Fungal evolution in the environmental reservoir
14:30 – 15:00 Fungal secondary metabolites and adaptation
Nicolas Papon, France
15:00 – 15:30 Genomic evolution of Aspergillus
Johanna Rhodes, UK
15:30 – 16:00 Proteomics to study evolution of Aspergillus
Vishu Aimanianda, France
16:00 – 16:30 Genomic evolution of Candida
Marie-Elisabeth Bougnoux, France
Foreign so please have a seat we are going to start and this afternoon we have a session fungal evolution in the environment Reservoir so it is the first time we use the name the word Reservoir as mentioned by Jack and we will start with Nicola papon he’s a professor in Orange University
Hospital in Orange University sorry and his leading a research group in Orange so Nicola are you in line with us um yes Nicola hey yes we can see you perfect so now we are waiting we are waiting for the screen okay foreign metabolites and adaptation thank you
Very much Nicola I know that it was not possible for you to be with us because Angie is not so far from Ren but uh thank you very much for this talk so very much I’m very happy to come but yes and just from a click on sign meeting in between uh
Give you some basic information sick metabolism my teacher at first off let me stop by your support introduction on metabolism within the tree of um metabolite on chemical with relatively complex sure you get toxin produced by some Corona you know but everything is Nicola yeah yes can you hear me yeah
There is a problem with your connection it seems to be so can you what do you think you have to move your microphone like this lower Nicola if you put your microphone in front of your shirt instead of around your neck the microphone will be lower try it
It’s been an obvious yeah this is better minutes second screen I got to control this same here it’s better yeah better Okay so it’s okay for you yes okay yes yes it’s okay for us thank you so secondary metabolite are produced by uh oh sorry by uh winding a metabolic
Pathway that are fueled by primary metabolism the production of a dorm metabolite um is usually played specific ranking from genius species to a strain concerning the general functions you must consider that secondary metabolites were involved as a main chemical actor for Intel specific defense competition and communication
For example here within the mean family we know now that the biosensis of iridoids specifically emerge in the genus yielding to the production of compounds that are attracted for fields and the fact that Phoenix turn around these grasses this prevent too much attack by herbivorous insects surprisingly we also know some example
Where secondary metabolite mediate infra-specific condition is specifically produced by the male to try to trigger the sixth sorrow maturation of the female and promote the laying of eggs which stands out as you may know at the main process but condition the pathophysiology of skitosomiasis in fact secondary metabolite were
Described in near to all Kingdom within the tree of life and their reperto is mostly rich in plants in bacteria in fungi and in a way also in animal but for Animals this essentially involve aquatic organism now let me give you one example of the prominent secondary metabolite pair
Kingdom to illustrate their distribution within the tree of life here maybe you know the vacancy was produced by some medicinal plants these compounds such as vanquistine van blastina are produced in need of these tropical species to defend against and these precious molecule also known as first line drug to trick cancer
A scoop on the right panel here published in nature six months ago van blasnin is now produced in engineer yeast in which researcher transpire in sacrament encoding dozens of enzymes for Van blasting a terrorist biosynthesis for illustrating bacterial secondary metabolism I think you know the prominent example of iron minting
Because this compound emerged as the most potent antiviral agent to fight any fantasies in Africa and this give rise of course to a Nobel Prize in medicine to you eight years ago and finally for animal let me introduce a paper in science three years ago by Dallas Andes and colleague reporting to
Obamacine as a new high independent antifungal compound that is notably really active against the emergency candida Oris okay so now we will enter the core of the subject with a specific feature on the fungal’s secondary metabolism fungal secondary metalli can be only detected in pluri cellular from B I mean
Mold and mushroom because genetic determinant of a bison digital yes absentitis for instance we know that 43 distinct secondary metabolites are produced by the more aspergillus fumigators secondary metabolite arise from complex biosensity groups that are directly fuel either by amino acid or acetically from glycolysis an important feature of these fungal
Biosynthetic Roots is that gem encoding enzyme for a dome a compound era are organized in cluster I mean a same region in chromosome and in this clusters another in the center we usually observe a core enzyme that Define the backbone of the compound and around this coronzine we find a combination of
Decorating enzyme Gene in the evolution of fungi the Gen cluster are mostly one second by vertical transfer and in a lesser extent by convergent evolution and horizontal Gene transcript the main families of fungal secondary metabolites include terpenzoids pollicated non-ribosomal peptides some Irene molecular and some over less frequent type
And first definitely the biocentesis of perpended is driven by corenzyme named terpene synthesis here on the right panel a scoop in the field recently published in nature discovering in from A New Path called terpenomic biosynthesis thanks to a new class of the functional tap and synthase include flavor that pay from for
Instance age cheese and Turpin within fungi also include trichotycines that are the main part of the dangerous exposure notably responsible for insanitary house syndromes mirror what happens with are also specific sub-coming with well-known in fungi such as the femaline produced by aspergillus femigatus and we used to treat microsphorism infection in b
