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cc >> Hi, everyone, welcome to Wednesday Nite at the Lab here at the UW-Madison Biotechnology Center. Today we have Mark Staudt talking about fungi, so I think there's gonna be a lot you're gonna learn today about the world of fungus. And just a little bit of background on Mark, he did his bachelor's in engineering, actually, at the University of Texas and worked as an engineer for a few years before he moved into the biology world and came here for graduate school. He is currently finishing up his graduate work in the lab of Christina Hull here at Wisconsin, and his work is looking at one of the fungal pathogens, cryptococcus. And he also is a fellow in the Nap House which is a house here on campus for graduate fellows in different areas, from arts to science, to interact. So, welcome him to Wednesday Nite at the Lab today. ( applause ) >> Thanks. Is this on? Volume okay? Okay, great. So, like Kathleen said, my name's Mark Staudt, I'm in the department of biomolecular chemistry, but I work with Christina Hull as my advisor, so I'm in the Hull lab. And today I want to talk to you about fungi, and really, how fungi, a lot of the good they do, but also some of the bad they do. Because like Kathleen was saying, I came to the biological side from an engineering background. So I worked in chemical plants down south for a while, and so for me, really, all I knew about fungi was maybe what a lot of you guys think about it. It's just this. You know, I looked at mushrooms, if I had one in my salad, if I saw one in the forest. That was a fungi, and that was probably the last I thought of it. Except for maybe, I don't remember if it was junior high or high school, but they actually, when they went through the classical five kingdoms, was the other time I saw fungi mentioned. It's unique that fungi actually used to be part of the plant kingdom, because a long time ago, they looked at fungi and they grew out of the soil, they didn't move, and they kind of looked like plants. At least, the mushrooms did. So they just grouped them in with plants. And it wasn't until later that fungi had their own kingdom. But now, actually, we estimate that actually 1.5 million species are in the fungal kingdom, so that's really a pretty large kingdom. And also with the advance in molecular technologies, what they've seen is that fungi are actually more closely related, and I'll try out this pointer with the mouse here. Okay. So when I'm trying to point to stuff, we're gonna be looking for this little black arrow up here. But they actually found that fungi are actually more closely related to the animal kingdom than they are to plants. And that's gonna come into play later in the talk, when I talk about how fungi are involved with our own bodies. Another reason I think that fungi possibly are maybe a little misunderstood is that they go by so many names. You can see a lot of them here, over on the left we have yeast, that a lot of people have probably cooked bread with, to make leavened bread. Over on the right we have these nasty-looking critters, we have mildew, mold. Down here we have mushrooms, which we're more familiar with, the Smurfs definitely love them to live in, make them homes. Over here we have a truffle, which actually, amazingly enough, is a lot of times worth more than its weight in gold. That they're so valuable to gourmet cooks. And this guy was probably the closest I could come to something that might look like a rust, a smut, or a chytrid. But we'll talk about those more later. The whole idea of this slide is just that when you hear these words, like mildew, mold, yeast, truffles, stuff like this, these are all fungi we're talking about. So whether you're just hearing them in the news, or just maybe talking among your friends, I probably talk a little bit more about them than you guys might, but if it comes up, these are all fungi we're talking about, all these different names. But for the purposes of this talk, I really want to get across what our relationship with fungi is. And kind of in three main areas. First, what our relationship with fungi is in the environment. Secondly, what it is in the food chain, and then finally, also what it is in our own bodies. But how fungi interacts in all these instances, like I said, both in good ways, and perhaps, in maybe not-so-positive ways. So first, looking at the environment. This is kind of an interesting tidbit. Again, going back to when I was in, I don't know if it was grade school, but back in the day, when they always talked about large organisms, they always looked at stuff like blue whales or redwood trees. Well, it turns out, a fungus, they're always forgotten about, the fungi, but we're actually the largest organism in the world. In the Blue Ridge mountains in Oregon, there's actually a single fungus that covers four square miles. I mean, that's pretty incredible, that's over 1600 football fields. That this one organism goes over. And you can see here, in this person's hand right here, those are the filaments that are going underneath the soil throughout these four square miles. And then at different points, these fruiting bodies, the mushrooms, actually pop up. But they've actually done genetic testing, that over this entire four square miles, this is all the same organism. Genetically the same, one enormous thing, so blue whales have got nothing on the fungus. But actually more importantly, that's an interesting tidbit, a more interesting, or more important thing that fungi do in the forest is actually two things. One, they form mycorrhizae that help plants grow, and the second is they actually decompose a lot of the organic materials in the forests around the world. But first to get to what mycorrhizae are, over here on the left you can see, these are, the bottom two pictures are actually, the yellowish parts are actually the roots, and the mycorrhizae are the symbiotic relationship between plants, plant roots, and the fungi. What symbiotic means is that they both are helping each other out. The plant provides food for the fungus in the form of sugars, and then the fungus helps to be able to get together a lot of minerals, particularly nitrogen, as well as water, to help the plants grow better. Then you can see up top, in that picture with the two plants, these are actually a redwood sapling, and on the left, this smaller one, was grown after they removed all the mycorrhizae, removed the fungi interaction from this plant. And on the right was a normal one that was growing with this fungal interaction. And you can see that the one on the right is a much more robust plant, you see a lot better growth. And it's been estimated that about 90% of the plants on earth have these interactions with this fungi, has this mycorrhizae. And so you can imagine the impact worldwide of these, of this interaction with the fungi that it has on all plant life here on earth. The other thing on the right side that we see fungi doing is actually decomposing all this organic matter. You can see different states of it, this one is called "white rot," its Latin name is "phanerochaete." Then we see different ones on degrading wood, and fungi as we'll talk about a little bit, it doesn't know the difference whether that wood is your house or whether it's a tree. It's still gonna decompose it one way or the other. And while this may not be a super attractive thing to look at, it's actually hugely important, because what it does is, the fungi recycle this carbon