>> Tomolo Steen: Thank you so much for coming. I'm Tomolo Steen at Science, Technology and the Business Division. And today's event is cosponsored with [Inaudible] Health Services. We have been organizing lectures, health-related lectures together with Dr. Charles and Miss Lively [phonetic] is in the back there. And this month is men's health, and we thought about what would be a good topic. And this is actually, you know, for anybody. It's helpful for men and women about fungi. And so Dr. Calderone is really internationally known for his study on the candida. And he actually textbook on that topic. It's a big thick book, isn't it? And so if you, you know, want to read more, just to let me know. I'm happy to give you a reference. And just today is just end of this Men's Health Month closing lecture. Let me introduce Dr. Calderone. Dr. Richard Calderone is chair of the Department of Microbiology and Immunology at the Georgetown Medical Center. And also, he's a director of the MS Program on the Biomedical Medical Science Policy and Advocacy. So there is students come from a variety of background one, to learn about science policy before going to medical school or PhD, they often take our program. It's a one-year program. And so he's a director of that program. And his research originated the candida, and that was a focus, but he expanded a lot more research, and especially the drug targeting for the treatment of the fungus and sicknesses. And now, he's looking at microbiomes, skin microbiomes. So microbiome, people think often got microbiome, and we sponsor the lecture on got microbiome cases. But he is now going to talk about skin microbiome and also the fungus infections. So before further ado, please join me welcoming Dr. Calderone. [ Applause ] >> Richard Calderone: Tomolo, thank you. Is this the one. This one. Okay. Well, thank you for the introduction. It's nice being here. You mentioned microbiome. We're not going to talk about microbiome. We don't have enough data yet to even. But what's interesting about the subject itself is that, I think it points to the importance of teaching also, because you'd be surprised at how much you learn by teaching. You don't walk into a classroom with all the information. You got to go dig it out. It's not up here. And you learn things. And one of the things that I'll talk about today is something that I initially learned by giving a lecture to master's degree and PhD students. And I think that, I firmly believe that. I mean, well, it's a way of learning, I think. And teaching is a way of learning. You know, I think that's a message for all of you that are interested in doing teaching or are teaching. And I'm sure you know what I'm saying. So I'll skip to the story today. I want to, so I've been at Georgetown 45 years. I know I look like, what 29, 39-years-old? I'm not. Forty-five years, and so interests change and all of that. Doing much more teaching in the department now, because it used to be almost strictly a PhD, postdocs, but now it's PhD and postdocs, not as many, but more master's degree students. They're fun to work with, and I see one or two, I thought I saw, yes, I see one for sure, that was in the policy program. So anyway, okay, enough of that. So here's what I want to talk about today, and it sounds a little bit odd, and maybe according to the title you read in your abstract. But we're going to do these kinds of things. But at the end I wanted to talk about some of the interesting things going on with environmental fungi and environmental organisms. So just a brief discussion of that. And then a paper that was released in this year, 2019, a science article on the disappearance of biodiversity among amphibians. There's nothing, I don't know anything about amphibians. I mean, okay, I know what an amphibian is. But I think the important thing is just this concept of loss of diversity, biodiversity. And it's due to a fungus. And that fungus is something called a chytrid. In actuality, it's so simple compared, the structure is so simple compared to what we normally look at under a microscope for fungal pathogens that it's a very unique kind of organism. The classification may be fungus, maybe not fungus, that kind of thing. So it's sort of one of these intermediate. But I thought I would bring it along and try to give you an idea of impact of environmental fungi also at the very end. And of course, that's what I do is up above. So here are some introductory comments. I'm sorry, it looks so detailed. So there's about 150 species of fungi that cause disease, infectious diseases, in people. And these diseases vary. They could be infections, diseases of cutaneous tissue and mucosal, lung or blood-borne pathogens that either get inhaled or are part of the normal microbiota of people. They cause a serious number of allergies of various kinds, various kinds of organisms, and they can be global infections, or they can be endemic. Some of the really important respiratory disease pathogens are things you probably never heard of, blastomycosis, maybe you have, histoplasmosis, coccidioidomycosis. Those are all diseases that are endemic to the US and parts of Africa, parts of South America. But really heavily concentrated epidemics. They cause epidemics and serious lung infections in a number of people. If I made it sound interesting, now, see, I'm not going to talk anymore about that. Nevertheless, that's part of the whole thing. Antifungals. We do a lot with that kind of thing, and it's mostly development, searching for new antifungals. And I'll get into why we are doing that. But I sort of summarized it here. There's resistance to the antifungals, just like bacteria. There is something called drug-drug interactions, which I'll get to. And there's even toxicities. You have to understand fungi are more like people than bacteria are like people, right, where you [inaudible] and so are fungi. And so what you run into is some toxicity. So if you have a drug that kills fungi, you might have toxicity associated with it in people that are receiving that drug. So I won't say much about immunity also, but there is immunity that's established, very strong immunity. It's usually cell-mediated immunity. There is one vaccine that has been developed, and it's gone