MycoTalks S6 E3: Eric Dang and Minh-Hong Nguyen

Hello everyone and welcome to the November edition of micro talks. Uh it's

my pleasure to be here and uh I will co-chair this with my with uh Jay calls.

So we have two fantastic speakers today.

Eric Dank from the Reagan Institute of U Mass General MIT and Harvard and he will talk about barrier tissue immunity to

pathogenic fungi and we have Ming Nong Guen from uh University of Pittsburgh who will be our second speaker. So the

micro talks uh is u uh is a series that we started now uh

several years back. Uh the philosophy is uh diversity in career stage gender geography field. We have two 30 minutes

geography field. We have two 30 minutes micro talks. We keep this format. It's

micro talks. We keep this format. It's

been successful so far. 30 minutes of in-depth discussion and after that we have uh Q&A after the second presentation. So please uh put your uh

presentation. So please uh put your uh uh your questions in the Q&A sections and we will read them and ask our speakers.

uh uh the January micro talk will be by uh uh two presenters being Zai and Bern

Hub and then we have the February one by by George Dippy and Tamara During. Um I

want to also uh advertise here another uh series of talks Michael clinics uh

which uh we also started uh one year ago and uh there will be a December edition December 11th and for those of you who

are interested in clinical micology please join that. Uh so without further ado, I'll start uh with our uh

presenting our first speaker.

So uh that's Dr. Eric Dung.

Uh so uh Eric uh performed his uh graduate work at the University of California, San Francisco in the

laboratory of uh Jason's sister. there

he studied oxyister and cholesterol metabolism in uh regulating microfage inflammatory responses.

He received his uh PhD in 2018 and uh soon after he joined the laboratory of Hiton Matani at UCSF for his post-doal

work where we where he did some very similar sim similar work using genetic approaches where he studied the mechanisms of immune system manipulation

by cryptocus neoiformans uh and uh uh he had some very uh seinal findings in TH2 immunity in the lungs.

Uh it's a fantastic study. In 2022,

uh Eric joined uh tenure track position at NI ND in the laboratory of host immunity and microbiome. And then in

2025, he moved at the Reagan Institute uh in Cambridge, Massachusetts where he holds an academic appointment in the department of iminology at Harvard

Medical School. uh his group focuses on

Medical School. uh his group focuses on understanding the cross talk between fungal pathogens uh and commensiles and the mamalian

host. And with that uh we are looking

host. And with that uh we are looking forward to Eric's presentation.

All righty. Is that the right screen share mode? There we go. I think that

share mode? There we go. I think that works.

>> Yes, looks like it.

>> Thanks so much for the introduction, Ian. Uh and thank you to the organizing

Ian. Uh and thank you to the organizing committee for uh this opportunity to present. Um I I wanted to show a sort of

present. Um I I wanted to show a sort of new story from the lab uh that's sort of focused a bit more on the intestine when a lot of what we've been doing is in the lung.

So uh I don't I usually give an opening slide with a bit more of a fungal pitch, but I think for the global mco community, everyone's already drinking the Kool-Aid. uh but I just want to open

the Kool-Aid. uh but I just want to open up by making this point that I I think we all appreciate that fungi are a present but ever growing source of human

morbidity and mortality and beyond pure amunosuppression I think there's an interesting concept that's emerging in the field which is that fungal infections can result from maladaptive immune decision-m

and what I mean by that is that we've now sort of appreciated in immunology over the last 30 years that immune responses have to be exquisitly tailored to the appropriate pathogen that's the

invading insult where we turn on selective classes of responses that try to clear those selective agents and so each of these uh uh imunological arms has a physiological role. So type one

immunity is generally characterized by interferon gamma producing lymphosytes that try to ramp up cell autonomous killing mechanisms to control the growth of intracellular bacteria, viruses,

fungi as well as tumors. Type two

immunity is characterized by the production of 13 and four from gata 3 posites and this acts to protect against parasites and allergens through induction of the classic weep and sweep

response that tries to expel these noxious agents. And then lastly there's

noxious agents. And then lastly there's type three immunity which is characterized by mostly IL is 17 production and this has been very clearly shown to provide protection against extracellular bacteria and

fungi. Now when I say that uh infection

fungi. Now when I say that uh infection can be driven by maladaptive decision-making what I mean is that it's now appreciated that beyond just purely immunosuppression that turning on the

inappropriate class of response can actively uh predispose to to fungal colonization or invasive disease and I think one of the best examples of this comes from my colleague at NIH Mahalisanakis where what he has

beautifully shown is that in addition to 17 deficiency over production of interferent gamma particularly in the skin actively promote promotes colonization of mucutaneous candidasis.

There's also data that's been present in the field for you many decades now which shows that a number of environmental fungi have this predilction to turn on type two immunity in the lungs when they're inhaled. And this flavor of

they're inhaled. And this flavor of immune response is actively imunosuppressive to the ability of the host to clear the microorganism. And so

one of the major questions that my lab is really interested in is trying to understand how the immune system makes decisions to turn on physiological versus maladaptive immune responses in

the face of fungal exposure.

We're particularly interested in this uh uh process of type 2 inflammation turned on by fungi. And you know, one thing that we debate a lot in the lab is how much of this predilction to turn on type

2 immunity is a pathogen-driven response, meaning the host is being manipulated versus this being a conserved evolutionary response. And I

think that the idea that it's pathogen driven would be kind of neat, particularly for things that we think about, but I view it that if you actually look more at the broad literature, this is a a much more

conserved uh functionality. And so it's very clear that environmental molds, so things like ultraria, penicyium, aspiggillis, when these are inhaled, they predispose to type 2 inflammation.

And you know, many asthmatic patients up to 20 to 80% depending on the studies show that there's sensitization against fungal allergens. There's also some

fungal allergens. There's also some really uh interesting emerging data on commensal or at least gut fungi that when these uh show dispiosis particularly in early life windows that

this can predispose to type 2 inflammation both locally but also have distal effects that enhances airway inflammation. And that's actually a bit

inflammation. And that's actually a bit of what I'm going to be talking about today.

And then lastly, there are these environmental yeasts that can drive type 2 immunity, I think, through these conserved uh initiation pathways, but then some of them have molecules that

can hijack this response. But I think broadly this is not a a microorganism directed pathway. This is this is a host

directed pathway. This is this is a host directed pathway. And so uh largely what

directed pathway. And so uh largely what my lab is interested in is sort of looking at the interface of micology and immunology to try to understand the inputs and outputs of fungal induced

type 2 inflammation. And we approach this from three general questions. So

one is trying to understand the repertoire of fungal cues that influence host immunity. Two is trying to

host immunity. Two is trying to understand how type 2 inflammation is initiated in response to fungi. And then

lastly, we're interested in understanding when type two responses are turned on, why is this maladaptive?

And so what I want to talk about today is is an ongoing project that sort of merges question one and two.

And our sort of lead into this was something that I just alluded to, which was some of these human epidemiologic studies that have linked fungal dispiosis to asthma sensitization.

Some of this comes from uh uh studies that have correlated asthma uh incidents with maternal or early life antibiotic use. And there's been hypotheses that

use. And there's been hypotheses that that may be due to depletion of bacteria and enrichment of fungi. There have also been some nice studies done by Brett Finley's group in Vancouver where

they've looked at uh cohorts of kids that either developed asthma or not. And

one thing that they found is that kids that develop asthma later in life showed early fungal dispiosis in the guts around age 2 to five. And then again I just want to bring back to this this

point which is that you know a lot of asthma patients do show sensitization to fungal allergens. And so from this sort

fungal allergens. And so from this sort of human backbone one thing that's been interesting to think about is can this be mimicked using animal models.