that otherwise would be dead and not useful, it recycles it back into the environment. So that organisms can then use that carbon to grow new organisms, to grow us and other plants and animals. And then the other side of that is also, if you think about it, if fungi didn't degrade all this stuff and rot it all down, we would be under, I'm pretty sure, literally miles and miles of dead organisms. Realistically, we wouldn't be here, right? But if we were, we would be under miles of dead plants, dead animals, and everything else if the fungi weren't here to decompose all that and return it back into the environment. So with this idea that fungi degrade a lot of stuff, you may have heard a lot of talk about biofuels. Because one of the enzymes, or the proteins that these organisms have to be able to break down this wood are also going to be useful in the making of biofuels because they're able to break down this plant material into other, smaller chemical structures. And while you've probably also heard, there are potential pitfalls with biofuels. You know, we have to do this right, we have to look at what we're doing and think about it. And a lot of that work to make better technologies, to mature this technology that we're gonna get the actual benefit we want out of it, and to design some of the plants themselves that will help with this. A lot of that work is being done right here on campus, here at this Great Lakes Bioenergy Research Center. This is actually a really big grant out of the Department of Defense that the University of Wisconsin got, partly because of our strength within the scientific community but also because Wisconsin is uniquely positioned with so much farmland and so many different types of crops, to be able to look at and take advantage of some of the attributes of these different plants. So these enzymes that degrade stuff will also become important, hopefully looking toward the biofuels, and hopefully getting a little bit of energy independence within the U.S. and the world altogether. And so you can see, I'm probably a little biased in favor of the fungi, but you know, like I said, they also do bad things, although I would propose at least on some of these that fungi don't know better. Like I said, they don't know if it's your house, they're just decomposing, they're rotting stuff, they're just doing their thing. So it happens to be your house, inconvenient for you. But you can imagine situations where this becomes a larger problem than normal. I'm sure you guys have heard a lot about the different hurricanes that have been hitting the Gulf coast. I think this is a picture of Katrina, but I'm actually particularly intimated with the hurricanes in the Gulf coast because, let's see, where's my pointer? It's easier for me to look at the screen, but if you can see it up there, my hometown is about right here, if y'all can see. So all these hurricanes have been running over my hometown for the last two or three years. In fact, Rita was the one that passed directly over my hometown and my house. In fact, we had flooding in our house, we had all these different kinds of rots, and mildews building up. So it does cause a large problem when you see areas, of course, we were very lucky, everything's gotten back together, everybody was okay, but you can see, and you've seen pictures in the news of New Orleans. Where they were under so much water, and then, under these conditions, damp, heated conditions, the mildew and fungi just move on in and start rotting this stuff down, they're taking care of business. From the side of New Orleans, kind of a bad thing. So another place that, it's pretty amazing that fungi are actually working, they're actually degrading the astronaut's house. The Mir space station actually has fungus growing on this bottom left panel, on the outside of the space station, which is really amazing. They didn't think anything was able to grow out in space, it's super cold, what are they using for energy, all these type of things. And they assume it just got carried from earth up there, but some studies have shown, when you might think, how do they get their energy to be able to grow in space? I mean, there's nothing really there, they don't have soil, they don't have other plants or animals. There's actually some studies that show, or at least suggest, that they might actually be able to use ionizing radiation to actually use that as an energy source to produce their energy. So there's ionizing radiation out in space. Another place they see fungi growing where they wouldn't necessarily expect anything to grow is in the old reactors at Chernobyl, the big nuclear meltdown that happened in Russia, or I guess, Soviet Union at that time. I'm not sure if it actually was in Russia, but over in the Soviet Union, they had a big meltdown with a nuclear reactor, and in that nuclear reactor is this unbelievably flourishing, huge fungal colony. And so this ionizing radiation idea is quite controversial, so it hasn't by any means been proven, but it's gonna be an interesting story to see how these organisms are actually surviving in places that you would never expect to find life. But I think it just goes to show how widespread fungi are. And their abilities to adapt to different situations. Oh, and when thinking of bad things, I guess the one group that could really hate on the fungi are the amphibians. They're getting a pretty raw deal here. Initially amphibians, I'm not sure if you guys may have read in some of the papers a while back, but amphibians are really going through a lot of problems, they're having, they're being wiped out in a lot of locations all over the world. But they're having all these developmental problems. Here you can see, one's growing without legs, this one doesn't have an eye, lots of problems in their development. These are even ones that make it that far. You can imagine a lot of them just die in their development and don't even make it to at least a partially formed organism. And at first, they thought what was happening to the amphibians was something kind of like the canary in the coal mine, the idea that the amphibians' skin, they absorb chemicals, sorry, but they absorb chemicals directly through their skin. So they thought maybe it was pollutants being released out into the environment, and that these amphibians were our first canary in the coal mine, our first signal that, "Oh goodness, we've put too much pollution into the environment," something bad's happening here. But actually when scientists went and looked at what was happening, the pollution levels at these different locations actually wasn't that bad, it was nothing that would harm the amphibians. But what they did find were these chytrids, and this is a chytrid right here. That's a group of fungi, it's often classified as what is thought to be the oldest group of fungi. So evolutionary speaking, one of the primary fungi that other ones were derived from. But one of the unique characteristics of this chytrid is that it has motile gametes, or spores, so they can actually swim. So you can think, in a water environment where these amphibians are, they can easily get to the amphibians and then infect them and cause all these developmental problems. So now that they've kind of identified that the chytrids are what's causing all these problems, that in fact it wasn't the pollution. They're trying to figure out why this chytrid is an increasing problem. As well as possible solutions. Some of the ideas of why it might be a problem