So let’s go for the second problem family of fungal secondary metabolite I mean polycatids foreign of these compounds are produced by multi-modular enzyme refill to our publicated syntax their metal system is one of the most famous fungal pro-inflammatory collocative and for instance we have recentation in our laboratory
Skeletosporium which is a main mode that colonizes the lung of patients with cystic types don’t regulate the biosynthesis of this thermometer system to evade the post immune system and to promote a long-term colonization in this patient most of the so-called mycotoxin also belongs the collocated family in Foundry
There are highly toxic for cattle and pneumonolf and they are produced by several mold but develop directly ingrained stock used for peeling livestock and as pollicated at the last a publicated example we can cite loracastatin which is produced by some Ward and as a powerful inhibitor of a specific enzyme of methylated pathway
Is blocking nature the bank synthesis of Essentials steroids in microbial competitor and as you may know Statin are also in human health a green drug to treat eye blonde cholesterol the NRP the backbones are produced also by multimodel enzyme stunt taste the first example of course is penicillin historically identified by
Claiming in the modern penicillin Chrysler genome and you see here on the historical historical petri dish that along the identification of this famous antibiotics the over excellent example increase restrain the family of nlps is cyclosporine a and this compound as you may not remain one of the most used anti-cut scenery immunosuppressant in
Solid organ transplant patients as the the main example you can cite for hybrid molecules we can cite of course and introduce echelocandings such as the gas performing which is a semi-synthetic natural product initially extracted from the ascami city and no widely used to treat deep seated candidiasis and finally some others uh anecdotical
Type of fungus 600 metabolite include a for instance in the alkaloids produced by the right air Goods and well known for their pharmaceutical properties and a last example of secondary metabolites obvious third time is a tremorganic mycotoxin which descript the gabaergic amino acid neurotransmission and these induces filmoring when you
Ingest continuity to it so now what about the functions known for second derivative light in fungi basically 600 meter light are involved in fungal development for example if you knock out the PK sport Gene in Solaria macrospora you will lose the capacity to produce periodicia and if you on the
Right or Express this Gene you will observe a huge freaking body in this tree we also know for a while that pigment such as melon protects of fungi from damaging UV radiation are above all you know broadly used by fungal species as different chemical and weapons again microbial competitors
To infect plant and also to find insect for instance it’s important to keep in mind that all experiment in the literature investigating the anti-microbial activity of extract are finely biased why in terms of concentration if you compare to real ecological concentration and it is highly likely that physiologically relevant concentration of a specific
Secondary metaboli with function only as a signal rather than a weapon in the nature the proof that secondary metabolite uh defense and weapons in fungi in nature is very easy to observe in your laboratory for instance if you treat aspirations with Aquino community so another product you will detect many
Changes in the regulation of signal transduction paths we’re leading to over expression of Gene encoding enzyme involved in many different bowsenic pathway for secondary metabolizing response to this input signal in most of the fungi iron acquisition is also mediated by a secondary metabolite for instance in our lab we recently demonstrated that
In scheduling once again in the number of patients with seismic parenthesis these More Produce NRP type cider forearm that scavenge iron from pseudonymous another interesting question in the field is how hunger producers of toxin can prevent how self-in expectation because of course most of the time the produce
Secondary metabolite as a direct Target density in the fungal producer so for instance Euro in pink in Fusion that produce the t2 toxin well-known a specific pump is important in cluster immediate the influx of the toxic compound outside of the stairs in some other cases the fungal producer expresses a detoxify enzyme that
Converts the toxin to a non-toxic derivative this is your case of a Glide routine for instance was used by aspergillus film glitches or the possibility many more species use gen duplication for amplifying the Target and prevent stealth intoxication this will get all some more about produce statins and sometimes this chain amplification
Is also coupled with the selection of mutation in the Target sequence for promoting the resistance because these second eyes of corn is less susceptible to the toxin important fact here this mean that browsing fungal genome for this type of resistance Gene is one of the most powerful strategy for drug discovery
And finally to slide concerning future development in drug Discovery during the last weekend you know a secondary metabolite Discovery has been mainly supported by the explosion in the number of fungal genome available and cook a bit with a powerful online software with with recently led to the identification of Android of Newfoundland Byzantine
Cluster once the new generator is identified we benefit now from terrific advance from the synthetic biology field for characterizing but also for processing at I scale the corresponding natural product notably by engineering two main maintenance so as a conclusion we can remind that only few hundred Gene clusters are