And I think from there it's it's it's fitting to get introduced by Illyn because it was a study from from his group uh and in parallel some work from David Underh Hill that initially sort of got at this. And so what they published

was this this really cool experiment where they put mice on fluconazol drinking water to try to deplete gut bacteria and then come in with different house dustmite challenges to then drive

a type 2 response in the lungs. And what

they found was that giving mice luconazol actually resulted in worsened allergic airway disease which sort of might argue that fungi are actually protective in this context. But when

they actually looked uh at the gut composition of fungi, what was clear is that uh you get depletion of fluconosol sensitive strains like candida. But you

actually get expansion of fluconosol resistant fungi. One example being here

resistant fungi. One example being here as aspergillus amstiladami in green. But

then really one that shows almost no colonization that then expands dramatically was the species called Williamia sebi or also now known as wimmia melacola. And then what was

wimmia melacola. And then what was subsequently shown is that if you just monocolonize with Williamia and then give house dustmite that gut colonization by wimmia is sufficient to

enhance an airway eosinophilic response.

And so our sort of starting framework then is trying to understand not these distal effects per se but how are the fungi initially being sex sensed in the gut in order to initiate type 2

inflammation.

And so some of this then comes to this paradigm now of epithelial alarmin production as the uh upstream driver of type 2 initiation. And so what's appreciated is the response to allergen

challenge epithelial cells make a trio of innate type 2 cytoines. One of the classic ones being isle 33 also tslp and

then the newer player is 25. Each of

these three will signal to resident lymphosytes or myoid cells in the tissue and this serves to initiate type two responses.

One thing that we were sort of thinking about on these alarm side is you know how do you get this cross talk between the gut and the lung particularly because these resident lymphosytes like innate lymphoid cells are thought to be

non-resirculatory and there was this paper from Ron Germaine's group uh uh in 2018 where they had confirmed that at homeostasis so if you stain for ILC2s here in red uh

in an unchallenged mouse they stay in the tissue they don't exchange between parabions and you see they very rarely show up in these cyan uh villis lactals which are the draining lymphatics.

But upon injection of isle 25 one of these epithelial alarms you see uh ILC2 expansion and then they start showing up in these lactals where they eress from the tissue and then if you look by

flowcytometry in the blood normally they're almost non-existent in the blood because they're non-recirculatory but after 25 challenge they start showing up in the blood and they also start showing

up in distal organs like the lung and so this idea of epithelial alarm production might be a mechanism whereby you now drive systemic recirculation of lymphosytes that are normally tissue

resident and that can enhance distal airway responses and that's actually uh been followed up by a very recent paper where they showed that if you colonize with titriconous murus in the gut this

drives recirculation of C2s and that effect has an enhancement on housemite asthma in the lung but again for sort of the questions we were thinking about then if if 25 can have this

functionality well who are the cells actually making 25 and how does that work? And this kind of comes to some

work? And this kind of comes to some really interesting new epithelial biology that's emerged over the last decade which has found that uh tough cells this very uh rare and and

interesting cell type in the intestine are the major source of isle 25 in vivo.

So there were three papers that came out about 10 years ago that all found this.

This is the data from Rich Lockxley's paper where they made an IS25 reporter mouse and found that isle 25 reporter staining here in red is very rare in the intestinal epithelium and very

selectively shows up as co-staining with DCLK1 positive cells and not any other cell as part of the secrettory lineage and DCLK1 is known as this very specific

marker of tough cells.

These are very underststudied cells for for most of their uh uh lifetime that they've been known by by humans. So they

were actually first identified in the 1950s. And you can see from these uh you

1950s. And you can see from these uh you know microraphs why they're called this.

They extend these the projectiles out into the lumen that are highly enriched for sensory GPCRs and they sample the lumen of the gut looking for sensory inputs that drives their deolarization.

These cells are very uh related to taste cells in the tongue.

The reason why they are considered to be cousins of taste cells is that they share expression of this sensory G-proin subunit called gustoucusen that couples to groin coupled receptors that sample

the lumen of the gut. And so one of the best examples then is that susenate a metabolite will activate this GPCR sustenate receptor on the surface of the tough cell. This drives depolarization

tough cell. This drives depolarization of the tough cell through gustoucusen that ultimately results in secretion of 25. And if we back out to the tissue

25. And if we back out to the tissue level analysis of this, what is essentially is now argued as a summary of the literature is that protozoans so

particularly tryonus secrete extracellular sustenate. This is

detected by the tough cell to drive 25 production. This signals to these local

production. This signals to these local ILC2s that then start secretreting 13 and 5. Those cytoines have pleotropic

and 5. Those cytoines have pleotropic targets. But one really interesting

targets. But one really interesting outcome is that 13 will signal back to the LGR5 positive epithelial stem cells to ramp up further tough cell differentiation. So this sort of creates

differentiation. So this sort of creates a feed forward loop whereby tough cell activation turns on ILC's that then further expands tough cells to enhance functionality.

And so one of the things we were interested then in is you know if we think about these paradigms that are known for for fungal sensing uh you know the the the a lot of the work that's been done in sort of beautiful classic

studies by Gordon Brown and many many others is the idea that cell wall sugar detection by cype lectins will turn on a number of cytoines that can drive type one or three inflammation. It's also

clear from a number of studies including some recent work looking at alteria pore forforming toxins as well as fungal proteiases you know work from Ilon has done this as well as Darren Weisner when he was in Bruce Klein's lab and and many

other groups have shown that damage uh of the epithelium by proteasis or toxins can result in alarmin production that can depending on the context drive either type three or type two

inflammation. But our starting point

inflammation. But our starting point here was well there's really nothing known about whether or not fungi have interfaces with tough cell activation.

And so does this circuit apply in the context of fungal sensing. And so given this feed forward loop that's known to exist, we started with a really simple question which is that if we use as a

model since this is known to drive uh these interorgan effects, cania colonization drive tough cell expansion or activation in in mice? And so here we're we're colonizing uh animals and

looking in the small intestine to start since this is where most of the tough cell literature has been done. So you

can see if we stain for DCLK1 there's these rare tough cells in in the intestinal epithelium. And when we

intestinal epithelium. And when we looked in the small intestine which again is where most of the parasite literature is looked we didn't see anything. We were initially a little bit

anything. We were initially a little bit disappointed, but we continued and scanned along the intestinal tract and one thing that we've noticed that comes up very reproducibly is that we actually

get this surprising specific tough cell expansion upon colonization in the colon. And we do think part of that is

colon. And we do think part of that is that it seems to more preferentially colonize in that location. But this is surprising for a reason that I'll I'll circle back to.

We then also made uh tissue extracts from the colons to look at cytoine production. We can see when we colonize

production. We can see when we colonize we get local induction of type 2 cytoines that are usually ILC2 derived like IL5 and I13.

If we then wanted to ask are these actually tough cell dependent and so it's been known from a number of these differentiation studies uh of of intestinal epithelial development that

PU2F3 is the master transcription factor for tough cell differentiation. So what

we've done here is now uh do oral gavage withia into either wild type littermate controls or p2f3 knockout mice which are tough cell deficient. And when we do that we get not not a complete in the

case of 5 but we get a a a measurable reduction in 5 production when we get rid of tough cells and then we actually get a more complete reduction of 13. So

we do think that there is functional tough cell contributions to this local type two cytoine induction.

Now I'd sort of mentioned that this colon observation is surprising and and the reason why is that if you circle back to this model of a feed forward

loop whereby tough cells activate ILC2s and then that ramps up more tough cell differentiation.

There was a paper a follow-up paper from Rich Lockxley's lab where they asked what happens if we just hyperactivate ILC2s by deleting this negative regulator A20. And when they did that,

regulator A20. And when they did that, so you now make a hyperactivated ILC2.