are possible climate change, is a possible warming of the earth making different environmental niches that chytrids can now move into, where normally they wouldn't be there, but now because of some shift in either the rainfall or the temperature, they can now inhabit these environments which then cause very detrimental effects to the amphibians. Another idea is also that just humans, like we do a lot, even we've seen, Kathleen mentioned the Swine flu. How quickly, because we travel everywhere across the globe so quickly, either in our planes, in our boats, everything we do, even what's stuck to our shoes, we transport these things. Organisms, microorganisms especially, everywhere. And is it actually human, unintentional human interactions that are spreading these chytrids to different populations and endangering amphibians worldwide. So hopefully they'll figure something out, but a lot of research is going into trying to figures out what these chytrids are doing, how we can stop it, and hopefully not have the amphibians kind of wiped off the face of the earth. So like I said, those are some bad things, but we're back to coming into looking at a different relationship with fungi. So we kind of talked about it in the environment, you can think of kind of the next step as we're moving to ourselves, as it comes out of the environment, as it comes to us through the food chain. And again, for me at least, and I should also say, I should've said this in the beginning, but if you have any questions or any thoughts, you can throw them out any time, so you don't have to save them for the end or anything, just go ahead and throw them out. But at least at this point, we're moving into the food chain. So you can think of, it's directly coming from the environment, we grow things, we eat it. And again for me, the first thing I thought of, before I moved into the lab that studied fungi, was these guys. You know, fungi that we eat. And I love this little quote I stumbled across, that "All mushrooms are edible, but some only once," unfortunately, so you gotta watch what you're doing. Actually, out of all these, all of them are edible more than once, except for this guy over in the corner, this is actually called the "death cap" mushroom. You can probably guess why, because it's poisonous and it will kill you. So you can see, though, it doesn't look much different. This pointer's killing me, but it doesn't look much different than these button cap mushrooms that you see in the grocery store all the time. So while there aren't a whole lot of species of fungi that are necessarily that poisonous to human, they're relatively abundant in a forest, if you weren't an expert, you definitely wouldn't want to go out there and start picking things and licking them or throwing them in your salad, because you never know what you might end up with. So definitely leave it to the experts. And speaking of experts, humans spend a lot of time and money, it's a fairly large industry, I didn't get an economic figure, but I believe it's at least in the billions, actually farming mushrooms. And this actually took a lot of work, it's difficult to recreate the conditions to have a good fungus farm. But while humans have been working on this for a relatively short time, and have had reasonable success with it, ants have actually been doing it a long time. Which is a pretty amazing thing to me. Oops, I skipped a slide. So these are leaf cutter ants, and they're in the tropics, down in South America. And what they do is they cut up all these leaves and they bring them all back to their nest, and then the different take on ant farms, they actually have farms of fungi that they grow. Then you can see here, they kind of have a little cycle going, that the ants provide fertilizer and feed the fungi with these plants and bringing back the organic material for them to grow and then the ants actually eat the fungus, so again, another symbiotic relationship that they're helping each other out. And actually, in this pretty neat food web, it actually gets more complex, there's actually bacteria involved that will help protect the fungi and then the ants actually provide some of this bacteria. And it gets to be a pretty intricate and pretty amazing web of food chain. And actually, here on campus, a lot of the work was done here by Dr. Cameron Currie, in the Department of Bacteriology. He's done some amazing work on this food web right here and actually has gotten, I think at least one or two papers in "Science," so some really interesting and really stuff that nobody expected when they were looking at these things. But bottom line, we're not the only ones that farm fungi. Ants have beat us to it for a long time. And so not only are fungi part of the food chain themselves, you know, we eat mushrooms, so they're directly part of it. But they're also required for the production of many foods. You know, even here in Wisconsin, where would as Wisconsin, as a transplant, if you didn't have beer and you didn't have cheese, I mean, what would Wisconsin be? I don't know, a bunch of cows, I guess, that don't give milk. But that's definitely the two characteristics I noticed when I came here, cheese curds, good beer, you gotta love it. But fungi aren't involved in making all cheeses, actually, but they are involved in making what you think of as the moldy, or the smelly cheeses, maybe some of the more gourmet cheeses, and it's actually the molds they put in there, so stuff like Roquefort or blue cheese. Those actually are putting fungi in, to make them more pungent. And then, of course, like I said at the beginning, a lot of you guys are probably familiar with yeast, that we use in bread and everything, to form food. As well as fermentation products, beer, you guys are familiar with, as well as wine, also uses fungi. And one at least I wasn't familiar with was soy sauce, is also a fermentation product from fungi. And actually, Kikkoman is a producer of soy sauce. And again, I didn't even know soy sauce was involved with fungi till just a couple years ago, but here in the new microbial sciences building, which is right over this way on Linden, if you pass it on campus. They actually gave a couple million dollars to have a fermentation lab, the Kikkoman Fermentation Lab here on campus. So they're still actively looking for ways to improve these things, even though these ideas of fermentation with fungi, especially beer and wine, have been around for millennia, actually. But some of the new ways, like I said, these have been here for a long time. Some of the newer ways that we're also looking at fungi indirectly getting into the food system is actually what we call "mico-proteins," so "mico" is just "fungal," so fungal proteins. But they're starting to develop some of these as meat alternatives. Whether people want to do it for health reasons, possibly for sustainability reasons, and possibly just for taste. We actually are getting a lot of mico-proteins, one brand is Quorn, spelled "Q-U-O-R-N. They make a lot of stuff, they'll make some stuff like tofu, that you can just throw on the stir fry, but they'll also make chicken nuggets you can fry up, chicken nuggets. The fake chicken, obviously, it's mico-proteins. But two things, with health it's good, but also, you can actually grow these in large vats, and I think especially as this technology develops, it's a food source that, especially compared to meat, which is rather hard on the environment, resource-wise, could actually probably provide a