Characterized to date and on the buses but more usually are more um 30 to 70 gem cluster per species and but to take 20 000 more are described there are more over one million of cluster to be discovered in the future and this is obviously an underestimation anyway I
Hope I commit you in that from the secondary metabolism will continue to Boom as an excited field of basic and applied research for years I thank you very much for your attention [Applause] thank you very much nicorazzo the connection was good no problem okay and thank you for your fantastic talk
Metabolites secondary metabolizer less known than uh other topics so I I think it is really important to to have this conference within a session on Evolution and on adaptation so thank you very much if is there one question otherwise it is just a time for Joanna roads after so
No okay thank you very much uh Nicola and we move to the next uh the next talk thank you very much for being here you are working in the lab of um Matt Fisher and we are very happy to have you regarding uh your topic and evolution of aspergillus thank you genomic evolution
Brilliant so we’re going to be diving down into the genetics lesson fantastic talks and um thanks to Nikolai for his talk as well which I will actually touch upon in mine so um I’m working on a anti-fungal resistance project um with Matt Fisher but I’m also based
In Paul verbay’s lab in Redbird um say in the Netherlands and he will be given a talk in the next session um next there we go so uh just disclosure uh funding from welcome Tristan I’ve also received an honorarium from the Gilead Sciences so aspergillus is a genus is really
Varied it’s found in a diverse set of environments both terrestrial and also aquatic as well we’re thinking about aspergillus sadawi which causes uh this sort of infection in in fan corals and just basically eats them away and then of course we also know um aspergillus infections in humans and
Then also in in Plants as well so it’s kind of a really sort of interesting organism set of organisms it can just cause so many opportunistic infections such a wide variety of environments and it’s a an efficient safrotrope and as you can see it’s it looks phenotypically
Quite varied so let’s have a look at some of these different aspergillus species so aspergillus flavors I kind of like this one it kind of looks like if you’ve cut a kiwi in half and and that’s what it reminds me of but I wouldn’t lick it I wouldn’t eat it that’s for
Sure it’s opportunistic and it’s a destructive agricultural pest that contaminates crops with what’s called aflatoxin which Nicolaus just sort of hinted on um and it causes croppy old losses but it can also um enter the the food the consumption uh um uh food cycle and cause human death
In in rare cases but also hepatocellular carcinoma and it’s estimated there could be anywhere between 25 000 and 155 000 cases worldwide each year attributed to aflatoxin ingestion I’m sure we all know about aspergillus fumigartis um listed as a critical priority fungal pathogen on the who list that came out
Last Autumn there’s High rates of azol antifungal drug resistance infection and this varies depending on the country because a different surveillance techniques and programs so anything between four to seven and eighty eight percent and it causes invasive infections particularly invasive aspergillosis okay Asperger’s Niger or Niger is widely used in the food industry
Um for enzyme production and can sometimes be the cause of some cases of pneumonia and again like similar to Flavors it can secrete these toxins these mycotoxin called ocherotoxin which causes problems with the kidneys if ingested and hazardous to human health and the last one I want to touch on was
Aspergillus nigilance so this is really being kind of like the model organism and Asperger’s fumigas has kind of taken over in terms of genetics but aspergillus nigilance was was one of the first that people were really looking at in terms of genetics and genomics and it’s considered an important model for
Eukaryotic genetics and cell biology and one of the kind of candid drugs is actually derived from a fermentation product of a variety of this as well but what is in a name so where does it where does aspergillus the name actually come from It’s actually kind of interesting so it’s first described
About 300 years ago by a priest and botanist Antonio Michelli and aspergillus actually refers to the asexual life cycle so fungi can actually undergo asexual life cycles when the conditions aren’t really favorable and it will actually produce clones of itself but they also have the ability to reproduce sexually produce more
Diversity because they have mating types so basically the phenotypic diversity of sexual fruiting bodies is greater and different genre describe the resexual cycle so for instance Asperger’s fumigators is the asexual whereas near sartoria fumigata is the sexual and this paper that came out a decade ago can you believe
Um it just shows the the variety the diversity of aspergillus as a Genus um using penicillium chrysogenum among FBI is out groups and this is just basically using one reference genome per species just to give you an idea and in the in the column the First Column you
Might not be able to make out the numbers but these are the genome sizes and then the next column is the associated Gene content and if you can just about make it out the genome size is range from about 20 28 to 40 million base pairs and the gene content is
Anything from about twelve nine thousand to fourteen thousand and what’s neat about aspergillus is that they all seem to arrange into eight chromosomes and a mitochondria there might there might be some sort of um deviations from that but generally speaking that’s what they what they do
Um and there’s actually a quarter of a million genomes publicly available for the aspergillus genus that’s more than cryptococcus and candida it’s not quite as much as saccharomycia service here which has over a million but it’s still a lot actually the aspirinus genus is well placed to be a