You if you look in the controls, there's very few tough cells. If you look in those hyperactivated mice, you get tough cell hyperlasia in the small intestine.

But when they looked across other tissues, you actually don't see that effect happening in the large intestine like the colon. And follow-up studies that they've done, if you inject 4 or 13

systemically, you again get small intestinal expansion, but not colonic expansion. So there's been this question

expansion. So there's been this question about how much this feed forward loop even applies in in the in the colon and whether or not type two cytoines are sufficient or even necessary to do this.

So we asked then given that we're seeing this sort of rare phenomenon of colonic tough cell expansion in in this context is it stat 6 dependent or dependent on

type 2 cytoines and so is 4 and 13 need to signal through the transcription factor stat 6 to to drive this phenotype. So we asked what happens if

phenotype. So we asked what happens if we get rid of stat 6 in the system and when we do this uh comparing stat 6 knockup mice we don't see any loss of this tough cell expansion. So if we

compare back to again wild type litmate controls they see this you get this colonic tough cell hyperplasia and we don't see any difference when we delete stat 6. So this is acting via a very

stat 6. So this is acting via a very different circuit than what's been described in the small intestine.

So what we're seeing then is that when we colonize with this fungus, we're getting colonic tough cell hyperplasia that's stat 6 independent. So it's a non-cononical expansion circuit. And so

obviously the question is well what's driving this? And uh the hypothesis that

driving this? And uh the hypothesis that we had was rather than using an epithelial immune expansion circuit, couldia be secretreting something that's directly influencing stem cell

differentiation.

So to get at this, we we've moved into organoid culture systems where we can grow crypts. And so now look at the

grow crypts. And so now look at the effect of exogenous molecules in the absence of immune cells or neurons. And

using that system we can use positive controls. So add in type two cytoines

controls. So add in type two cytoines which are known to give you tough cell expansion and an organoid culture. And

then take fungal supernatance and ask what we see. So what we've done here now is make a organoid culture from the mouse with uh just control. And you can see if we stain for DCLK1 there's almost

no tough cells in just a normal organoid. If we do a positive control

organoid. If we do a positive control and treat these with IL4, you get tough cell expansion in the organoid culture.

If we then just use diluted uh uh yeast culture media as a control, that again doesn't do anything. But if we take conditioned supernatants from grown fungus, those are actually sufficient to

drive this tough cell differentiation in the organoid culture. And since we'd seen in vivo that this effect was stat 6 uh independent, we asked whether or not the same thing was happening in this

system. So we've grown stat 6 knockout

system. So we've grown stat 6 knockout organoid cultures. If we uh again give

organoid cultures. If we uh again give PBS, we see nothing. Now if we give is 4, which should require stat 6, we don't see tough cell expansion. And then if we give condition uh non-conditioned media,

we see a few tough cells. But these

tough cell hyperplasia is maintained in a stat 6 knockout when we give conditioned supernance from the fungus.

So what we're seeing then is sort of again a mimic of the invivo scenario but what we found is that there might be a bioactive molecule that's directly inducing tough cell differentiation which leads to the obvious question of

well what is the nature of of this secreted activity and so you know you can go through the list of uh different macroolelecules uh but we started off asking is it maybe proteinatious and I'll say we got a little bit lucky here

we'd initially seen that the activity was heat sensitive and then also we found that if we uh treat these supernants with protein a coupled aggro speeds that that reduces a lot of this

tough cell induction capacity.

If we uh look at silver stains off the supernatance of these grown cultures, you can see that there's a number of uh proteins that are released into the media. Um you know, depending on who you

media. Um you know, depending on who you ask, this is either a lot or a little.

We were at least just happy to see that it wasn't a smear, which gave us some confidence that we could maybe try to purify it. And so we started off then

purify it. And so we started off then with an old school technique which is using ammonium sulfate precipitation cuts uh to basically ask where we can crash out activity and resolubilize it.

And so I'm not showing the bioassays here but we've done 40 to 50% 50 to 60 and 60 to 70% cuts. We don't see any activity in 40 to 50 but we maintain it

in the higher concentrations. So we used those uh sort of uh that that cut range as an initial purification scheme. And

so what we were hoping to do then is use a biochemical approach to see if we could identify that the protein coming from these supernants. So the approach in general is to take 40 to 50% ammonium

sulfate precipitates, resolubilize those pellets, dialize and then run on size exclusion columns, take the illusion fractions off the column, put them onto organoids, measure tough cell induction

capacity, and if we can get enough specific activity, try to identify the protein by mass spec. And so what I'm showing you now are just uh the the illusion fraction uh analyses off the column. We we we didn't do every single

column. We we we didn't do every single one because uh Hungu the postto would have killed me but we at least did uh uh you know sequential five fractions off the column. And so what you can see here

the column. And so what you can see here then is this I'm not showing you every fraction off the column but if you look here uh 36 through 40 have no tough cell induction capacity. the next set of

induction capacity. the next set of fractions now activity starts coming on that's maintained or even more so in 47 through 52 and then in the next set that activity drops away and there's actually

the same amount of protein across all these fractions. So we're at least

these fractions. So we're at least getting some specific uh uh uh uh localizations off the column where we're seeing selective tough cell induction capacity.

So as a starting point then we we basically have taken the schematic where we you know we've found that the activity is proteinatious. We've done

sort of an initial, you know, semi crude system with ammonium sulfate cuts followed by sizing. And then we basically just took these two active pools versus two inactive pools for mass

spec. And then additionally, we ran

spec. And then additionally, we ran silver stains off of these fractions from the column. And again, we got a little lucky here. But what we find is that in the two active pools, so 41

through 46 and 4752, there was this one band that came screaming high in the two active fractions. So we additionally cut

active fractions. So we additionally cut that out and did mass spec on that band.

And when we cross compared the the pulled mass spec to this band and then also filtered based off of proteins that have a signal peptide and in a certain size range that we knew from some cutoff

filter experiments. We basically got

filter experiments. We basically got this one protein that's called I4 Y5L4 which is a 39 kilodolton protein with a signal peptide.

So from there we wanted to see if this protein is sufficient to induce tough cells in organoid cultures. So we cloned the uh cDNA put it into a mamalian expression vector with an IL2 signal

peptide and a histag uh expressed this and then uh enriched for using nickel na aguros. And if we then take the

aguros. And if we then take the recompant protein and put on organoid cultures we find that that is sufficient to induce tough cell induction in the organoid. So we do think this is the the

organoid. So we do think this is the the the likely candidate from there. So

obviously you know what everyone's going to ask is what what's the predicted function of I4 Y5 L4. Uh and this this wasn't that difficult. It actually comes up right away on Unipro when you look at

the uh when you look at the protein and it it encodes a predicted secreted asparagenase which was surprising to us.

Um but asparagenases have been well studied particularly E.coli coli derived versions and what they're known to do is catalyze the conversion of lsparagene

into aspartic acid and ammonia. Uh this

has been well studied because of its uh use as a chemotherapeutic for acute lympositic leukemia. Um and so people

lympositic leukemia. Um and so people have done mutagenesis across the ecoli derived version which is used therapeutically and it has this well-escribed active site uh that requires these two thrienines here. If

you mutate those to alanines, you kill catalytic activity. And you can see that

catalytic activity. And you can see that motif is highly conserved in this uh willmia derived protein.

So we wanted to then ask is this a selective functionality of these fungal derived family of proteins or can we see a similar phenomenon with an asparagenase coming coming from

anywhere? And so because the ecoli

anywhere? And so because the ecoli version is commercially available, we just purchase this and ask what we see.

