Fungi: You Can’t Live With ’em or Without ’em


>>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 ]

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