Model genius for our genomics and genetics investigations for eukaryotics um in general but also aspergillus and fungi so let’s get into the nitty-gritty genomics um I know some of you might actually be too young to remember this but most of us probably do less than a quarter of a
Century ago the human genome was published and it cost 95 million US Dollars now thankfully it’s a lot cheaper it’s only a thousand dollars ish to sequence the human genome and with the falling cost in June in in sequencing a whole genome for humans it
Also falls for fungi as well so we can take advantage of that so we have sequenced more fungi because it’s more accessible and because it’s cheaper and the beauty of genomics is that we can use it for a range of things but certain things that we need to take into
Consideration first firstly the DNA extraction method so if we want to do a short read sequencing so Illumina you need to do a certain extraction method if and if you want to do a long read using nanopore Minion or pack bio you need to do a slightly different
Extraction method and it also matters when you’re doing your extraction so as I mentioned there are sexual life cycles asexual life cycles when are you going to take your DNA extraction should you extract from the Spore or the hyphy or the fruiting body and there’s all these
Things and the majority of the time actually the extraction is just done whatever is there on the lab bench at the time which perhaps isn’t so great so we could actually see different results in our sequence data depending on what we when we do the extraction
But the beauty of the genetic data is that we can do genome assemblies and structural studies to look at actually what is in these genomes functional annotation pathogenic microbiology to look at what are the resistance mechanisms that are conferring drug resistance and then comparative genomic analysis within and then also between species
So there’s remarkable genomic sequence diversity within aspergillus there’s a few more fungal species included here but aspergillus as a genus is kind of just this bit here and from the majority of the ice that’s included here there’s just one genome but a few of them actually have some more so two genomes
Four genomes for fusarium and then when we get into aspergillus we’re actually starting to see um a few more being sequins but mainly just just one uh representative uh genome there so it’s actually as diverse as our own phylum the divert the vertebrates um but the close relatives fumigatus and
Fisher eye are as divergent as humans and mice so it’s a heck of a lot of diversity within the genus and although the genomes are quite stable it’s generally ranging from that 28 to 40 million base pairs they are not what we would call plastic genomes I know that
Some people do think that aspergillus genomes are plastic they’re not as plastic as other fungal genomes and they do have several similar characteristics but there are a lot of differences and we think that these differences well we know that they are there are differences because of this it’s because of
Horizontal Gene transfer and that’s what these arrows are symbolizing here so the big Arrow here symbolizes a big uh horizontal Gene transfer event from aspergillus Netherlands into podospora and then other various events occurring so now that Gene genome sequencing has become more accessible we can sequence more genomes than ever before and
There’s these really big genome data sets available and being published so actually it is worthwhile doing pan genome analysis this is where we can take all of the isolates within a species or a clade and we can look at what genes are common to all of them and then what are not common
Accessories so we’re just using this little this little diagram I’ve created here so the yellow Gene and the blue Gene would be what’s called core because they’re present in all three isolates the purple and the Pink jeans would be in the accessory genome and this little turquoise Gene here
Would also be in the accessory genome would it be called a unique Gene because it’s only found in one isolate so with this analysis we can actually reveal more diversity so when we look at the aspergillus genus as a whole it actually um the pan genome shows that there’s way
More diversity than we thought and there’s just under 63 000 Gene families across 36 aspergillus species and only six percent are found in all genomes that’s incredible so this diagram here we’ve got the gene count in the red fairly similar across the species same for the core genome and
It’s the species specific genes where we see the diversity anything from 182 for aspergillus Niger Niger all the way up to 3 000 here for aspergillus ellipticus so we can actually see quite a range and that means if we sequence only one or two or three just a handful per species
We’re missing out on so much diversity what we’ve also seen with panjumina pangenome analysis is that there’s evidence of Gene loss duplication and extensive horizontal Gene transfer and we think that this is what is driving the diversity within this genus so let’s have a look at aspergillus fumigators specifically because this is
Where a lot of the research has been carried out the aspergillus reference genome was created about 20 20 30 years ago um there are multiple there’s this two there’s two that sort of the main ones and they generally agree in that the they contain about 10 000 genes over
Eight chromosomes in a mitochondria that equals about 29 million base pairs and then over the last sort of 18 months there’s been three studies looking at pan genome analysis and it’s revealed way more diversity than we thought and what was really nice is that these three studies all included the same isolates