And so what we've done here again is make an organoid culture treat with control and then here now is treating with the ecoli derived sparagnase. And

you can see we again get this beautiful tough cell hyperlasia coming up in the organoid culture. We then wanted to ask

organoid culture. We then wanted to ask questions about whether this is potentially due to substrate depletion or production of a product. And so what we've done now is either add back in a

spartate alone or tried to rescue the asparagenase treatment with supplemental asparagene. And what we see is that when

asparagene. And what we see is that when we spike back in asparagene into these cultures that has this rescue effect on the action of asparagenase whereas if we put in a spartate on its own that has no

effect. So we think preliminarily that

effect. So we think preliminarily that this is due to asparagene depletion.

To get at that a bit more specifically, what we've uh done now is actually take the organoid culture media and reconstitute it from powder that doesn't have amino acids, which then lets us

titrate the amount of asparagene in there. And what we found is that if we

there. And what we found is that if we titrate the amount of asparagene in the organoid culture, that if we bring this down to lower concentrations, that that is again sufficient to turn on this

induction program.

So uh what I want to just sort of back out then and and and talk about with these uh uh way we're thinking about this data is you know I mentioned earlier in the talk that there have been

these traditional modalities of fungal detection. The classic one being shell

detection. The classic one being shell sh cell wall sugar detection through cype lectins. This turns on a number of

cype lectins. This turns on a number of inflammatory cytoines that will initiate type one or three inflammation. There's

also been this paradigm of protease or performing toxin that essentially causes damage responses in epithelium and this results in release of alarmins that can

turn on type 2. In the case of parasites in tough cells, the argument has been that active metabolite production so the classic example being susinate is detected by tough cells to cause

depolarization and initiate type 2 responses. What we're positing initially

responses. What we're positing initially is that there's sort of a a new modality here which is not active metabolite production but potentially metabolite or nutrient and in this case amino acid

depletion might be a cue that says that there's something there that we don't want because it's competing away nutrients and this acts as a trigger of tough cell expansion and activation that turns on the weep and sweep response.

And some of the things that we're interested in understanding now are how is this actually sensed from a cell biological perspective? There's not a

biological perspective? There's not a ton known about sparagene sensing, although some of the pathways that are out there argue for uh GCN2 based detection that can shut off mtorque one.

We're exploring uh those sort of arms. There's also potential roles for amino acid transporters or we don't want to hang our hat on it but you know given a lot of the uh beautiful work you know

controversial but from David Sabatini with specific amino acid sensors like the cestrin or castor pathways that all coupled to tour perhaps there are unknown asparagene sensors that are

coupling into these pathways so um our model then is we're thinking about nutrient depletion as a mechanism to sense fungi that sort of acts in parallel to this active metabolite

sensing pathway that occurs for parasites and some of the ongoing efforts that we have now are trying to generate asparagenase deficient fungal strains in collaboration with Rob Kramer

at Dartmouth to test whether this can uh is necessary uh in vivo. Uh we're also asking whether or not uh some of these pathways can uh are required for that distal airway enhancement effect. And

then we're also really interested in asking questions about how specific this is to asparagene itself versus and it just so happens that fungi make an asparagenase but perhaps depletion of any given amino acid could drive a

similar effect. And so these are some of

similar effect. And so these are some of the broader questions that we're trying to follow up on now in in active ongoing studies.

So uh with that I want to wrap up uh and thank my lab which has sort of uh you know been an interesting roller coaster of the LA roller coaster over the last year dealing with a transition from the NIH up to Boston. I want to make a

particular shout out to Hong Yu Wu a very talented posttock in the lab who's really single-handedly pioneered this whole project which is a really new interesting area for the lab. I want to thank all my colleagues back at the NIH

as well as people that I'm getting to know here at the Reagan. Uh, and with that I'll wrap up and I'm happy to answer questions in the chat and then I'll also follow up the Q&A at the end.

Thanks so much.

>> Thank you Dr. Deng. That's great. Um, so

it's my uh pleasure to introduce my uh colleague and and second speaker of the micro talk session that's Dr. Hong Nin from University of Pittsburgh. Hong, I

think you can go ahead and bring up your camera. Perfect. Um, so Dr. Nwin is a

camera. Perfect. Um, so Dr. Nwin is a tenure professor of medicine at the University of Pittsburgh and uh co-directs the center for healthcare micology and fungal genomics uh and also

serves as director of the uh transplant infectious disease um program uh as well as the XCR pathogen laboratory at University of Pittsburgh Medical Center.

Um so Dr. Nin has a long-standing interest in both fungal and also multi-drugresistant bacterial uh infections um including mechanisms of antifungal drug resistance and fungal

diagnostics um and uh uh she's recently applied a long reach sequencing using the Oxford anapore uh technology to um

to understand um uh isolates that that uh are capable isolates of Canada that are capable of causing bloodstream uh infection and so her uh uh title of her

talk today is within host genotypic and phenotypic diversity of Canada causing bloodstream infections. Um thanks so

bloodstream infections. Um thanks so much uh uh Dr. Nuin for uh uh giving us a seminar today.

>> Thank you Dr. Co for the uh introduction and thank you um uh the committee for uh inviting me uh to give the talk today.

Can you see my slide?

>> Yeah, just need to go to um swap. Yeah.

>> Okay.

Hey, is it okay now?

Okay, great. Good afternoon everyone.

So, um the long-standing belief is that most uh bloodstream infection and steroid infections stem from a single organism that mean that there is an

extend expansion of the single strain that successfully pass through the bottleneck as I shown here. This single

uh organism hypothesis actually has shaped both the diagnostic workflow and clinical decision m for uh for over 10

decades now.

Um however uh there have been uh uh uh some problem because many uh uh microbiology lab actually all of the

common practice of the micro the clinical microbiology lab is typically to select a single representative colony

from the uh plate uh for uh uh identification and antifungal susceptibility testing. The clinician

susceptibility testing. The clinician then in turn based their therapeutic decisions on the characteristic of this single presume representative isolate.

Uh over the past decade or so there have been emerging evidence that challenges this longstanding uh uh hypothesis.

In recent study we have shown that blood culture on patient with candida bloodstream infection can harbor distinct strength. They are clon but

distinct strength. They are clon but genotypically and phenotypically distinct. Importantly, some of these

distinct. Importantly, some of these strain may differ in antifungal susceptibility and yet missed by the clinical microbiology lab. Data to date

suggests that a single colony may not adequately capture the true complexity of the infection carrying major implication on the for the diagnosis

treatment and our understanding of uh candida pathogenesis.

So uh my hypothesis for following this type of the study are number one positive blood culture are comprised not with a single train but a mixed

population of genotypically and phenotypically distinct microorganisms. This is particularly true and relevant

for GI commens like a candida. Second,

the antifungal treatment failure can sometime be explained by the emergence or expansion of an already existent in the blood uh of the varian sh with

diminish antifungal susceptibility and the rationale behind these hypothesis are very straightforward.

One is that the single colony or lineage is not representative of the entire population of the much broader microbial

community. And then the genetic and

community. And then the genetic and phenotypic diversity is very well recognized for over a decade at site of

colonization or chronic infection like a patient with citic fibrosis colonizing with uh pseudomonus when the patient can

be uh infected with sephon but antibiotic uh uh susceptibility difference and also for microacterium tuberculosis.

And lastly once the candida enter the bloodstream from wherever the site they come from it is exposed to the immune defense and sometime antifungal therapy.

This pressure can drive a rapid selection and expansion of adaptive varants within resulting in a mixed population with differences in virulence

stress tolerance and antifungal susceptibility through testing.

So uh before I present our new data I want to briefly highlight the two recent uh publication from our group that laid the groundwork for the current

presentation.

So uh I start out with candida ambigans.