lot of food and a lot of protein resources for possibly a lot of potentially impoverished nations around the world. So right now it's seen more as, at least in the US, it's marketed more as a high-end product, like, you'd probably have to go to Whole Foods to find Quorn. But potentially, and hopefully, it will actually be able to impact, like I said, possibly non-nourished or impoverished nations down the road because it can be grown in such large amounts, and fairly cheaply. And then I think, too, just because fungi get a bad name, they had to come up with some clever way to call it "Quorn" or something, they're not gonna tell you you're eating fungus, right? They're gonna come up with a clever marketing scheme and hopefully sell it to you that way. But, hopefully people will get over that, also. So those are some of the good ways, of course now we're flipping back to the bad side. Of course, fungi can also mess up the storage and production of many foods. I'm sure all of us here have left bread out too long, that we get some of this mold, Aspergillus, on it. Aspergillus are these white, kind of fuzzy things, and they sometimes get green in the center. But that's a fungi, just, again, come in, left your bread out, it's gonna take care of business, it's gonna start taking it down. So that's what's happening with those guys. And then, also, even in the production of food. You can see here, like on strawberries, that's actually a powdery mildew on them, but of course, it's very bad for strawberry production. Over here we have barley that's been infected with ergot, which also is another plant pathogen. And over here might be the best example of fungi, with my talk title that I had to make up quick, is it good or is it bad? This is actually --- is the name of the fungus that is infected, and this is a cornstalk, and these little things are individual kernels of corn, that normally would be those nice little yellow kernels of corn that have been infected with this fungus, and are actually filled with spores, and fungus. And sometimes to the point of bursting. So when we get this in American fields, this is a bad thing. But actually, in Mexico, this is considered a delicacy. So they actually, in fact, in Mexico stores, you can find it actually canned here in the US. You probably won't find fresh-- Goodness, did I do something? I don't know what happened. Okay, as long as everybody's okay, I'm good. But I think I was saying that you're gonna have trouble finding fresh, and I'm not even gonna try to say the word, I would butcher it. It's some Aztec word. If you ask me after, I could email it to you. But if I try to say it, it would be not-recognizable. But it's a delicacy in Mexico, whereas here, it's considered a very bad thing. And actually, all of these, we spend a lot of time and energy spraying our crops, to try to prevent these fungal pathogens from infecting our different crops. And hopefully getting them to the food market without any of these things happening. Before I move on, there was just another little side note, interesting over here, on ergot, is actually a fungus that, actually, LSD can come from ergot, that's where they get it. So you can decide whether it's good or bad when you think back to the '60s, Lucy in the Sky with Diamonds, a bunch of that stuff was floating around. But also, another interesting historical side note, was the idea of witches and witch trials. There's been a fairly convincing, I haven't read a lot about it, but it looked like they, I don't want to say "did their homework," but it looks fairly convincing. When they go back and look at historical records, a lot of the witch trials actually merge with these expected times of ergot infestation of barley. So one of the side effects, if you eat barley that's infested with this fungus, is you would go into hallucinations, like LSD, you would go into convulsions, a lot of these things that might be associated with witches. And they've looked at the times of the water fall through history and stuff, and the prices of barley that likely was going through an ergot infestation, and worldwide, in Salem and in other places in Europe, they found a pretty good correlation that what was likely happening was ergot was infecting this barley and then people started having these symptoms of convulsions and hallucinations, everybody thought witches. All of a sudden, bad things are happening, and a witch hunt starts. So fungi can even impact history, indirectly, through some of their actions. But back to the food chain, not only can it mess up the production of some of these crops, but it can also affect these crops through what we call secondary metabolize. Basically things as they grow that seep out of the organism and into the area. So it's not the organism itself, but it's things that are released from the organism. And this toxin over here, called "Aflatoxin," actually comes from this Aspergillus flavus, which can grow on corn. But actually, Aflatoxin is one of the nastiest ones of these, it's actually, I don't think they've come out with anything more, it used to be the most carcinogenic thing known to man. And definitely, if they've come up with something else, it's definitely in the top things of carcinogenic compounds. And this can affect food supplies when it goes into feed stocks for animals. In fact, one year, I don't remember the date or anything, but they talk about turkey X disease, I think it was over in England. I forget if it was in the east coast of the United States as well. But they didn't know what was killing all these turkeys, all these turkeys were dying everywhere, you could hardly get a turkey for Thanksgiving. But they finally found, it was actually the Aflatoxin that had gotten into the feed stocks, the grain that they were giving to the animals, and it was killing all the animals. So luckily that was a while back, before you get too worried, because the other thing that might more directly enter the human food chain is through peanut butter. Actually, Aspergillus grows quite well on the dunes, and peanuts. So if they aren't stored properly, you could get Aspergillus growing, which, as it grows, one of its byproducts is Aflatoxin. But luckily we're well aware of this. Food storage has improved tremendously and they test for this is in all our food products, so it's nothing that we really need to worry about, other than to be aware and make sure that the proper precautions are taken. >> Was that the one that got the Jif peanut butter? >> I think that was salmonella, from what I remember in the news, which is a different organism. And luckily so, because Aflatoxin, I don't know if it would've been worse directly, salmonella may hit you faster, Aflatoxin may hit you later. But luckily it wasn't Aflatoxin. But it is something to be aware of, when you read these things, that hopefully all these food producers are doing their jobs, and most of them do do a very good job of keeping track because anything, when you make it in large quantities, things can happen, like the salmonella, like Aflatoxin. Yes, sir. >> Are you aware of any cancer outbreaks attached to Aflatoxin? >> Yeah, he had asked if I'm aware of any cancer outbreaks with Aflatoxin. And I don't think there are, because they were aware that it was so cancerous, in fact, I think it was unusual, that's why they called it turkey X disease for a while, because it didn't even cross their mind, because they knew to store it properly, and I guess something happened in the mismanagement of that