As a subset of their Pangea pange analysis and then they added additional ones for their studies perhaps specifically in a geographical region or ones that if collected perhaps a little bit more clinical or more Environmental so as you can see the mean genome sizes are roughly the same for the free
Studies but we actually found quite significant differences so Lotus Lofgren found the most you found fifteen thousand uh genes for her pangino in in our study we’ve had about 13 000 genes and Amelia Barber found just under 11 000 genes so actually we can find massive differences and it’s important
To note that the mean genome size is the mean genome size that means that the range is something else and I can’t speak for Lotus or Amelia but what we are seeing with our mean with our range of genome sizes some of the aspergillus FEMA artists uh genomes are up to 70 million
Base pairs and the gene content is off the chart we you know it’s new stuff that we’ve never seen before and it’s really quite exciting we wouldn’t have found that if we haven’t done pandem analysis and what we all agree on in these three studies is that the core genome was
Enriched of housekeeping functions but the exciting stuff is in the accessory genome and this really really does Link in quite nicely with the previous talk secondary metabolism is a big part and secondary metabolism is a significant factor for the delineation of aspergillus as a whole but it’s also linked to azol antifungal
Drug resistance so let’s just touch on secondary metabolism in aspergillus as a Genus yes it’s commonly unique to individual species and there’s a higher number of secondary metabolism Gene clusters in aspergillus than there is in penicillium so it’s just under 3 000 in over 37 aspergillus species and the Dynamics of secondary metabolism
Suggests that horizontal Gene transfers we’re coming back to that horizontal Gene transfer is um is basically the mechanism um of of what’s driving the evolution here um and this is really apparent because if you look at this phylogen you hear you can see that there are quite some distinct aspergillus species so for
Instance flavors is down here and it’s very different say to the aspergillus species up here there’s one secondary metabolism Gene cluster that was found in five really distinct species and the only way it could be present is through horizontal Gene transfer when we looked at um secondary metabolism in Asperger’s
Fumigartis it was really quite interesting um because we looked at what loci in the genome we’re approaching fixation and this means basically is the loci found in all the isolates in that population and if they were we would expect to see it write a DOT right at the top of the
Of the plot um and what was interesting is that we saw for this FST we saw like these waves across the genome and these um High regions of FST mirrored significant Gene phenotype associations from a g-west study which linked loci to eye triconazole drug resistance and these regions these these Peaks and
These shoulders these significant loci were actually involved in secondary metabolism so basically what we think is happening is horizontal Gene transfer is basically literally moving segments of the genome comparing resistance to azol and Azle antifungal drugs and then also they are linked to secondary metabolism as well
We took 28 Gene deletion mutants from the coven knockout collection and Screen them for for growth on media containing each connazole and we saw that there were two isolates that were not able to grow one that had sip 51a in octave and one that had ABCA knocked out
And I’ll come back to that in a minute so what is actually driving the evolution we’ve got a hair we’ve got a horizontal Gene transfer as the hair but what is driving it so it’s selection pressure which is basically external agents that are just basically causing this change they could
Cause a negative change so for the trait to basically die out or positive change that causes the increase in the trade and positive doesn’t mean it’s positive for us it means it’s positive for aspergillus and it’s just basically this whole mismatch of stuff we’ve got change in agrochemical use changing that risk
Groups changing climate changing by Auto change in virulence and then also the way that we as humans use resources so we’re spraying chemicals environmentally in agriculture but also in Horticulture animal husbandry we’re composting which is actually a significant factor as well but we’re also using the same chemicals in
Agriculture that we’re using in the clinic as well and all of this together is creating pressure in aspergillus fumigatus drug resistance mechanisms are primarily centered on sit51a and that was the the gene that was knocked out in the ice That Couldn’t grow in orangeconazole which is what we’d expect to see
And it has a multitude of resistance mechanisms that are pretty unique raspages and the structural polymorphisms tandem repeats of of sequences in the promoter region the most prominent being a tan and repeat of 34 base pairs this causes over-expression and then also duplication events and we see drug-resistant aspages
Fumigartis in the environment at high levels we would expect to see it quite high in agricultural settings so say for like arable farming but actually it’s only about one percent we see more drug resistance in urban flower beds um and that’s at 14 and we also see a heck of a
Lot of drug resistance in air samples and this does follow a seasonality pattern we are more likely to find drug resistant Aspergers fumigarts in the spring than say summer and winter there we go and this mirrors clinically as well so if we’re finding it in the
Soil we’re finding it in the air then we as people are breathing it in and Azle resistance was present in 13 of isolates um from cystic fibrosis patients and the about 27 of them had this tandem repeat uh 34 mechanism as well and what’s really interesting is that these azle-resistant isolates have a