It was uh recently uh um uh uh uh uh published in the general infectious disease and the work is driven mainly by

uh one of our research faculty uh Dr. Hassan Patrin. So the method is very

Hassan Patrin. So the method is very simple. We obtain the blood culture from

simple. We obtain the blood culture from patient with uh candidmia. We squeeze it on the blood ager plate and also uh uh

chrome ager uh plate and pick uh uh uh uh up to 96 colonies and save it for future uh whole genome sequence. So you

know for candida we selected sample from four distinct patients as short here.

Three of these patient had persistent candidmia ranging from 3 days to 10 and 13 days respectively. One patient just

have a short uh uh uh run of candidmia left for only one day. For this patient with a single blood culture we obtain 10

sw and uh uh and subject them to honome sequence. The other three patients we

sequence. The other three patients we did five strain from the ignition blood culture uh and then we go to the last uh

positive blood culture and uh and tested the the the five colonies on there. All

of these undergo uh underwent the illuminina and one single isolate that we obtained from the microlab that was tested by for susceptibility also uh

underwent long group sequencing.

So long story short here uh the strain from individ patients are colorcoded here. You can see that uh you know this

here. You can see that uh you know this train cluster together within the patient but are far distant from the other patient just telling us that we

didn't did not have an outbreak going on at our hospital. When we look at the small scale gener genome variant it it

is uh the the uh the finding was un uh was not very impressive. The amount of snip and indel range anywhere between

zero to 42. None of these mutation associated with antifungal resistance.

However, when we look at the large uh large scale genome by looking at Y map here, you can see that uh we see an

employee we see uh loss of halo zygosity and also gene copy number variant. For

the uh purpose of of this review, I just mention uh briefly about one patient which is uh patient MN. The MRI uh came from the ignition blood culture and the

NRA came from the blood culture isolated 13 day later on uh various antifungal therapy. So looking at the Y map uh here

therapy. So looking at the Y map uh here you can see that there is a adoploy at chromosome 7. So for the first set of

chromosome 7. So for the first set of blood culture four out of five had the uh uh tricommy in the uh chromosome 7 or

the uh follow up uh blood culture under persistent blood culture we no longer see this kind of tricom. So whether we

look at the phenotype, the tricom lost the uh uh uh ability to form hy on the hyphold in uh induction plate and it

also has less uh bofilm formation than the uboid uh that that uh mainly recover after the therapy.

So we further did a phenotype by testing uh a competitive uh infection of the tricom and the u uh uplo stren. We use

incubation in human blood xvivo. We also

test in the mouse GI colonization by testing stone and also invasive in the GI mucosa as well as the uh disseminated

model of candidasis. And what we show was that all o over all of these testing the tricom strain are more fit in this

condition. And although the stren did

condition. And although the stren did not make much uh high fee in the uh uh in vitro in vivo in the murine kidney model here we did see some high fee but

it's much less than uh what was seen with the yuplo shred.

Then we look at for the susceptibility all of these uh Tricom and you approach train of own susceptibone to achinandin

with very similar echininoand mic.

However, when we look at the tolerance, tolerance is defined as the ability for the fungus to grow on antifung. But when

we test the uh output uh mi of the organism output, the mic remain the same in the susceptible range. Then we found that the upuplo actually were more

tolerant to aot canine tested. We used

the um blood plate used the refraction of a growth assay and supra m assay to uh evaluate the tolerant uh like uh uh

presented by uh Dr. Burman's lab. So you

know in vitro at least the upuploid are more tolerant uh to the aocandon.

Then what happened in mice? So uh we treated the mice uh with the makeup fungen and then uh uh uh we could go

treated with um uh both theloid and the uh and the tricom strain. What we saw here was that without mikafin the uh uh

tricom is more fit like shown here but with the echinoand then the pro swen become more tolerant and more fit in the

mice treated with mafen.

So let's go back to the patient map.

Then day zero is when the first blood cone uh uh uh was obtained and became positive and then we had subsequent blood culture up to day 13. What you can

see here on the original uh blood coneure over two twoird of the uh of the blood cone were do by the fit uh tricom

7 shrine one day and then 3 days after function uh therapy the tricom just uh almost totally disappear down to about

seven to and 2% respectively and then now the blood cone is a one of the uh um

Uloid organism and it this scenario uh validate our invitual study is that upuploid became more tolerant with gasping exposure and just persisted in

blood.

So in conclusion then what we saw was that within a a single culture of the patient we saw different phenotype and

also uh uh um uh uh genotype differences and echininoand intolerance may account for at least some of the treatment failure in uh our patient.

So for the second paper it was uh published about uh a year and a half ago uh in uh uh nature communication and we did very similar study but we

concentrate on candidaca and you know in this study we used 10 different patients and you know the finding was very similar except for the

following um um uh that you know in Canada we did not exhibit the large scale genome uh uh genomic changes like

anubloy like we uh we saw in Canada and began instead what we found was that within host diversity it was driven by small scale variant for snip and indel

that correlated with antifung resistance. So out of these two uh of

resistance. So out of these two uh of the 10 patient two fail gluconosone therapy and then uh on the subsequent blood conure we obtain fluconosone

resistant. Then we went back to the

resistant. Then we went back to the ignition uh blood culture and we found a small uh percentage of colony of fluconosone resistant colony that were

already uh resistant there and and the uh anti the mechanism of antifung resistance or mutation within the 011

gene in one patient and then uh the elux uh pdf1 and md1 in the other patient. So

together then this uh uh uh result challenge the current single organism hypothesis. If validated then the

hypothesis. If validated then the finding suggests that both the clinical and the microlab practice may need to consider for a microbial population and

it may change the pathogenesis of invasive candidasis.

to share with you. As you know here you as you all know Canada or is an emerging uh multitrack resistant pathogen unlike a candida ambigance cla and other

candida species except for Canada parapsilosis these tend to be an exogenous rather than the GI commen and

so on. So it it it prim primarily the

so on. So it it it prim primarily the skin colonizer and this is the uh uh the setting for multiple outbreaks uh

throughout the world and you know uh the outbreak thus far tell us that most of these Canada always generally clon

however for the past two years they have been increasing in small report that showed within host diversity uh uh So you know people found mix so

different plate uh in single patient and one of the patient actually had snip variant uh uh over time and the snip difference and indel are more isolate uh

from the same patient and then in one patient resistant developed to uh take aidocandin and ephotinb and the

resistance were found to be uh due to uh mutation uh in the minor sub subop population.

So, uh I'm going to show you data on uh two different uh uh cases that uh we saw the same the the first uh patient is a

Canada or panchimmia from a patient that was transferred to our hospital from Nevada and we all know that Nevada have had an outbreak of a clay 3 candida or

he came in and then developed a bloodstream infection. So uh we put the

bloodstream infection. So uh we put the line uh the patient did just well responded to the echinoand therapy and

survive because of the clonal nature of the candida or uh we decided just to pick the one randomly chosen shin uh the

the morphology is different. So we pick as many different morphology as we can and all of these underwent along with P

sequencing. So long story short, you can

sequencing. So long story short, you can see here all of these uh uh strength from the patient are own uh uh uh very

very uh genomically related and related to the uh clay 3. However, in three out of these 41 uh 41 trends, we found a

possibone circular chromosome that were we are still uh working on it to uh uh to prove and also in six out of these 40

uh one train they had uh chromosome 3 anuplo and the anuplo was confirmed by uh uh five different gene that are

located in chromosome 3 and then you know uh for the phenotype we saw that the uh the diploid candida oris had much

less uh uh uh uh aggregation compared with the uh uh uh uploid and also has

much less um uh adherence uh using the PMI sweats medium and also in co collaboration with Janet Net in

Wisconsin we also did the model uh in skin uh adherence and the same pattern that was was uh true uh in all three models.