through a lot of preclinical experimentation and trials. And then has moved on to the FDA. I think it's in phase two now. And let's see, a vaccine which is to something called recurrent vulvovaginal candidiasis. And so that's one of the, I think, the good products that's come out of all the basic science that's been funded. I want to talk about incidents also, because I think to make your case about the importance of fungal infections, you need to know how much, you need to tell people about how much there is. How much disease is there? So I'm going to show you some of that data. Growth forms, if there's 150 fungi, there's 150 different forms, growth forms, and that's exaggeration. But I think you get the idea that you're dealing with a lot of organisms, many of them are very serious, cause mortality. Many cause morbidity. And most of them come from the environment. Things like aspergillus, I'm sure you've heard of aspergillus somewhere along. Yeah, so that's an environmental. The ones I just mentioned, these endemic mycoses fungal disease that you see in this country are also aerosol of spores in the environment are unhailed, etcetera, etcetera. Candida, which is what we work with, is the exception. It is part of us, I would say 85% of the people in this room, 85 to 90% are carrying the organism. Most of them, most of you carry it in the gut or maybe commensals of the oral cavity or vaginal mucosa or the skin. It's there, and it causes infections, mainly in people that have underlying diseases also, and we'll talk about that. Okay, so just as a real rapid look at. Mycoses means fungal disease. And here they are. They can be chronic. They can be acute diseases. They cause morbidity and/or mortality. And there are all the sites of the infections. I put some of those site locations of infections in green, and those are the ones, those are the sites that are infected by candida, the organism we work with. There are many fungi environmental that produce toxic diseases. And, you know, one of the most common ones is something called aspertoxin, which is produced by an aspergillus. And it's not peculiar to developing countries. It is in developing countries. It's also in the US, and it's a billion-dollar industry just to prevent this kind of thing from happening and trying to develop a resistance and that kind of thing. And then molds and allergies would be another big area. Okay, incidents. I think the ball got rolling a bit better in terms of interest and mycoses, fungal diseases, when people began doing the incidents data. And I have to mention one group. They're called GAFFI, and GAFFI stands for Global Action Fund for Fungal Infections. What they do it's a database of incidents and how much mortality, how much morbidity and epidemiology. So they published that data, but they also provide information on lab diagnostics and also treatments. So, I think the important thing is that this was the initiator of a lot of the incidents. And now, it's just, you see incidents data coming from all countries. And I'll give you some of that data in just a few minutes. And I guess the underlying thing is there's a lot of fungal disease which is going on. And so let's take a look at that. I'm sorry for the small print, but this was a paper that was published in Science Translational Medicine in 2012. And what they looked at was the ten most significant invasive fungal infections. So these are people that would be infected by oral, acquisition, aerosols and end up on the lung and become invasive and systemic invasion. Or they might be things like candida. There's candida right there, which is part of us, as I said, infections occur. So these are all what are call opportunistic infections, meaning that there's something else going on in the patient. And that something else could be surgery. It could be stem cell transplant. It could be radiation from chemotherapy, which makes people susceptible to a number of things, not just fungi but bacteria also. And so these are distributed everywhere. And what you have are numbers now. Now estimated life-threatening infections per year at that location. So aspergillus, about 200,000 cases. Candida about 400,000 cases of significant morbidity mortality infections. Here's cryptococcus, one million cases per year. And we'll talk a little bit about is that everywhere, or is it only in certain parts of the country, and we'll get into that. And then mucormycosis less, but important, mostly in diabetic patients that were acidotic. In other words, not treating their diabetes so well. Pneumocystis is another fungus which is quite, quite common. And then, so that's the first group. And notice what I've circled here is the morbidity. And one of the problems I think in looking at incidents is that, especially in this type of incidents, is you have to look at studies, data studies, that speak to the service the patient is in the hospital. Is the patient in intensive care unit? Is the patient in cardiology, dermatology or whatever? That's a big, big factor that determines how much infections there are. So that's why there's variation like candida, 46 to 75%. If you were looking at children in intensive care units, it would be up around 75%. But in other services not very high. So the data has to reflect where the infections are occurring. It also will vary depending upon the hospital. You can go to a website and look up, I'm not picking on New York, you understand, but you can go to New York, and they list all the hospitals in the state of New York. And they also list which ones have the highest and which ones have the lowest rates of infection. So, you have to understand that there's variation even in hospitals and, of course, globally, there's variation. The other large group, the endemic, are things that I mentioned. Mycoses, dimorphic mycoses, blastomycosis, cocci, and histoplasma. And the numbers are much less. You can see that the first three are found in the US. And they do have significant mortality. And these are the three that do cause epidemics. You don't, there are others that cause epidemics, but of all of these, you don't really see epidemics in Canada. It can occur, but it's rare. And the other hand, histo and coccidioidomycosis see a lot of it. Okay, so that's a global