grain. But normally they're quite aware of it, so they take precautions to prevent it. And as well, with the animals, where they have seen some Aflatoxin breakouts, it doesn't get to the point of cancer, it more directly kills them, I'm not sure of the mechanism, but that's why all the turkeys died, it wasn't necessarily from cancer. Interesting question. Yeah? >> Is Aflatoxin just a cancer problem? >> You know, I should know more about that. He was asking if Aflatoxin is only a cancer problem. I know it does tend to go to the liver, our liver is kind of used as a filter for a lot of toxins that go through our body, so that's where it builds up, that's where it causes a lot of these problems. I'm not sure if there's an acute, I haven't read of any acute Aflatoxin poisonings in humans. But then, I haven't read a whole lot about it, to be honest. >> I used to work for like, Best Foods, and Skippy peanut butter. This guy I worked with, he won the golden peanut award. Anyway, what happens is, to every peanut they put a light on it, and if it fluoresces, it gets kicked out. >> Amazing, yeah. >> So that's back in 1970 or before. >> Yeah, that's a great way to do it. It's actually, I wasn't necessarily going to mention it, but it kind of makes you think in the same way, of actually, in some ways, because these large productions, a lot of times we associate organic with safer stuff, so not to necessarily scare you or anything, but in some ways when you have the peanuts that are just stored in those bins in organic places, that just sit there for maybe months, how long, that could have stuff growing on them. They don't go through the same tests, like you said, when they check every peanut that goes through this place. So some people might argue that when you make your fresh peanut butter, you actually run a little higher risk than you would from actually buying Jif or some different type of commercial peanut butter. That's pretty interesting stuff, the way they keep track of all that. Anything else? Good questions, keep them coming. Because now we're gonna actually move into the stuff that's probably nearest and dearest to my heart, I guess, hopefully not literally, but it's fungi in the human body. And here are, again, the good and the bad. These fungal toxins that just a second ago I was telling you how cancerous they can be, and how they can harm a lot of animals. They can also do a world of good for humans. Alexander Fleming, most of you guys have probably heard about, really discovered what I think would be called the first miracle drug, penicillin. And he found it by looking at fungi, it was one of those accidental discoveries, he was about to throw out a dish and he actually noticed that the fungi had actually killed some of the bacteria on that dish before he tossed it out. He went back and looked at it, and penicillin is what he found. But these secondary metabolites, that I was talking about, these things that some of these fungi excrete, a lot of them are used as defense mechanisms, and since a lot of these fungi are growing in the soil, one of the things they're at least exposed to often is actually bacteria. So some of these secondary metabolites, while some can be bad, some actually kill bacteria because that would be a natural competitor in the environment. And that's exactly where we get a lot of our antibiotics, where penicillin came from, was from a fungi. And I don't think it'd be overstating to say that probably the majority of us in this room are probably here because of penicillin, either, whether it affected us directly or whether it possibly affected our parents or grandparents, especially through the wars and stuff like that. It's saved really countless, countless lives. And even with the idea of antibacterial resistance, which is building up, penicillin itself, and the derivatives that have made it more potent, penicillin with small adjustments to it, it's still doing a world of good, and, I mean, basically every day we get in the hospital. When you think surgery, it used to be a really scary proposition. Anything that happens on the battlefield. Bacteria will come in and they will just start decimating the human body. Now we can actually kill these things, which is really amazing. Along with antibacterials, which, like I said, could be argued as one of the biggest revolutions in medicine we've had, there's other drugs that come from these secondary metabolites. Cyclosporin A, is one that can help suppress immune system, if you ever had an organ transplant or something like this, to make sure your body doesn't reject it, they can give you that. There's ergot alkaloids, again are coming from that plant ergot where you get LSD, so it can also be used, I'm not sure quite what for, I guess an alkaloid, but I don't the actual chemistry behind it. But they can also be used to treat migraines, so it does have this affect on the brain. And then also statins, some of you guys may be familiar with, as used in cardiovascular-type situations. So a lot of good comes from these toxins as well. And not only are fungi able to produce these secondary metabolites just through their normal growth, we've now reached a point in our knowledge and technology that we can actually use fungi basically as little factories so that we can genetically engineer them and use them to make therapeutics that we need in large quantities. So insulin is a great example of this. Insulin used to be collected from actual animals. They would grind up organs, and collect insulin, so you can imagine this made insulin very expensive as well as, they had a lot of problems with purity, and stuff like this, getting back to this, when you're doing it in large quantities, who knows what happens when you're grinding up all these organs, especially if that animal may have been infected with something else. So now that we have the technology to use fungi to engineer a system where the fungi to grow, this is just an example of essentially what would be a bioreactor, a reactor that has living things within it. We can actually produce insulin, as an example here, we can produce large quantities of insulin, basically, I don't know that it's limited, you can probably produce as much as you want as long as there's a market for it. You can produce it cheaply, at least as compared to harvesting it from animals. So much, much cheaper, and it's also at a much greater purity, so you don't have to worry about what you're putting into your body as much. So this was really great, especially now in the nation, where we think of the rise of type 2 diabetes. That this, and I'm sure future therapeutics as well, that we can actually engineer these organisms to make this for us in large quantities at a fairly cost-effective rate. And then we kind of enter into maybe a neutral idea of fungi. Like bacteria, a lot of microorganisms, there are tons of microorganisms on us, so fungi are one of these as well, just like we have bacteria. There's been some estimates, actually, that by the time we die, there's actually more mass of microbes on us, or in us, on us and in us, than there actually is mass of human cells. So it kind of makes you almost reevaluate what it is to be human, if we're more microbes that are living with us and on us. But for the most part, they just live on us, no problem, they're just there, not causing good or bad things. But of course, I'm sure you guys are familiar, that you can have fungal disease. And fungal disease can affect anybody, of course, in fact, probably