Fitness advantage over drug susceptible isolates so there’s a different mechanism occurring here in terms of micro evolution so these isolates have advantage in acidic pH and also High histamine and histidine conditions which are the conditions that are primarily primarily found in the cystic fibrosis lung so we’re seeing not only selection pressure
From external external to us but from internal adaptation to the human host driving evolution by sequencing a heck of a lot of isolates over a thousand we can use a population genomics approach to see that azle-resistant aspergillus fumergartis has spread globally and increased in prevalence over time so that tr34 drug
Resistance mechanism is here in blue and what we can see is that the majority of the isolates are actually in this bottom section of the phylogeny and then the azos settable isolates are in the top part of the phylogeny when we map this on this same phyology onto resistance to agricultural
Fungicides we see that the pattern is the same the majority of the resistance mechanisms are in this bottom part of the thyroid phylogeny and the susceptible ones are in the top and what’s interesting is this contains a mixture of both clinical and environmental isolates so these clinical isolates they’re only going to acquire
Isolates that are resistant to environmental fungicides by breathing them in they are not going to be treated with an environmental fungicide so patients are acquiring infections from the environment and these infections are hand drug class resistant the Pang genome for the aspergillus fumigators actually revealed additional information so what we saw was that
There were some drug susceptible isolate on a different genetic background they were in that that group at the top of the phylogeny where we’d expect to see susceptible isolates and they didn’t contain any known mutations in sit51a I want the pan genome analysis revealed was that there were genes in the
Accessory con and the accessory genome that basically point to Alternative resistance mechanisms associated with secondary metabolism that are driving a different mechanism for resistance so resistance in fumigartis at least is definitely more complex than we thought but what about mechanisms in other aspergilli so this is a paper uh by Emil glucosala
He’s currently in New chatel and he’s moving over to America this fall to take up a tenure track position and he’s done extensive work on what’s called Starships um he’s a massive Star Trek fan so that’s why uh they’ve been called that and he’s done this analysis over
Multiple fungi but he’s found these Starships in aspergillus flavors and starships are these huge mobile elements that facilitate variation in gene content and they basically just pick up and move they’re like transposable elements they are about 110 KB long on average and they share conserve components and carry diverse arrays of
Accessory genes and they’re a newly discovered route of evolution that can be transmitted not only through horizontal Gene transfer but also vertical transmission as well other mechanisms include transposable elements and repeat induced point or rip mutations so I’m thinking to the work of clutterbook who sadly passed away last Autumn but his work
Um in in rip mutations in fungi is unparalleled and also really hard to understand it’s quite dense but it is good and then we have seen the activation of transposable elements in stress conditions in various aspergillus species and that the transposable elements are involved in the regulation again of secondary metabolism it keeps
Cropping up which means it must be important in aspergillus nigilance so to summarize we’ve learned that sequencing one genome per species is insufficient because it really doesn’t give us the depth of knowledge about diversity and we miss a heck of a lot of Gene content Pang genome and population genomics
Analysis offers way more opportunity to identify diversity and and diversity associated with drug resistance as well aspergillus genomes are not plastic but they are evolving and this is because of pressure from us as humans in the anthropocene activity Azle resistance and aspergillus fumigartis at least is linked to
Secondary metabolism and on the same background as the Azo resistance we’re also seen as resistance to agricultural fungicides as well and we can also see adaptation of fumigart as to certain patient cohorts in this microevolution at like the loci level in cystic fibrosis but also in other respiratory conditions I didn’t
Mention it but we don’t see it in covid in Kappa patients whether that changes um remains to be seen by a doubt it would there are various pressures driving evolution in fumigartis and fumigartus is responding in many ways and we’re not even keeping up we are we are woefully falling behind but what
Can we expect from other aspergillus species there’s a bit of a sort of like a disjointed a way that we approach analysis in the Asperger’s genus as a whole there seems to be a certain level of analyzes that continue in fumig artists in a different sort of analyzes
That occur in the other aspergillus species and they’re finding out different things it’d be really good if we kind of work together and mesh them together and see what we can find out um thank you so much for your attention this work would not be possible without
All of these people and I hopefully have given you a nice summary of where we are with the state of genomics and aspergillus [Applause] so thank you so much Joanna for This brilliant talk very nice slides um do you have a question for Joanna even if it’s very
I appreciate it was very diving in so no question is stupid so it’s like yes you want yeah thank you thank you very much for the very nice talk I read with great interest great interest your question paper about the outbreak of a specialization in non-flying parrots oh yeah as a veteran
Yeah I love many long numbers very nice story yeah I had a question about the potential microevolution uh regarding the the host so for example in birds so any information yeah so it’s a bit of an odd one because the Asperger’s fumigators outbreak in the kakapo was actually quite different to what we