study. So one time I was invited to go to Mexico to give a talk, and I said I think I'll look up the incidents of fungal infections in Mexico. And so unfortunately, this is all abbreviated. RVVC is a vaginal infection, and the R stands for the fact that it's a recurrent disease. So a woman infected, there's a chance that she will have four infections per year perhaps, and that qualifies categorization as a recurrent vulvovaginal candidiasis. So here's the burden, and it's two million so and so cases in that the rate, usually incidence data is explained as the rate per 100,000 people. And so there it is, quite high, mortality and [inaudible]. And this is an aspergillus infection. This is another aspergillus infection. And that incidence is fairly high. A fungal keratitis eye infections, there it is. Invasive candidiasis 8.6 rate. The rate mortality as I mentioned. And serious fungal infections, which is what they classify in Mexico, everything else except what I mentioned they put in a category called serious fungal infections whereas cryptococcus and coccidioidomycosis are serious infections too. But this is like other things, and you can see it's quite. And here is coccyon [phonetic]. I just told you it was in the US, but it does have a location. It resides in the deserts of New Mexico and so on and so on. So here is the total fungal burden, and I think I got the right term. It's 2,749,159 patients have fungal infections. And the interesting thing is if you look at tuberculosis, there it is, 21. So the rate is quite different, isn't it? And so what it points to is that there's a lot of fungal infections. And that was the preceding study here, as many or more die from invasive fungal infections than drug-resistant tuberculosis or even malaria, especially childhood malaria. It's much greater. So, okay. So that's one country. But what I, I also put this chart together, and it's a combination of data from two different references. So what I did was to compare countries as to, again, specific data, rights of infection for 100,000. So the diseases, I've mentioned these to you already. But the countries, you can see it's really going to be quite different. So, for example, oral candidiasis, which is very common, not so much in this country anymore but in other parts of the world, it's very common in HIV. Its infections, and so 769, that's the rate per 100,000, Uruguay, 74. So about ten times higher in Kenya. And I can go through these things, and you might figure out what in fact, why is this so? Well, it's going to depend upon the specific underlying disease. So if the patient has HIV/AIDS, it's going to be a lot of oral infection. So that's part of it. But income is a very big factor in terms of healthcare. And usually these countries, especially the developing countries, do not have as good facilities in terms of diagnostics, determining what the infection is and also access to antifungals. But I don't want to go through all of this, but here is, where is it right here, streptococcal meningitis, again, HIV/AIDS. And if you look at these ratios, it's 162-fold higher in Kenya. And again, it's because of the association with HIV/AIDS. So I guess Uruguay, you know, a country that is making great progress in healthcare. So you begin to see different kinds of infections. You begin to see blood-borne infections and lung infections occurring. In what kind of patients? Well, transplants. Kenya, I'm sure transplants are done rarely. I don't have any numbers to prove that, but I would imagine that a country with greater wealth is going to be doing that kind of thing. So cancer treatment, put it all together and you see that. For example, candidemia is much higher in Uruguay versus Kenya. Okay, so enough of that sort of thing. But I still think that's important, and it's really good to see more published papers on data from a variety of countries, so you can get a real good picture of how it differs. But that's the important point here, it differs. It differs whether you're living in Kenya versus living in Uruguay or the United States or so on. And it bothers me, we train medical students, been doing that for a lot of years, and we have case presentation where they come in and listen. So they'll hear about meningitis caused by bacteria. And I'm sitting here thinking, wait a minute, what about cryptococcus? You know, that causes more than a lot of the bacteria. But in Georgetown University Hospital, they don't see cryptococcus. Why? Because it's under control pretty much. And that's what you want to happen for all of these countries. And of course, that's not possible, at least right now. But anyway. So this is what we work on. And it's polymorphic. That simply means it has a lot of different growth forms. It's part of, again, as a normal human microbiota. And it causes skin mucosal blood-borne invasive kinds of infections. There is, I'm not going to go through this. I think the only thing to mention, I'm not going to go through all these pictures of diseases. I'll flash them through very quickly, but I think the important thing is when you look at risk factors, number one, that's very important for clinical infections caused by candida to occur. There has to be a risk involved. And so obesity, poor hygiene, diabetes, antibiotic treatment and oral contraceptives. Antibiotic treatment, that's a repeating pattern. You have to think of candida in the gut, okay. It's down there not by itself. It's there with thousands and hundred thousands, millions of bacteria, and if the patient is taking an antibiotic, what happens is, of course, the bacterial population decreases, but the fungal population increases. Right? Because fungi are not bothered by tetracycline, ampicillin or anything like that. And so they increase. And when you have that condition, you change that equilibrium. And that's kind of like basic microbiome study. We were talking about this 50 years ago, maybe not 50 years ago. But nevertheless, okay. So that's one type of candida. Here is oral. I mentioned HIV. HIV, diabetics, broad spectrum antibiotics, again. And so, I want to mention one drug, and it's a compound called fluconazole. That was developed in the 1990s. Why? Because everybody that's HIV/AIDS was developing oral candidiasis. So, the