The case two is more complicated and uh very interesting to me. Uh this is a case of candida or chest infection after

lung transplant. So uh uh at the

lung transplant. So uh uh at the transplant we routinely uh did the BAL uh uh uh so you know it can target the

anti antibacterial antifung with laxis and we found by surprise that the bl was colonized with Canada or two days later

the patient developed infected a chest hematoma that it turned out to be a big deal because the patient subsequently developed a persistent infection that uh

uh involve the entire mediastinum and cause a mediastinitis.

So we got we we have sequence uh the the strain that were colonized in the pal and uh and the the two disease

associated stren but for today talk I just going to present to you the data that we have from the uh b itself. So

this work uh was done by Shiai Chang from our lab. What Shiaoi did was he did the whole genome sequence. This time uh

uh we did illuminina uh uh and then uh uh we did a longwidth sequencing for one of the index train and as you can see

here all of these shrin were clon very very closely related genomically and it's also related to the plate one uh uh

uh shrin that we obtained from the CDC.

So you know they are very close known.

Then uh you know you using white map uh on the entire 20 trends. We found only one that are totally different than the other one and and the founding the

finding is when we first look at it it looked like a disease an employee. But

the headache headache stopped because when we look at the uh number of copy uh uh uh uh variant here actually these uh

uh what's so called an body actually is not because uh the copy number is non integer it less than two. So it's between 1.3 to

1.5. When we saw this the first question

1.5. When we saw this the first question came to our mind is that are we dealing with the mixed population of candida or in a single strain which we call A1

here.

So you know uh before we do everything further we check for co rate and you know the A1 again A1 is we think that there is a a mixture of a lot of

different stren in there and A2 which seem to us that it's only uh one strain the growth rate is the same however when we carry uh the uh the lifespan up to

about 40 days then you know the uh the S1 the one with the uh unemployed like on web map actually uh uh had less

uh growth benefit than the the S2.

So to look at the uh uh uh possibility of mix colony then we spread the A1 uh which has a the copy number uh variant

compared with A2 and you know we waited for 4 days and you know so it's different to tell but you can see from here there are larger colonies along

with the smaller colonies and the larger colony here are different and larger than what we see here in A2 and A2 look

like it's a homogeneous uh lawn of of uh similar colonies. Then we did again but

similar colonies. Then we did again but this time we stopped the incubation at two days and it became very clear they are small and large colony here. So you

know we try to isolate the large colony and the small colony uh to have a single uh colony so we can do further phenotype.

However, we've we spread it for five five different uh passage. We continue

to see uh a mixed uh uh phenotype. So

from the large colony, we see the majority of large colony but you can see here here a smaller colony and in the small colony we have a large colony as

well and despite the five uh uh uh passages. So we get naked large to large

passages. So we get naked large to large and we get large to continue to spread we continue have to have the mixed holiday. So the question that we ask is

holiday. So the question that we ask is and then you know when we look at these uh so the question we ask is it switching

phenomenon that we are seeing. So what

we see here is in the small. So this is we pass it twice. We can you can see here compared with the large colony the

small colony has a very irregular uh border compared with the large colony that it's mainly smooth order.

So to look for the uh uh switching we put this isolate on the fluxin uh B plate and you can see the large the A2

is Millie white and then the A1 is a pink then you know we put more colonies on that and then now it's clear that we are dealing truly with the switching uh

uh from the uh the colonies that we obtained in vivo. So in the large big is that mean larger colony uh it it's uh and you know in our hand at day seven

all the colony look pink. So you know these are smooth colony but on the law uh on the uh A1 from the uh after tooth

passage you see small you see large pink and we also see uh uh white pink and on top of that we have the sector uh uh

morphology that we see both pink and and and uh and white. Then uh you know from the small colony uh we look under the

microscoscope and the small colony actually give us the high the larger cell size than the

large colony on the plate. It give us a smaller cell size and this is the A2 and in both of these uh uh uh microscope

slide you can see a mixture of large and small as well. So you know since uh uh large colony has been linked to ploy uh

differences the next question we ask are we dealing with gloy difference.

So you know uh we get these isolate that we obtain from day eight day 30 and day 50 and and we subjected to illuminina uh

sequencing. As you can see here,

sequencing. As you can see here, whatever the uh copy number variant here that we saw in S1, it disappear. But

whatever on the white side of the chromosome, it possess and it seem like from day eight forward, we have a stable

uh organism that you know the genotype hasn't changed. So we look for uh

hasn't changed. So we look for uh deploy. So we use two shrine, one from

deploy. So we use two shrine, one from the CDC given to us by the CDC and and and the other one uh by uh by our

colleague uh uh Robert um I forgot his last name. I'm so sorry. uh but you know

last name. I'm so sorry. uh but you know as you we can hear the for the for the deployed then you see two peak one is at the 1 C

peak and the 2C peak and it's about 9K at the location of about uh 19 uh K here

and for the uh tetraloid the the the uh the flow sure shift uh uh uh to the right and it's about 20K so it's the

bone of the 10K and 40K which is bone of the 20k. So what happened to our isolet

the 20k. So what happened to our isolet looking at A2 which is look uh all the chromosome look the same. So as you can

see here there is a peak at five a peak at at about eight. So this is half of what the deploy that I show you from here. So this is a hloid type of stren

here. So this is a hloid type of stren then we look at the the T8. This is the stable uh uh strength that we got after

multip 8 day uh uh um uh passage. You

can see that uh the two peak at around 10 and 20. So this for sure is the deploy and the A1 is somewhere between

uh the uh uh hloid and the deployed and you know you can see a smooth peak here uh of of the uh C1 and then you know the

the the larger peak later. So we think that the A1 uh based on the flow is a true mix of the uh organism and then we

run some uh susceptibility to make function. the A2 is more uh resistant uh

function. the A2 is more uh resistant uh than the A1 and you know whether it's large or small it more susceptible than

the A2 and these uh uh uh uh bloody differences show that you know it has less bio formation than uh for the uh

regular hoid A2. So in conclusion, candida or race just like albbeans and glabrada exhibit uh genetic and phenotypic diversity with the within the

clinical culture. In patient one we saw

clinical culture. In patient one we saw chromosome 3 anuplo either fragment fragmented segmented or their own chromosome 3. Patient two, we saw a

chromosome 3. Patient two, we saw a mixed cell population of at least two carotype that are distinct uh and you know it associated with phenotypic

switching and differences in morphology, lifespan and bioformation.

So uh altogether then positive culture may be comprised mixed population of genetically and phenotypically Canada species. Type of the primant prominent

species. Type of the primant prominent predominant genetic diversity at least in our study may differ between Canada species. In Canada and beans we saw

species. In Canada and beans we saw mainly anes L and large scale genome variant. Candida Claprada we saw mainly

variant. Candida Claprada we saw mainly snip and indel and candida or we saw switch phenotype and ployy differences.

Thank you very much for your attention and I would like to thank uh my lab who had did uh have have done all the work that I present today. So uh thank you

again for your attention.

All right, thank you. Thank you for the fantastic presentation. Uh, and now uh

fantastic presentation. Uh, and now uh it's the most fun time here uh Q&A. Uh,

so the way we going to do that, I will start with uh, uh, questions to Eric Dank and I will be combining questions that are in the same direction. So maybe

sometimes we have two questions answer in one shot. Uh and uh please for all the participants please add your questions to the Q&A. Uh so I think

we'll alternate with uh Jay after that.

So uh the first question to Eric. So

many people actually are complimenting you both for the talk and for your new position. But here uh you can see that

position. But here uh you can see that in the chat who is saying what. But we

will go specifically about the questions now. So uh couple of questions are uh

now. So uh couple of questions are uh very similar. So people are very

very similar. So people are very interested because she saw actually uh as perase in E.coli. So the questions are you know how conserved that is a

cross fungal species is one of the questions. The other one is more uh you

questions. The other one is more uh you know if you look at species similar to Wimmia what is the situation there? So

uh the cons how conserv is the phenomena.