everybody in this room at some point in your life will run across some form of fungal disease, either you or somebody you know. These are just some of the common ones, there's hundreds of species that can cause fungal disease. Ringworm, you see a lot of time on kids through grade school, also, athlete's foot, you can see a bit of. This is an extreme case of athlete's foot, hopefully none of us have to deal with one that bad. But luckily we can treat these. As well as, you can see, this guy has some toe fungus, which, if you've ever seen those Lamisil commercials, evidently this guy is toe fungus right here, and he flips back your toenail and dives on in, according to the little cartoons in the commercial, just starts having a party, right? So that, I guess, is toenail fungus. But luckily these are topical-type infections that we can treat fairly easily and fairly effectively. What gets a little more difficult is systemic fungal infections. These are things like fungemia, fungal pneumonia, fungal meningitis. And this is just one example of fungal pneumonia, you can see, this is an X-ray of somebody's lungs. So what you would want to see in the lung cavity right here is a nice black lung cavity, you don't want to see anything in there, it should just be clear. But you can see all this white fuzzy stuff, there it is, you see all this white fuzzy stuff going all through the lungs. So this person, it's likely an indication of a fungal infection, or at least some infection within the lungs. And chances are this person probably isn't doing very well. I'll talk about that in a second, but fungi overall are emerging pathogens. When you look at hospital acquired infections, infections that you get once you're in the hospital, about 10% of them are fungal infections. And the more concerning figure is what's right under that, is that 50% of these infections are actually fatal. You have a 50% mortality rate with these infections. And you know, when you, at least when I think of modern medicine, that figure is kind of astounding to me, it seems really, really high, that it could take out half of us, even with all the technologies and all the drugs and knowledge that we have so far. So why would it have such a high mortality rate? And it actually goes back to what I mentioned in one of my first couple slides, that when they looked at the molecular, the evolutionary position of the fungi, it's actually closer to animals and us than it is to a lot of other things, like plants and other parasites. So here, what's in that blue circle, you can see is highlighted. Right there. We have yeast, which are the fungi, again, we're getting into these multiple names for fungi. But it's right by humans. So what happens is when we look for drug targets, the bacteria are way over here, so we can find things that affect, like at the molecular level, at the proteins, because they're so different evolutionarily speaking, we can find things that will kill the bacteria quite easily. And because we're so far apart from them, they don't hurt the humans. But what happens, because of this close evolutionary distance between fungi and humans, is there's limited therapeutic options because really what you run into are, a lot of them actually target the fungal cell wall, which is one of the few differences between fungi and humans. But even those, they find the most effective fungal therapies actually also kill humans, or do a damage to humans. So it becomes a race. You gotta hopefully kill the fungus, before the drugs kill you, or the fungus kills you. That's a race nobody really wants to be in. It's not a good situation, so why we study this stuff is, we really want to see, and we think a key to treating these fungal diseases is understanding how fungi grow and how they develop. So we can find some of these targets, some of these differences because it's not obvious, because we're so close evolutionarily speaking, that we need to find differences between humans and fungi, between plants and fungi, so that we can find drugs and therapies that only affect the fungi and don't also affect us. And so, I need a transition here, sorry. In thinking of fungal pathogens for humans, one of the problems we run into when we want to study these is a lot of them aren't very amenable, they aren't very easy to work with in the lab. Some of them only grow on a host, whether it be a plant, whether it be an animal or human, so you can't work with them in the lab. Some of them you can work with in the lab, but we don't have very good molecular tools to be able to study and understand what's going on with these organisms. An exception to this, at least in my lab's humble opinion, is cryptococcus neoformans that we work on. It's great, and this is a picture of that beautiful beast here. So we see on the right here, is a large mother cell. And then budding off is a daughter cell, so one of its progeny. And you can see all these projections, are actually a capsule, and this capsule is one of the factors that help cryptococcus evade the immune system, and allow it to potentially be an opportunistic human fungal pathogen. But what's unique about cryptococcus as an opportunistic fungal pathogen, is we really have pretty good genetic tools to work with. We can culture it in the lab, so we can actually do a lot of work with this and learn a lot about it. And that's why it's one of the ideal fungal pathogen models. And when we look at this, just to give you a little bit of background on cryptococcus, when I say it's an opportunistic fungal pathogen, normally where crypto is most often found, and in fact, most fungi tend to be opportunistic pathogens, what that means is normally they're living in the environment. Cryptococcus has been associated with eucalyptus trees initially, it's been found on many other trees since then. If you think of trees, how they have bark, it actually is most often found in the hollows of the trees, where you think there might be some more detrius, more dead material for it to probably grow on, as well as on bird guano. But then again, it's not necessarily associated with the birds, the guano is just a good nutrient source for it, what we call a good substrate or medium for it to grow on. So here's some of the places where it's found in the environment. And normally that's probably where it's most often found, and where it stays, its natural habitat. But what we believe happens is that spores from these fungi are released into the environment and then we can inhale them into our lungs, into a host's lungs, whether they be human or animal. And because spores are small in size, they can be inhaled deep within the lungs, because normally, anything that goes into your lungs, we inhale millions and millions of spores, fungal spores, bacterial spores, every day and our body just clears it no problem. But when they get lodged deep it can cause an infection, and particularly if you're immune-compromised in some fashion. And once that infection is established, it can then disseminate the crypto to the central nervous system, where it causes meningitis, which is basically a swelling of your brain, which is uniformly fatal without treatment. When I say it affects immuno-compromised patients, like I said, crypto again, nothing here in the United States we worry about too much, it mainly affects populations such as AIDS populations, if you've had an organ transplant and you're on immuno-suppressants, you'd want to be careful. Some chemo therapies. For whatever reason, if your immune