disposing factor for oral candida, one of the disposing factors of HIV/AIDS. And so, rush, rush, rush. Companies were looking for antifungals. And a compound called fluconazole became the number one bestseller of billion dollar product. And so what happens, however, was that in that population of candida in the gut or in the mouth, the whatever, it's not just one species. There are many different candida that are there. And so what that drug was doing was selecting for other candida species. Why? Because those other candida species were resistant to fluconazole. Does that make sense? So candida, [inaudible] was not so resistant. But other species of candida are there also. And so you had a change in leadership. I don't know if that's the right word. You had a change in conditions that allowed other candida species to take over. And it resulted in development of other similar compounds, but you still have some of the same problems. Okay, and vulvovaginal. This, here is this RVVC I mentioned predisposing factors again. A number of them. And yet we know so little about immunity to vaginal candidiasis. There's been at least five or six theories why do women, why do some women get one infection and it disappears, never again. And others get four per year or more infections, and it's a chronic problem. What's the difference in immunity? We don't know. Okay. Staph infections are the leading cause of bloodstream infections in the US. BSI is bloodstream infections. So coagulates, negative staphylococcus is staphylococcus epidermidis and coagulase positive staph is staphylococcus aureus. And there's the total percent of bloodstream infections. And enterococcus is third and candida species is fourth on the list. Now, I would tell you to make any sense out of this, okay, so they're good numbers for BSI perhaps, but again, you're dealing with a number of underlying conditions that are associated with the frequency of staph or it's just the frequency of candida. And services, what unit of the hospital is treating these patients. So you have things like that that can change the numbers. But here's bloodstream infections. This is from a 2001 paper, and so here is the, so this is the proportion of bloodstream infections and accrued mortality. So here's the number of infections, the percent, and here is the mortality, crude mortality. So this is a coagulation negative staphylococcus that would be staph epidermis. Here is staph aureuses, enterococcus and candida. Look at the mortality that's associated with the candidiasis infection, even though it's less than the other three bacterial species, the fungus kills quite a few people. And here is another interesting point here. It's kind of, not so complex, but here are the pathogens that cause bloodstream infections. And here is something called the days between admission and an onset of bloodstream infections. So the person comes into the hospital, has surgery or maybe some other condition that requires treatment in the hospital, how soon, if they get infected, how soon did they become infected? So days between admission and onset, and you can see, E. coli, less than two weeks. I'm not going to go through all of these, but you get an idea that not all these organisms are equal in terms of how fast they develop in the hospital. And here's candida, candida you're a bit beyond three weeks to infection. Why? Why would that occur? What is the take home message on that? Well, why do they occur is because E. coli, staph aureus when it's in the blood, the physician gives the blood to the clinical lab, and you get a diagnosis pretty quickly. It grows from blood, patient blood. And as you go down here, when you get to candida, you can't find it because it's not growing as well in the blood of bacteria. And I'll give you an explanation for that. But you might be in that hospital for over three weeks before a diagnosis is made. A little bit of history that may go along with this. So it becomes positive, right, in three weeks. And the philosophy, even in the 1990s, was, I'd better get over here. The philosophy in the 1990s was oh, you've recovered candida from the bloodstream. You'd better do it again because it's probably a contaminate. Right? It's on your skin. Maybe in the IV catheter is in you, and you get an infection. The assumption was, it was just a contaminate. Redo the thing. So you're redoing it, and now you've got another three weeks to wait. So the truth is, that's wrong. You never assume candida and all these other organs, you never assume that they're there for the fun of it, that it doesn't mean anything. It's there because they're causing the infection. But that had to change. That philosophy with candida had to change to show that in fact as an important event, if you find the organism in the blood, okay. Think one more thing, when we look at the story, think about cost. If you're in the hospital three weeks or more versus less time, it's more expensive, more expensive, right. And so, that is a major problem with a lot of these infections, including candida is that they're much more difficult to find in the blood, and the second thing is that the longer you stay, the more it's going to cost the insurance company. The hospital is going to pay for some of this and you. And that's the way it works. So those are important considerations. Then the other thing that'll tie into what I just said about length of time before a culture becomes positive, because many of these pathogens, and I've given you two fungal, aspergillus, fumigatus and candida that form biofilms. And I want to show you a picture. Because the biofilm is a really very important part of the whole infectious process. What is a biofilm? It's a three-dimensional community of microorganisms embedded in polysaccharide of the pathogen and host that is attached to surfaces. What is it attached to? Well, it can occur in all medical indwelling devices, catheters, voice boxes, respiratory intubaters [phonetic], things that go on your nasal passage to improve oxygen. Replacements, heart, valve, knee, hip. Central nervous system, shunts, pacemakers, the whole thing. And so if the organisms, and we're not talking just about candida, if the organisms contaminate those things, you have to, the physician has to