>> Yeah. So, so asparagenases are expressed by almost all fungi. The the question is more whether or not they have one in the genome that has a signal peptide or not.

So, in in general molds, so you know most aspiggilis species, penicyium, fuzarium etc. etc. clasporium they all have signal peptide containing asparagenases. So, they tend to secrete

asparagenases. So, they tend to secrete quite a bit of activity. And this has been scanned a lot because because of this use as a cancer drug, people have been looking for different sources of asparagenases for a while. Um what I can

say is we've we've done some preliminary experiments taking soups from non-walemia species like certain aspergillis strains uh and we can see the same phenomenon happening there. Um

now in terms of global patterns about which ones are allergenic versus not I don't want to you know overstate that per se because there are other mechanisms to trigger things but there is this general pattern that molds which

we do know tend to be very type two triggering do have very conserved expression of these asparagenasis that are secreted.

>> Yeah. Okay. Very nice. Jay do you want to go or I >> Yeah. Uh yes, we have a a a question in

>> Yeah. Uh yes, we have a a a question in the the Q&A uh for uh Hong. Uh so the the question revolves around um since

these are clinical isolates um what what do you think the the role of you know prior fungal antifungal exposure is u or actually imological pressure although I

think a lot of these patients are probably um you know transplant uh recipients. So what do you think is for

recipients. So what do you think is for regulating this anloid state uh uh you know in vivo?

>> So for the anoploy in uh in Canada and beans. So our lab we started with Canada

beans. So our lab we started with Canada uh uh drugs and we saw all of these resistant uh uh popping out in the

ignition conure. So for candida ambigans

ignition conure. So for candida ambigans and candida always we specifically specifically pick the uh the the sample

from the patient without anticonent anti-fung for uh for the past three years. So you know the annubility that

years. So you know the annubility that we saw in Canada and began and in Canada always from uh people that have not been treated with antifungals.

>> Great. Thank you.

Yeah. So I will go with the next question which is actually linked to what Eric to what you actually started discussing here. So there is a

discussing here. So there is a suggestion that uh the spurge aspen circuit uh uh being co-opted as a way to

accommodate the fungus by a way of real estate expansion instead of pathogen clearance. Have you talked about that in

clearance. Have you talked about that in uh uh do you see overall enlargement of the column in >> so the enlargements are so we I'm trying

to think if we've actually measured it we haven't noticed that overtly right um you in terms of co-option you know it's always the debate about you know is it

micro driven or host driven um I mean I would I'm biased here but I I think because of the conservation across most species even ones that aren't

necessarily neessarily mamlon adapted.

Uh I I think this is more of a more ancient pathway for fungi. Probably

these asparagenesis seem to be very upregulated by nitrogen starvation and so it's a nitrogen scavenging mechanism.

Um that doesn't mean that it's doesn't play any role in adapting to host conditions potentially, but I don't I don't think it's a circuit that's evolved to colonize hosts. Um but I'm I'm perfectly willing to be proven wrong

on that. Um

on that. Um >> all right, so interesting things to do.

Uh Jay.

>> Yeah. Um Hong, what um how is an employee regulating econceptibility mechanistically do you think? I think it depends on which chromosome and which

gene uh uh it it affect. So you know uh on the for candida ambigance we know a lot about the genome uh because it has been curated so it help us for candida

or risk it tough because uh you know it it it virgin but I I think an employee uh does not uh imply that this is a tolerance or resistance it depend on

which chromosome and which gene uh is associated with the anoid.

>> Yeah. So I I don't know what uh for glucan synthetase one what what what chromosome is that on seven in in Canada or or or

do you think maybe there's other mechanisms of resistance that or susceptibility where um you know access access to the GSC1 enzyme um maybe you

know impeded by some other uh issues in the cell wall right that allow uh interaction. So, so for candida or the

interaction. So, so for candida or the majority of of the plate are resistant to fuganazone, right? So, and the resistance is mainly due to earth 11 in

candida or at least on play three it belong in the uh uh chromosome 3. uh

what we saw was that uh so now you know we are looking for the gene that um associated with the antibbody and and and and the adherence because that's

what we we are after because for the pathogenesis and you know um what we found was in Canada or it's ALS4 which

is on that chromosome and we did uh uh RNA seek the data I haven't shown because >> yeah we're going to ask you about Yeah. Yeah. Yeah. So, so it it's up

Yeah. Yeah. Yeah. So, so it it's up regulated. So, you know, again, it it

regulated. So, you know, again, it it tell us that it depends on which chromosome in which that that are involved >> and and do you see

have you had the opportunity to look at environmental isolates or or do you think there's something about the the bloodstream isolates specifically that um that that is allowing? I wish I can

>> at our center you know they with the microlab screen using PCR so you know it it the work and they're afraid of spreading and the work it just so

tedious so I don't have the uh the the the skin uh uh isolate but I got the screen uh skin from uh people in Chicago

and you know most of them are clon so there is something that you know once the the uh the candida always get into the human host. It is not to uh uh to

human host. It is not to uh uh to change.

>> Interesting.

Thank you.

>> You're welcome.

>> Right. So question for Eric. I will

combine two again here. Uh so the first question is uh uh if uh actually the tough cells in the colon versus the small intestine are different in terms

of sensing and uh uh in the small intestine organoids treated with their speres do tough cells expand and the second question is why only tough cells

do other cells also cell lineages also respond to to this mechanism?

>> Yep those are great those are great questions. So, so um you know, so

questions. So, so um you know, so there are differences in the colonic tough cells that have been described. So

there's this ATOH1 dependent expansion circuit that we still need to test.

Actually, I I think you know why the expansion is colon specific. So you

know, one argument would be it has people have looked at relative asparagene levels across the intestinal tract and they are lower in the colon compared to the the small intestine except in germfree where they're actually higher and just steady

everywhere. Um so so it is possible that

everywhere. Um so so it is possible that because of that lower concentration it's more sensitive to these uh enzyme activities. The the other argument might

activities. The the other argument might just be that it's um uh it colonizes there better and we we we do see that uh we we've done organoids from uh both

small intestine and colon uh and we see the exact same thing. So we don't think it's something specific about the stem cell programming that allows that to happen. What what's still curious is,

happen. What what's still curious is, you know, I spent all this uh mental exercise on why is 4 circuit doesn't apply to the colon in vivo, but it actually does work in organoid cultures.

So, and I've talked to, you know, Yakab Bon Moa and Rich Lockley about this, and it's not clear why because they've seen the same thing. So, you know, there's something in vivo where that's repressed, but then it can still be

observed in vitro and I think that's not understood, but that's not our main goal.

>> So, it might be not the cells, but the environment either or directly is doing something >> but it's not the cells themselves >> right um and then in terms of do we see other lineages yes that's a great

question because you know one you know there's this general secretary lineage that that kicks on and you do see all that coming on when you give 4 or13 um so we've looked at that so if we if we

do uh Williamia treatment or asparagenase treatment uh we've done PCR looking at uh lineage factors for panith uh goblet entraendocrine

uh so far and away the most induced um gene signature is tough cells. Uh you

get very little panith and goblet cell induction, but you do get a little bit of enterendocrine induction. So it's not 100% tough cell specific, but that's definitely the the the most specifically upregulated.

>> But the tough cells might be the sensors basically.

>> Yeah. Yeah. Yeah. Right.

>> If you look at all that, >> right?

>> Okay. Thanks.