system is suppressed, you'd have more worry about this. But again, we have good fungal treatments here in the US, we rarely have fatalities from cryptococcus here in the United States. It's without treatment that it's uniformly fatal. So you can imagine, actually in sub-Saharan Africa, where they have a large AIDS population, as well as a very difficult time getting fungal treatment in those areas, that this is a huge problem and actually kills a lot of people. And so in my lab, we're actually studying the sexual development of cryptococcus, because it leads to these spores, which have been shown to be infectious particles, and are believed to be a likely route of infection, that it would come through your lungs. Speed up a little bit here. But this is a sexual development process that we work on in the lab. So it starts off with two cells of different mating types. We call them "A" and "alpha," but you can think of them as male and female. So they see a mating partner of opposite type, so you have mate detection, and we do this on a media that, it's literally V8 that we go buy at the grocery store and we mix in with our agar, which is a bit unusual, but again, I think it comes down to, it's plant material. It's basically a lot of ground up vegetables and everything, and the fungi like it, they grow on it, so it's cheap and we use it, because a lot of things, once you call it scientific, all of a sudden the price goes up ten times. We just use straight V8 from the supermarket, like any of you guys would. Probably not for growing crypto, but you know. If you wanted to. But at this point, if we had the mate detection, so the two mating types have found each other, get together, the cells actually fuse, but the two nuclei for each cell actually stay independent and the nuclei are where all the genetic material of the organism is. And at this point, it actually undergoes a morphological transition into a filamentous stage, so it goes from growing as those budding youths I showed you in the first initial picture of crypto, and now it's growing as a filament. When it grows as this filament, it's called a "dikaryotic" filament, and dikaryotic just means two nuclei. "Di" is two, and "karyon" is nucleus, and so you have two nucleus filamented, the dikaryon. So that's all that word means. And it grows as this dikaryotic filament until some other signal, we haven't found out yet, it forms this ball and club structure called a basidium, where the nuclei finally fuse, genetic material can be exchanged, and then they form spores that are then released into the environment. And this is just kind of a microscopic view of this actually happening. One picture is missing at the end. But you can see here, these are spots of cells that the individual haploid cells, just one mating type, so that's no sexual development, you don't see any of these fuzzy filaments. But when the two get together, you can see these filaments coming off of that cell mass. And then when we take the microscopic look at it, you can actually see the filaments themselves, and there was a third picture, I guess, a fifth picture that actually showed the basidia, that you can actually take pictures of with the little spore chains coming off of it. But sorry, I think that one's missing for tonight. But besides spores, why should your tax dollars fund me and my colleagues studying the sexual development of cryptococcus? Well, as it turns out, who would've thought, but sexual development also makes people, too. So, what we see here, when we look at sexual development in humans, it's obviously a really complex process, but because it's so complex, that makes it really difficult to study. So what we have here is a fairly simple organism, a fairly simple system that we have the molecular tools to look at, those genetics I talked about, that actually goes through a morphological change from cell type to cell type and then also can form multi-cellular structures. So it's a unique way to possibly get at some of the developmental aspects, and again, because of evolution, we can study it in the simplest system and apply what we learn to hopefully learn things about higher organisms. Such as ourselves and other animals. Another way to think of it, this cell sexual development, is more spore-related, but it's actually that filaments allow spore dispersal. So you can think these guys are stuck either in their bird guano or whatever else, and they can't get these spores out of there. But when they can form these aerial filaments, you can see right here, it provides a little bit, just enough distance to get above this substrate, whatever they're on, to release these spores and provide better dispersal. And that's really what I kind of look at in my project. I want to look at this dikaryotic filament stage and see what helps it form, what kind of makes it tick, and the two reasons I just mentioned that I think this is important research is, one, it is a developmental transition, probably one of the simplest developmental transitions. And when I say simple, one that we can control and look at that actually has a morphological change, it goes from one growth state, when it's growing as those round buds to a filamentous state, so it's actually a morphological change. As well as its potential importance for spore dispersal. When I say I want to kind of find out what makes it tick, this is, I'm trying to decipher what the molecular control of this filamentous stage is, the dikaryon. And one way we do this is, I want to find out what proteins actually affect this within the cell. So if we can see, this is a yeast. This is a model organism that we know a whole lot about. So one way we look at this is we look at these organisms we know a lot about, and this is not to scale, but you can see, the blue dots are nuclei, and these little odd shapes I made represent proteins. And so what we can do is look into yeast where stuff is well-defined and have done a lot of work and, say, this one red triangle that's right here, in yeast they know it's important for the cytoskeleton integrity and nuclear movement. So the cytoskeleton is the same as our own skeleton, it provides integrity for the cell, as well as something to be able to move on, like you muscles, to be able to move on your skeletal structure, so it's kind of the same idea with the cell. But if we know that's what it's doing in this other organism that's been well studied, I want to go look for it in cryptococcus, the organism I work on, and find out, do we have one of these? And as well, what does it do? Particularly in that dikaryotic state that I'm most concerned about. And the protein I'm just gonna talk a little bit about today is Bim1, and it was named because it binds microtubules. And microtubules are just long filaments within the cell that form part of this cytoskeleton. You can think of them as kind of akin to bones, they're structures within the cell that provide integrity, like long cables. And so what we look at, this is just a pictorial representation of the two proteins, the yeast one that we already knew about, and then I did find a protein in cryptococcus neoformans that we compare what we call the sequence, when we look at its evolution. So these represent functional domains of the two proteins. And you can see by the red and the green boxes, the same functional domains show up in this cryptococcal protein that I found. So it's very likely it's doing a similar thing. That it's also involved in cytoskeleton integrity and nuclear migration. But