repeat. Remove the infected knee joint product and redo the whole thing. And so how much implantation is there? In the US, 1.1 million knee, hip devices are implanted per year. The infection rates are about 60% of implanted devices. Candida species account for about 20% of those infected devices. The other problem is that, remember I said that you've got an organism, but it's covered in polysaccharide. And so antibiotics don't penetrate too well. They don't get to the source of the organism. So, the belief is that biofilms contribute to drug resistance. You can have single candida alone or candida plus staphylococcus aureus causing a biofilm. And the biofilm seeds the bloodstream, like an indwelling catheter. So let's look at, what is the catheter? And in fact, this was invitro, but what you're looking at, this is low magnification. So here is the catheter that's been split open, and all this is biofilm. This is invitro, but to what extent do you see it in vivo, much less, but it's still important. And here is a higher magnification. Here again is the catheter, and this is all fungus, and you can see some filamentous forms and unicellular forms of the organism. So, what does this have to do with disease? How does it become blood borne? Okay, I'm sorry. How does it become blood borne? Well, a couple of different ways. So in this top of the slide here there's an intestine, and there was intestinal surgery. It gets sewn, yeah. But sometimes there's leakiness to that suture that's put in there. And the organism escapes, gets into the peritoneal cavity or it gets into the bloodstream in that way. So here is the organism colonizing the gut. Surgery, if it's not done entirely correctly, that's one of the ways it could get into the blood. But over here, it takes into account these biofilms. So here we have a catheter, and the catheter is contaminated with candida. And what happens is that it forms a biofilm. And from that biofilm, it can enter the bloodstream. So you've got a catheter with an organism growing on the catheter. If they find it, it's fine, then take it out and do it again. But nevertheless, that's another way it gets from, in this case, the skin, it's on the skin. It gets into the bloodstream. And so what it does, these organisms, once they're in the bloodstream, they visit different sites of the body. So they can go to the kidney, the organism can go to the spleen, to the liver, eye, lung, bone, etcetera. Remember the point that I made about how long it takes to find it in the bloodstream. And the reason is, it plays hide and go seek. It's in the bloodstream, but then it goes back into the tissue. Or it could be in the liver, in the liver, but then it goes back to the bloodstream. And then back to the liver. Or maybe even back to another tissue. But it all started with those biofilms that are formed. And so, you can't find it in the bloodstream simply because the organism is assuming different sites, and maybe it's not in the bloodstream so long. And so you miss it. And so this is the reason that when cultures become positive, assume that it's an infection. Maybe you're thinking, well maybe besides culturing, what else should they be using to diagnose it? There is a very, very good PCR technique, which is available, to find candida in these situations. The problem is the cost. And I've had people from a cancer center in Ohio State University saying they're not willing to pay for the cost of one of these PCR devices. We're talking maybe 100,000 just to do a couple of assays. So probably what's happening is there's an original lab somewhere that's do everything. And maybe it's not happening that way. Okay, I've sort of given you a gloomy picture here. And NIH has done a wonderful job of really getting money to the scientists to do the work that should be done and really determine what the ramifications are of fungal infections, not just candida. I just wanted to mention one other organism, and this is an organism that aspergillosis. And here's what it looks like in the soul. And all of these things are spores. An the spores get released and they grow in the soil, or they get up into the atmosphere. And aerosols then will take them back to the ground. If this is a hospital ward or an outside construction area where error id getting into hospital. These spores can cause infection also. And I really just showing you this to show the variety of infections for some of these organisms. So over here, frequency of aspergillus, this is frequency of aspergillus. And the thing to focus is on what happens if there is an immune dysfunction. Something is wrong with immunity. Or what happens in a healthy person? Or what happens in a hyperactive person, immune person? And so here you have this really high morbidity mortality type of aspergillus called acute invasive, 50,000 cases per year immune dysfunction. And at lower frequency, some acute infections but not nearly as bad as the acute invasive infections. And the so-called healthy population, this number of cases, and you get fungus, aerosols that get into the lung, and what they do is settle into tuberculosis cavities. If a patient had tuberculosis, there's a cavity that remains. The fungal spores come into the lung, gets into those cavities and grows within the cavities. Or chronic fibrosing or some of these, I can't really define them for you, locally invasive. But then you look at the other end of the spectrum, hyperimmune activity, allergic sinusitis, severe asthma, ABPA, which is allergic bronchopulmonary aspergillosis in cystic fibrosis patients. So, what you have is a completely different picture depending upon the immune status of the patient. I think it's important when you're studying these things to recognize that. Okay, the lab focus is on drug targets, antifungal drug targets. I mentioned resistance. I mentioned toxicity and drug-drug interaction, so that's the why. Why are we doing this, and why are many other labs doing it? These are the reasons. And so what we do is, remember I told you both people and fungi are eukaryotic, so there's not much difference between versus bacteria that are prokaryotic. So, what you have to find, if you're going to do this, and first, I guess, first I should develop a concept of what a target is. The