending on the genus, and Aspergilli all have eight chromosomes. What we could tell from sequencing it is that there would be areas of the chromosomes where we would recognize genes that we knew could produce toxins. And these red arrows show you all of the toxin gene clusters that are regulated by laeA. So it really regulates a lot. 

One of the gene clusters we wanted to find and investigate was a cluster of genes regulated by laeA that make this molecule. This molecule is called gliotoxin. Now, gliotoxin has been known for about 80 years now. And it was isolated from Aspergillus fumigatus and other fungi in the 1930s, 1920s and 1930s. And it was found to be a very toxic compound. Whenever you see a sulfur bond you might want to be a little leery about a compound. And what they found out is that this compound actually killed other fungi. Now we get into our interesting story here about how fungi will make compounds to kill other fungi. And so at first people were thinking about maybe we could use this gliotoxin as an antifungal to control fungal diseases in agricultural situations. However, it also kills humans very effectively, so that idea was trashed. 

What we were able to do by understanding the proteins likely to make something like this is find this cluster of genes regulated by laeA and Aspergillus fumigatus. And what we did is we deleted these genes, particularly the first gene gliZ. The gene gliZ is a type of protein called a transcription factor, and we knew it was highly likely that if you removed gliZ none of the other genes would work and you'd get no gliotoxin. And indeed that was the case. This is another thin layer chromatography plate, and we've grown the fungus and run the extracts to look for gliotoxin. This is standard. And here you see wild type, and here's gliotoxin plus a bunch of other things you don't really want to deal with. If you remove laeA, again you can see most of the compounds have gone. This time in this strain we removed gliZ only, and what you can see is gliotoxin is now absent from this thin layer chromatography. There's a few other compounds missing too, but most of them are still there. 

So gliZ is not global like laeA. It is specific for gliotoxin and gliotoxin derivatives. And this is just a complement. We took the gliZ mutant and added gliZ back to the fungus and gliotoxin is back. And what we found, this was not surprising because other people had shown this, it had been known that gliotoxin causes self death. Apoptotic death is when basically a cell commits suicide. And when mammalian cells are exposed to gliotoxin, they self-destruct. And what we were able to see here, this is just a graph where we took extracts from our wild type fungus and added them to neutrophils. Neutrophils are the other cells in our immune system that protect us from fungi and bacteria. And this is the percent death of the neutrophils over, I'm not even remembering how long we did this right now, but you can see about 50% of the neutrophils died when we added an extract from the wild type, but if you added extracts from the gliZ mutant, hardly any died at all. 

So indeed, gliotoxin, which we once thought of to use as an antifungal, is really a very serious contributor to invasive Aspergillosis. Now, I'm going to show you, though, how tricky the fungus is. Now we're back again to this particular gene cluster. All of these genes make proteins that are required to make gliotoxin. Now, I told you that people were thinking at one time, why not use gliotoxin as an antifungal? Because it kills other fungi. It does it very well. Why doesn't Aspergillus fumigatus kill itself with gliotoxin? Well, that's what is really very interesting, and you really have to give fungi a lot of credit here, is when a fungus makes a toxin that kills other fungi, it almost always has inside the cluster of genes making its toxin one protein that protects itself from its own toxin. And in the case of Aspergillus fumigatus, gliT encodes a protein that destroys gliotoxin. And what another lab did last year is they removed gliT from Aspergillus fumigatus, and then they compared the effect of gliotoxin when added in the middle of a petri dish in this mutant to wild type. And as you can see, pure gliotoxin does inhibit Aspergillus fumigatus a little bit but if you don't have gliT, it's really killing it. 

So this is a very common situation in fungi. All those toxic molecules that I've shown you, the fungus that produces it will have a protective device so the antifungal doesn't hurt it, just the other fungi. I'm going to end with one last story. And this is a new story in our lab that hasn't been published yet, but I think it will be shortly. And I'm continuing with Aspergillus fumigatus, the pathogenic Aspergillus, but we do this with others too. So we found gliotoxin, now we're back to the same schematic. Gliotoxin was actually this cluster. Well, we wanted to start looking at other clusters that are regulated by laeA to look at the compounds to see if they add to pathogenicity. And this is our latest one. We took one of those clusters and looked for the expression of genes. 

This is gene expression in Aspergillus fumigatus, and we find that in a wild type fungus we have a series of four genes in a row that turn on at about 18 or 24 hours after the fungus grows, but if you remove laeA, they're not expressed. What we have found, and this is a graduate student in my lab, is that those four genes make a red compound. You can see it's here in these strains. But when you remove the genes that make it, it disappears. And we found this compound, this red compound, to be a compound called endocrocin. It was not known that Aspergillus fumigatus made endocrocin. Endocrocin is known, though. Endocrocin belongs to a type of molecules called anthraquinones coming from fungi that are used in the dye making system. So these are swaths of cloth that have been dyed from different anthraquinones from fungi. Beautiful. 

And, indeed, Yun was able to show that several of these genes make this red compound, anthraquinone, and she is putting together a chemical pathway. This probably is interesting to maybe two or three of you. [LAUGHTER] So we'll go by that. But what is most intriguing with this, she wanted to ask, where is the anthraquinone made? This endocrocin? And if you remember, I showed you a few slides ago, that different compounds are made in different tissues. So what Yun did is she grew the fungus and then she would collect extracts from either the auger the fungus grows on, the middle part which had mycelium, the conidium stalks or just the conidia. It's the conidia of Aspergillus fumigatus that come into your lungs and cause the initial damage. And you can see her results where she looked at extracts from different parts of the fungus. And what we see, this is the endocrocin here, two different media, it is in the conidia that enter our lungs where most of this endocrocin is formed. And what she's doing right now is pathogenicity tests. She has made some strains where she removed a gene required for endocrocin and looked at survivability of these mutants she made compared to the wild type. 

This time we're using a fly, Drosophila model, and you can see from our initial data is that endocrocin also appears to be a pathogenicity factor because when you remove it, the flies survive longer. Well, that wraps up my talk today, but I really need to end by showing you the people who've done all the work over the years. I am, basically, just an observer now. I love going into the lab and looking at the fungi, but I don't do any of the work. These are just showing a lot of the people in my lab from this summer. Graduate students, undergraduate students, postdocs, visiting scientists. So these are the people that deserve all of the credit for what I just showed you. And that's it. [APPLAUSE]