Would see in the human host um really so there was some sort of genetic distinction there um and whether that is representative of aspergillus infections in animals in general remains to be seen um I’ve got I’ve got three genomes to look at from a dolphin and a a cow type
Thing and a moose to look at and see whether they are sort of more related um compared to humans but yeah so it’s it was a very distinct genotype yeah but in terms of microevolution it seemed to evolve less than what I would see in humans as well
So whether that’s because there’s less pressure maybe I don’t know or it’s just a very different background and it doesn’t have the same mutation rate doesn’t have the same clock basically I’m kind of more inclined to think it has a different clock yeah but the jury’s out on that one
We’ll see as many years time thank you yeah yeah okay thank you we asked one more question uh Joe thanks for great talks I have a question because you just mentioned the pen genome probably will be future Direction video analyze genomi data but if you want uh find out transmission Route for example
Um you can link the source and on patients so it’s a pan genomics will help um maybe uh maybe to an extent um you have to remember the aspergillus premium well aspergillus in general has a lot of recombination so how do you define a cut off as to what is a similar
Isolate in terms of um transmission um transmission software and methods and algorithms that are out there at the moment rely heavily on a clone or genome which is why it works so nicely on bacterial pathogens and then Canada Iris as well which is kind of the outlier for aspergillus it might be incredibly
Tricky and you will probably end up applying those methods or having a very arbitrary uh cut off um that doesn’t really make a lot of sense so would the pan genome help potentially if you kind of said okay well the core Gene content is high but the accessory Gene content is is low
Between these isolates whereas if they were very disparate and there was no transmission you might expect more accessory Gene content but that wouldn’t be a clear-cut answer I don’t think okay so it was a great pleasure to listen thank you to you Joanna thank you very much and we moved to
The proteomic approach after the genomic approach with vishu from Pastor Institute in Paris he’s uh heading the aspergillus unit so we should the floors is yours hello everyone first of all thank you John Pierre for inviting me to be a part of this Symposium here so I’m a basic researcher I don’t touch
Any patients I touch only mice okay so you see a lot of data regarding basic research in my presentation so the topic what I’m going to talk about is a proteomic to study evolution of aspergillus so I have nothing to disclose so when you look into proteomics you can
Look into two different aspects what happens to the fungal side when fungus changes its morphology from dormancy to germination but this is bit confusing as you see here if you use a different medium rpmi Brian minimal zapak and collagen the way you separate protein is completely different so it is
Chaotic so it’s very confusing but still we are working with this proteomic analysis how the proteomics changes from dormancy to germination so we are working with the two different fungal species our specialist fumigators and flavors probably this is not a good context to speak about today here
So I moved to proteomics from the host perspectives so what happens when fungus is there in normal or healthy human and when the infection starts so you know today’s stock will be mostly focused on a specialist fumigators because that is the focus of my lab but we also work
With aspergillus flavors so you all know owing to the small size and hydrophobicity of spores they can reach up to the level of lung alveoli so if the sport is reaching the macrophage there will be phagocytosis there will be cytokine production and there is recruitment of immune cells to the site
Of infection then finally fungus will be cleared in a normal condition but what happens if it is going somewhere else if it is not going reaching the macrophage so then then it has to face alveolar fluid so we had the question what it interacts with so for that we did a very simple
Experiment we took a ball sample Bronco alveolar lavage from control individuals so here I say control it is not healthy individuals because it’s a collection of Bal is a invasive protocol so we collected bulb from control where there was no fungal bacterial or viral infection so we optionized Cornelia we took out
Spores of cornedia and we extracted what is bound on the surface we did the proteomics so this is the long list of proteins what we saw so most of them are complement proteins so here you see two types of extraction what one is hydroxylamine extraction and the other
One is uh sodium thiocyanate so here this extraction will remove those which are covalently bound whereas here it is non-covalently bound so we have to focus on this one because there is direct strong interaction between Spore and the proteins so as the major components found were complement proteins we had the question
What it leads to so what we saw when there is an optionized spores they are all dispersed when there is optionization they become aggregated so whether it has any implication on phagocytosis yes if you see unoxidized spores they are taken up very less compared to optionized spores here what you see here
So as most of the proteins which popped up were complement proteins we had the question whether there is any implication of complement receptors so what happens when there is opsonization by complement it will be taken up by complement receptors because this is important here there is lot of