>> Yeah. So there's a followup uh Hong um about uh so you think that the anaploy has to be specific to the the chromosome or or even and in in this context of

copy number variation potentially locus specific um to confirm resistance. So

it's not like so the so anobbody is you know duplication of the chromosome right right >> so it can duplicate the gene so the the gene effect is amplified

>> but you know in in that chromosome they may have a transcriptional factors that act on other chromosome as well. So I

think that you know I have to rephrase it to make it very clear that you know uh uh the unemploy or might not uh uh lead to resistance. It depend on what

gene and what transcriptional factors are there on on on that also.

>> Yeah. And then um and what about Azol and the kind of canan resistance in the in the in the colony variance is uh how divergent is is it between the small

colony and variance in the in the large colony. So in the uh about candida or

colony. So in the uh about candida or >> yes >> okay candida or uh own of the shrine was

susceptible to aocandon but when you look at the mic the uh the small colony

tend to be even more susceptible so you know the the mic is like a 006 the other one is by e test it's go down to about

uh 06 or 008. So it it uh it it's even more susceptible and it's we did not see any resistance there at least not yet.

Uh we are still screening.

>> Okay. Thank you.

>> Okay. So uh there is another question for Eric which goes in the same uh spot I think. So somebody's wondering if you

I think. So somebody's wondering if you can actually measure aspergin in different parts of the uh gastrointestinal tract. So since all the

gastrointestinal tract. So since all the tough cells respond similarly, could it be basically the substrate?

>> Yeah. And that's that's what I was alluding to in the last question. So

it's been you know there there was this actually you were on that paper I there was a group from Cornell that that did that study where they were they were doing mass spec along the intestinal tract for for different amino acid concentrations. And you you can see

concentrations. And you you can see >> you and I have to read the question. So

we have to discuss here. [laughter]

>> But but what was seen in that in that paper is certainly that um you know the the asparagene levels are are higher in in the proximal small intestine then they drop as you go down to the colon.

They're still present there uh but it's at lower concentrations in in SPF mice and then if you put them in germfree conditions then everything shoots back up. So there you know there are these um

up. So there you know there are these um local modulations that are dependent on the the environment there. So you know again like back to what I was saying before you know the the specificity could have to do with that concentration

difference. It also could have to do

difference. It also could have to do with just the burden difference of where the bug is and you know we we haven't totally uncoupled that.

>> Yeah. Okay. Uh Jay do you go or I go with a second question?

>> Yeah. Well either way go ahead while you >> Okay. So I can go uh with a second

>> Okay. So I can go uh with a second question here. Uh it's still back to

question here. Uh it's still back to this you know evolutionary idea of what fungi might be expressing it. Uh so can

you say that dorphic fungi have the same mechanism of faction? Uh

>> dorphics. Yeah. Uh we haven't looked um and uh so we haven't done those experiments with like coxy or hysto or anything like that. Um, you know, we've done it with candida and some other

things and we don't see that at least in yeast form. We haven't tried it in

yeast form. We haven't tried it in pseudohify form because I'm worried the candida light we might have to do it in a ECU one knockout so the candida license doesn't just kill the cultures.

Uh, so I can't answer that yet. Um, but

you know it's possible. We just, you know, I think we're viewing this more as a model to start getting into that biology. But the question is, you know,

biology. But the question is, you know, as you start to extend across looking at different species, you know, where does where do the patterns start to emerge?

And I think that's going to be where it's going to get fun.

Yeah, thanks.

>> Yeah. Um, so we have a question for Dr. Cece from um the NIH clinical center who's done a lot of work on numacys genomics and uh his question is do you

know if the anoploity observed in the Canada or isolates are centromeir mediated?

>> I don't know.

>> Not sure. Yeah. So as a director of you know somebody who's uh done so much clinical gen clinical infectious disease as well as genomics over the last few

years um what what what do you think the emerging technologies in a clinical micology lab for determining resistance is is going to be now that we have all these tools like long read sequencing

and and also just traditional antimicrobial susceptibility. um do you

antimicrobial susceptibility. um do you see a role for um AI or artificial intelligence in in the micology in the clinical micology lab?

>> So now you know with the uh nadore uh one so you can sequence you get the the wizard back within uh a day. So you know it in much it much less work than doing

the antifungal susceptibility testing for echinoand uh you know it might be feasible and because of the FKS uh uh

it's very specific although now you know more and more data emerge that uh there there might be other uh secondary mechanism resistance of yet for fluosone

I think it would be very difficult because there has so many genes and you So uh some we even have to do uh uh uh

expression data uh to look for the resistance. So I think if it works it

resistance. So I think if it works it works uh uh for for the echininoand for now but it would be very difficult to uh

just to use the um um the genomics for for uh the Azone and you know for bacteria as well because that lab also

it just uh there's so much res resistance here I have to do it and you know we do beta lakam ndm plus abc CD uh

uh you know NDM oxa and you know those does not cover for chromosomal resistance right like uh organ so the uh

uh the the PCR or the by how but you know I I don't think it can ever replace the the susceptibility testing.

>> Yeah great thanks I actually have a question for Dr. Dang actually about um yeah so I think Bart Lamrech's lab showed several years ago that um that is

25 is also postr postulationally um activated by proteasis. So do do you think there's fungal proteasis that could not only cause tough cell

differentiation but also potentially um make is 25 more potent through um post-transational um enzyatic modifications.

Certainly. Was it 25 or was it 33 that he was arguing in that time?

>> Both, but I think 25 too.

>> Yeah. Yeah. So, I think it may be David Cory's work, but yeah, I think uh it's one of those. Yeah, that's right. Um

yeah, no, that that's certainly possible. And there could be sort of

possible. And there could be sort of two-step models here, right? Where, you

know, there's the initial production, but then the proteasis amplify and, you know, I mean, I's recent Kazakhstania work too, you know, kind of shows that these Kazakhstania Syrian proteasis can

cleave 33 to provide more activation. So

that sort of might be a you know conserved pattern that's emerging in that area right >> it's very hard to prove though in vivo >> yeah yeah yeah yeah uses >> and you know and you know speaking of

bard I mean he also has some data out there you know that there's the question of cleaving to more activate some of these alarmins but then there's the other question of is there any specificity to inactivating more type

one polarizing cytoines >> and I actually think there's some data kind of emerging on that on that topic too in in that realm Yeah, I mean at least in the miring model I mean numacyus actually drives a

strong type two response in the lung you know with and in surgery of vargas has shown that in um in humans where he's looked at lungs of infants that were you know unfortunate had you know

unfortunate demise due to something else like a motor vehicle accident um in that the pneumysis fungal burn is really correlated with with goblet cell hyperlasia in those human lungs so um >> right

>> but I but I'm not aware of the mechanism of how pneumys you regulates a type two response >> right Right. Relative contribution is about 33 versus 25.

>> Right. Right. I mean in general tough cells they're they're not very common in the lower airways. Uh right

>> they are there in the trachea. Right. Um

but but again that's mostly been looked at in the context of these type 2 cytoine expansion circuits which don't seem to apply in the lung but you know we haven't looked at all there in that organ yet. And you know what you think

organ yet. And you know what you think about things like aspiggillis species that definitely do secrete esperogenasis is any of that playing a role in in non-type 2 cytoine dependent expansion

would be kind of interesting to look at.

>> Yeah, that would be very cool and you're collaborating with the right person to make those mutants >> rightilus. So that will be fun. Uh so

>> rightilus. So that will be fun. Uh so

with that uh we we're at the end of our u micro talks series for November. Uh

thanks for the speakers that was very stimulating talks and discussion after that and always fun to co-chair with Jay. Uh and thank you all and we will

Jay. Uh and thank you all and we will see you in December with uh other chairs and other speakers.

>> Thank you. Okay.

>> Thank you. Bye. Bye.

>> Bye.