target is the part, in this case, part of the fungus that you're targeting. Membrane of the fungus, the cell wall of the fungus, nucleus of the fungus, whatever you're doing, that would be the target. What does the drug react with? That's called a target. And what you want to find is using bioinformatics is to identify fungal specific targets, fungal specific. In other words, you're eliminating, you're reducing the possibility that in fact, there could be toxicity because the targets are similar, people and fungi. A good example of that is our membranes have a steroid called cholesterol. It's part of the membrane. Fungi do not have cholesterol, but they have something called ergosterol, very similar. And so those compounds that inhibit ergosterol synthesis, the fungal sterol, those that inhibit that also can be inhibitory to cholesterol. And so that's what results as toxicity. So, you need to do this kind of thing first. We'll get into some of that later. And then what you want to do is validate the importance of that target to the pathogenesis of candida. Is it a target that's not really needed for disease? Is it a target that yes, it is needed for disease? So you have to do that. And this requires a lot of molecular biology research. What you have to be able to do is construct single gene mutants of this organism. You take away one gene of the 6,400 genes that candida has, you take one away by molecular process and you say okay, it's missing that gene. What is different about the organism? Is it no longer causing disease in mice? Is it, you know, so that's what you're looking for. You're looking for a gene that not only is fungal specific but also is important to the disease process. So that's very important. And so these mutants are constructed, and you can assay for virulence. Does it kill mice? Does it not kill mice? Changes at the cellular level. Does it change the cell wall? Does it change the cell membrane? Does it change anything you want to look at? Or subcellular level, and we've looked at biochemical properties of these mutants, the protein, gene arrays, RNA sequencing, polysaccharide signal pathways, just to get a really good foundation of what, if that target is important, what is it doing? What is that drug doing? Where is the target that it's acting against? Okay, so why are we interested in drugs, resistance? And so what we're showing here is a fungal cell, and this is a susceptible cell, a fungus, candida, [inaudible] that's susceptible to a drug. And here is the drug. And this is the membrane here, and the drug comes into the cell and binds to its target, which is ergosterol. And the organism is inhibited or even dies, okay. So that's susceptible cell. Notice in susceptible cells there are these things called efflux pumps, and one is called MDR and one is called CDR. And so the drug comes in and gets pumped out. Okay, that's good for the fungus. Because the drug's coming in and gets pumped out. Here is the pathway. We can get into the origin of these pumps. It's kind of an interesting thing but really no time. But I want to just compare what you see in a susceptible to what you see in a resistant. The first thing you see is that there's many more efflux pumps. This is called overexpression. So this one has two. This one has 10 or 20 different efflux pumps, just to show you. So the drug in a resistance all comes in and gets pumped out by these different kinds of resistant pumps. But there are other ways that becomes resistant. For example, here's the target as part of that ergosterol synthesis. If there is the target, and the differences in the picture is to show you that the resistance cell, there's more target. It just overproduces target. And so therefore, there's not enough drug. In a susceptible cell you've got target, and you got drug. In a resistance cell, you've got more target, maybe the same amount of drug. So it's proportionally different. And the other thing is that you get point mutations. Somewhere in here it says point mutations. So, all of these things are occurring. And here's what I meant about drug-drug interactions. This is true not just for candida, for any infectious disease, bacteria included. So what I'm showing you here is here is a liver, and in that liver is an enzyme called CYP3A4, and it stands for cytochrome P450, and that's because the cytochrome absorbs light at P450. So it's a liver enzyme, and it's designed to eliminate the drug. So here is a profile. So the patient is on statin, and here is the concentration of this indicated over time. And notice that upon delivery of the drug, statin increases but then decreases over time. Now that's what's supposed to happen. Can't accumulate it, because then you get toxicity. Okay, right. That's the way it should work. But suppose that patient has a fungal infection. So he's not on or would be on, or she, statin plus azole. So what happens here is that there's competition for that enzyme. It's not just statin, but it's a fungal, it's a drug which is degraded by the same protein in the liver. And so what you have in this case, and over here it just, it's lowercase, to indicate this is the way it's supposed to be, and this is the way it's not. And because of activity of this particular enzyme. So what the profile you get is something like this. Statin, the patient's on statin, he or she is on azole also, and so the statin concentration increases and so does the azole concentration. And notice that it's not illuminated as rapidly. This is called drug-drug interaction. Both of those drugs bind to the same enzyme and liver that leads to their degradation. Now, the physician can find this information very, very quickly. Just go online, statin, don't use azole it says. The problem, so it's not that. It's not difficult to find drug-drug interactions. What is difficult is, I forgot what was difficult, yeah, oh, what is difficult, the difficulty is that you've lost, there's only three different groups of antifungals, right. Only three different groups. I mentioned that. And so you've eliminated the use of one of those three groups of antifungals because of drug-drug interaction. So now you're down to two groups of antifungals. Okay. All right. And I think that's enough for this. To look at the properties of