the yeast that was originally identified doesn't have this filamentous growth stage, so I want to see how does this protein that is probably likely functioning in a similar manner, how does it impact the filaments themselves? So one way we look at this, I should've mentioned on this other slide, when we look at proteins, like we can actually see the nuclei in a cell, we can look on a microscope and see those. But proteins are much, much smaller. I think if I did the calculations semi-right, trying to look at a protein would be like trying to look at an individual blade of grass on a football field from the stands or something. So it's something you can't directly look at, you can't just look and see what these guys are doing. So we have to find indirect means to look at it. So once this protein was identified, this is where, since we have the molecular tools in this organism, we actually removed this gene from the organism, so it's kind of like reverse engineering. We take it out, we see what happens. And then likely, whatever caused the phenotypes we see, something that's different from the normal, was probably caused by the gene we removed. So what I did was, I created strains of cryptococcus that didn't have this Bim1 protein, the gene for it, so you have no protein. Then I wanted to see what happened in sexual development. So this is our assay, at least this part of it is pretty simple, it follows the sexual development slide I talked about earlier. You mix the opposite mating types, you put them on the V8 medium we like, you put them in the dark for 48 hours, we debate whether it really needs to be in the dark, right, do you need mood music or anything, stuff like this. But some people swear it works better in the dark, so we stick with it. We put them in the dark for 48 hours, and then this is what we see. This would just be like a cartoon representation of a petri dish with haploid cells on top and cells that were mated together with opposite mating types on the bottom. And again, these are the pictures you saw a little bit earlier that, when you don't have a mating partner, you don't get any filaments. But when you do you see robust filamentation. And so on the next slide, what I'll be showing you is actually the edge of these spots of cells, magnified probably about 200 times under the microscope. So to orient you to what mating is, successful or not, again, these are the haploid cells, so you can see here, no filamentation. And looking up at it under the microscope, you can see there's no filaments here, you just see a smooth line of cells. But when we look at a wild type cross, so the wild type mating partners, when they get together, you see robust filamentation, a lot of aerial filaments growing out all over the place. But when I look at the deletion cross, when I've taken this gene out, the Bim1 gene is now gone, we see filaments, but all of a sudden this filaments can't grow as high or as aerial as what's seen in the wild type strains. But even though these do go on to complete sexual development, they go through the basidia that I mentioned that you form spores, they can't form the long filaments that we see in the wild type. So once I saw this, I wanted to take a little closer look at what's going on. And these are actually fluorescent images. Hopefully you guys can see this, because I don't know what to touch to turn down the lights, but if you can see the blue, it's outlining the cell structure, so that's the outline of the cell, and in the green are the nuclei within the cell. So you can see again, in the wild type crosses, you have nice, elongated filaments that look structurally intact, have nice integrity, but when we look over here at the deletion crosses, again, the cell, it's actually the cell wall that's staying blue, so the outline of the cell, all of a sudden these filaments don't look right. They're all floppy, they don't have any integrity, so something is definitely wrong with these guys. And based on what we saw in that yeast, other organism, we think it's likely to be microtubule mediated, that these microtubules within the cytoskeleton are causing this defect in the filament. And one way to look at this indirectly, is we take an inhibitor of these microtubules, and we want to see if these strains that don't have this gene, that don't have Bim1, are they more sensitive to these microtubule inhibitors? So what we have here is just cells in a single dilution, so there's less cells in each spot as you move down, and you can see when you don't have any inhibitor, everything grows just fine. But when you put in this microtubule inhibitor, in these strains that don't have Bim1, all of a sudden you see a large reduction in the growth. So this is an indirect indication that it can't handle the microtubule stress. Likely because the microtubules aren't forming as they are in wild type cells. So with this data we have a working model with this, and a lot of data I didn't necessarily get into today, but what we have here is the filaments, those dikaryotic filaments, with the wild type. If these are the microtubules as we think of the cables, you get nice long cables that form nice elongated cells that have solid integrity and can get off the substrate to possibly release their spores into the environment. And we can see that both in the cartoon diagram as well as what I showed in the fluorescent microscopy. However when we delete Bim1, what we think is happening is that these microtubules can't grow as long and they aren't directed properly. So all of a sudden you have filaments that can't form well, and end up being floppy and can't get off the substrate. And so with that, that kind of wraps up the talk, I just wanted to hit a few last summary points that hopefully I got across in this talk today. One, that fungi are essential for life on earth. And they're an integral part of many food chains as well as many industries. And that while fungal disease can be devastating for plants and animals, it's really the fungal models that we study, help us to understand this disease and hopefully will bring us new therapeutics in the future to be able to deal with the fungi as they become an increasing problem in these hospital and other situations. And as a final take home, I know you guys are probably a sympathetic audience, but I do have to plug that it really is the basic understanding of these organisms that lead to the scientific advances. And often in ways that you never expect. And I can't emphasize enough how much this is true and in fact, if we have time afterwards, I can probably get into another example of our individual organism, another therapeutic that came out if, that a guy wasn't looking for at all. He was just looking at cryptococcus trying to learn what happens, and lo and behold, the same way Fleming found penicillin, it's just a Eureka type moment. So with that, I'd just like to acknowledge my lab. Dr Hull, she's right here, and she's a great person to work for. In fact, if you ever get a chance to talk to her, she knows and loves fungi much more than me, I like them, but she's crazy about fungi. And just in general a really amazing person to talk to if you ever get the chance. And those are the rest of us in the lab, the grad students, undergrads, technicians. So we all help each other out. As well as the funding sources that have helped me as well as the lab to make a lot of this research possible. So that's all I have, if you guys have some questions, I'd love to hear them. ( applause )