these mutants that you make, you have to use something that's called reverse genetics. And it's a bit more difficult in candida because it has two genes. So it's a diploid. Every gene is duplicated, so it's a diploid organism too. And so this bar here represents the targeted gene that you're looking at. And here are the two copies in candida because of that carbon, I'm sorry, of the target gene. And so what you have to do is make something referred to as cassettes. So here's the five-prime end of that gene. Here is the three-prime end. In the middle of it is the candida because histidine one gene. And so you transform this into wild type, and that wild type does not have a His1 gene, and it does not have a Lou2 gene. So those are auxotrophs for the organism. So what you do then, transform it, and now you've converted one of these alleles to that knock out structure, that microcassette. And so, now it becomes His-positive. So up here, the parental strain could not grow in the absence of histidine. But in this particular one, now that strain has a histidine gene and can grow. So you can select for histidine resistance. But remember you have a second allele to target. And so here, it uses the principle of a leucine effect. And so here is that same cassette in which, of candida, which you have a candida Lou2 gene, and you transform that. And so you've made, therefore, a mutation in that target gene in both alleles of the target gene. And it's a long and hard process to do, but you have to do it. So, what you're talking about is, for example here, percent survival. Well, this is a mouse study, I forget, it might have been some of our data, so this is a mouse study. It's percent survival over days. Here is the diploid. This is the wild type strain, and it kills the mice very quickly. Has both copies of that particular gene, both alleles. Here's one that has a single copy. You have to have that control also. And notice the killing is, or survival, is about half of the wild type. And here is the strain where you've taken out both alleles, deleted both. So you get this nice activity occurring that's gene dosage related. And so this is what you're going to use. So finally, getting to the work we do, it was to develop antifungal drug targets. And the two that we worked on first of all was the, something called a histidine kinase. This is a protein that's found in bacteria in fungi, not mammals, back to the bio informatics again. The idea of specificity, fungal only. And the histidine kinases are important in cell sensing and biofilms. And those mutants that I just showed you, how you make them, in a mouse model, in a histidine kinase gene lacking that particular gene, the mutant are avirulent. So you want to see compounds that can inhibit those histidine kinases. Because if you inhibit, the histidine kinase, the strains, at least in mice are going to be avirulent. That's a good target. The second set, which we're not going to get to today, was other, the same example but in terms of not found in the human mitochondrial, these are mitochondrial subunit proteins, complex, the electron transport chain. Again fungal-specific or candida-specific. Mutants are avirulent. And we're seeking compounds, and we're all hopeful NIH is going to be happy with our work. So here's, I don't want to go through all of this, but just, here is a histidine kinase. Here is the protein here. It's a very complex protein. And the piece that you're looking are phosphotransfers. So there's an input signal. And it could be in it. It could be carbon dioxide. It could be salts, or example. It could be blood. It could be almost anything that gets sensed by the organism, sugar sources. And so you get phosphotransfer to a second region of that protein, and then transferred to another protein, which is called a histone containing phosphotransfer. And then transfer to a third protein. This is what we worked on. We've made mutants in this particular protein, mutants in this particular protein, and also in that particular. Now, I think it's important just, phosphotransfer is common to human cells also. Very, very common, just as. But these phosphotransfers are histidines. Or they're aspartic acid. And those types of phosphorylation do not occur in humans. So the mechanism which is different is the protein itself is different not found in people, but also the types of phosphorylation, fungi use different amino acids for those phosphotransfers. So you got a couple good things going for you. Compound discovery. This is, I'll go through this fairly rapidly. I don't know, how much time do we have here? >> Tomolo Steen: Almost done. >> Richard Calderone: Almost done. Okay, then I'll skip a lot. So this is what we've done with the Wichita State University Department of Chemistry. Bill Groutas was the person who would make lots of synthetic compounds. We would go through the process of screening all of these compounds to find ones that were useful. How much time, ten? Ten minutes? >> Tomolo Steen: Yeah. >> Richard Calderone: Okay, so here is, this is something, the type of compound, it's called a scaffold. And what you do is modify this scaffold. You can use something called structure activity relationships. You do MICs. How good is the drug? How inhibitory, how fungicidal it is. You can do all of that kind of stuff. We came up with four compounds that are in patent. And one is going on for further evaluation. So I'm going to skip over the screen we use, unfortunately, but we don't have time. It's a screen which is the genetic screen. And what you do, it's labor-intensive. You've got, use the strain of saccharomyces cerevisiae. Not a pathogen, but it's very similar to candida. It has 6,000 genes. You have to make a library of a mutant in each one of those genes. And that's what we have. So we have a library that you can buy actually of saccharomyces that have a mutation in each gene. Some of those mutations will be the cause of something called haploinsufficiency. They're not, you know, if you took Babe Ruth and removed one of his legs, he wouldn't be as good in terms of hitting a homerun. It's the same kind of idea. Which of those strains that have a loss of one of the two genes, which one is susceptible to a compound? So out of 6,000 genes, I think we isolated, and we could identify 13 genes that were sensitive, much more sensitive, to compound than wild type cells. And I don't want to spend a lot of time, but some are fungal-specific. Some are broadly concerned, which causes problems. And what gets inhibited is something called the kinetochore. So here, you'll recognize this, the blue is chromosomes. The green is a spindle fiber during chromosome separation. And these pink things are these kinetochores. And the kinetochores make sure that the chromosomes bind to the spindle fibers. That's the duty of the kinetochore. And so that's what was being affected by one of those compounds that I just mentioned. There was an activity, and that activity clustered, and genes that had the same functions. And this is what you use. So yeah, here's 13 hypersensitive mutants. We used something call Fun Spec, and that clusters the, which of those 6,000 mutants is affected by the compound? And are they similar, or are they clustered in some way? And so you do that, and that's what we did. Okay. We also use a repurpose compound. The library and, repurposing means that, in fact this isn't NIH, but other places also, National Cancer Institute, what it is is a library of thousands of compounds that have an activity but have never been tested against fungi or bacteria. So some might be antidepressants. Some might be anticancer compounds that are sitting in that library. Are they only antidepressants? Are they only anticancer? Do they have other activities. So that's what is referred to as repurpose compounds. And that's been pretty useful to people. So here's the way I look at the whole thing of drug discovery. It's tough work. And here's Calderone and a postdoc where the road to success, and you've got the speed bump, and the speed bump is R0A funding And it's a tough world out there, folks. Let me tell you, but we're trying. And so the postdoc is saying to me, wow, that's the biggest speed bump I've ever seen. Okay, enough for the fun. The people that have done the work. Now, real, do we have time to go through these last couple of things? No. Okay, let me, I talked to much. So the first example is decomposition of forest litter. And this particular study was done in Chernobyl. And what they did was to look at the importance of microorganisms and decomposition of forest litter. And they used various places in and around Chernobyl that had different dosages in soil from Chernobyl. Here is the fellow that did the work, and what he has in his hand are sacks of leaves. And the leaves are from a bunch of different deciduous trees. And he takes these sacks, and he puts them in different sites, nylon mesh bags, and they are extremely small pores. So he wants to eliminate earthworms getting into those bags. He wants to eliminate any other kinds of vertebrates or invertebrates that are going to get in there and mess around and destroy the data. And so he puts them in bags, the leaves. And the environmental sites were similar in temperature. The moisture of the bags were in 52 sites around Chernobyl. There it is. And this is what they found. When they looked at the proportion of decomposition, after nine months, at different sites with different background radiation, you can see that the proportion of decomposition decreases as the amount of radiation increases. So I think that's a really nice paper, and hopefully, it will stimulate other things. Here is something which is a little bit worse, much worse, to define. And this is something that was published, some of the data from 2006, but I'll get to a paper of 2019. The fungal catastrophic infection of amphibians caused by chytrid fungus, I am not going to even to mention that name. But there it is. That's the fungus. It's a chytrid. And here is what it looks like. It's unicellular. It produces these things called, here's a motile spore, and the modal spore gets bigger and forms something called a sporangia, and then those sporangia release more motile spores. So this happens in water. And so that's the whole cycle of the organism. And here is the data. And this is from a 2019 paper. And what they looked at was amphibian populations in North America, of Central America, Europe, South America, so Brazil, Africa. And they looked at a number of different species. And the color-coding indicates severity of decline, 20%, 20% to 90%, I can't read that really, 90%. It's presumed, yeah, you can read it, extinct. Presumed extinct and extinct. And so you look at this, and here's, they looked at ten different species of frogs in North America and so on, and you can see the results are amazing in terms of what's happened to those amphibian populations. So the summary is a decline of at least 501 amphibian species over the past half century has occurred, including 90 presumed extinctions. Only 12% shows signs of recovery, 39% ongoing decline. The greatest recorded loss of biodiversity is attributed to a disease. And the other thing which is becoming associated with this is temperature of water. So this is an aquatic fungi. The temperature water is increasing, global warming, etcetera. And they think this is part of the thing, so, I'll stop. Thank you. [ Applause ] >> Tomolo Steen: Some questions? Okay. >> I was amazed how much infection, you hear about how much infection occurs in different hospitals. But I was amazed that devices have been planted or implanted means, etcetera, are not protected from the fungi. How come it happens? I am not quite sure. I thought sterilization of instruments during surgery or, you know, the catheters and all that are, they do not have, they do not carry the fungi -- >> Tomolo Steen: Richard, Richard, you have to come over, Richard, you have to come over here. >> Richard Calderone: I say it's a good question. I think there are a lot of factors involved in that, and it seems to me that it's also a factor of what the organism is. You know, that's one of the things. You know, other things, you just don't have that problem, other organisms. So it's a bit complex. Yeah. But for some reason, it likes to, you know, I'll stop there. >> Tomolo Steen: Sorry, the time is actually limited. So you can informally talk to Professor Calderone after this. So please join me in thanking him again. [ Applause ]