Emphasis on dectane one dectin one is the receptor which phagocytose fungal spores but here what we saw was when we blocked complement receptors complement receptors cr3 or cr4 what we saw was there was significant decrease in phagocytosis so there was 60 65 percent decrease in phagocytosis when we blocked
Complement receptors but I would like to warn here even unoxidized spores are taken up that means complement receptors are not only the receptors involved in phagocytosis so we did another set of experiment so the normal route of inhalation is through International root so when we took wild
Type and C3 knockout my C3 is the central molecule in complement Activation so what we saw was there was no difference between wild type and C3 knockout mice on the other hand if you inject suppose through intravenous root of course we saw difference in survival
So this is the survival curve so we use different concentrations or different numbers of spores as you see in all different ah Moi there was a difference in Wild type and complement seats mice so what we saw was there was decrease in phagocytosis of spores in C3 knockout mice
Both by neutrophils and monocytes compared to Wild type mice so when we saw complement system is implicated in phagocytosis and clearance we had the question what is the difference if we optionize spores with serum and bulk because in in vitro system most of the time we use
Serum not bulb because it is difficult to get bad you clearly see here tnf Alpha was more produced il-6 was more produced or il-8 was more produced if you optionize power with serum on the other hand if you optionize spores with ball there is more production of oil 1
Beta so there is a pathway difference if you optionize spores with either spores or but so this led us to question is there any difference between the optional opsonization pattern of course we saw difference in the optionization so some proteins were only present only binding to spores when you optimize with serum
Or some proteins were only present when you optimize spores with Bal so one of the major or interesting point here interesting protein was surfactant protein d That’s because surfactant protein D is the major humoral immune component present in the lung alveoli it is the main function of surfactants
Is to prevent the collapse of the lung alveoli during respiration but surfactant protein D is also a c type lectin it is having lectin property it has a pattern recognition receptor property so so this is the structure it has a carbohydrate recognition domain the there is a collagenous region and we saw
That surfactant protein D is directly interacting with spores we proved it by immunolabling as well as by facts analysis so what is the implication of optionization by surfactant protein D so it was increasing phagocytosis and it was increasing Ross production so before Nikola was saying if there is a
Polyketide it tries to inhibit or it tries to reduce the production of Ross but when Spore is covered by melanin but over that if there is SPD binding it increases the production of Ross and increases the clearance of spores so we proved it by animal study also we used wild type or
SPD and occult mice as you see here most of the cytokines were less produced in case of knockout mice so that was about spores but we also had the question what it does if fungus is already germinating so if you cultivate spores in the presence or absence you see there is clear
Difference in the germination rate so there was 60 percent reduction in Spore germination in presence of surfactant protein d so this is the morphological defect control it is nicely producing hyphae but if you add SPD there is defect in the surface of halfway so here I would
Like to point 30 micrograms per ml the physiological concentration of SPD is 50 to 90 micrograms in bulb so we are much below the level of physiological concentration so it was this binding or alteration of the surface of hyphae was also affecting the cytokine production it was causing immunomodulation most importantly if you
Add SPD to oriconazole combination treatment you see increased efficacy of antifungal drug so that was mainly because if you use control this is calcophlore white staining which binds to the cell wall chitin so here control it was not going inside but if you treat with SPD calcophlore white is going
Inside that means that is pore formation in heifel cell wall if you treat it with SPD on the other hand that was about healthy condition we looked at infectious condition so what we saw was this is the volcano plot there was upregulated and down regulated proteins so there were eight humoral immune factors
Completely absent in infected conditions so here we took the ball from 10 patients four were colonized three were IPA and three were CPA so interestingly SPD C1 Cube equal into and Mass 2 cfhr 2 and 5 MRC and C8 are the proteins which are completely absent on the other
Hand in the Infectious condition when tracks in three was highly present so the question here now is whether we can use these humoral immune factors as diagnostic markers that is one possibility so we also had the question why pentrexin 3 is highly present here that’s because pentraxine 3 is not
Binding to dominant spores directly it binds to swollen Spore or germinating Spore so probably there is no inter interacting partner here on the other hand when we optimized first Spore with SPD then we added pentrexin 3 that was increased binding that means The Binding partner is missing in infectious condition
So here SPD alone when it is optionized it is producing high level of cytokine but when SPD over that if you add pentoxine3 there is lower amount of cell token production so probably this is a kind of regulatory mechanism because initially there is SPD so SPD is binding to spores there is
Pro-inflammatory immune reaction on the other other hand once it is produced then there is a production of pentraxine 3 it binds to SPD then there is less production of cytokine it’s a kind of Tolerance mechanism so there are lot of studies on correlation with the infection and all
These humoral immune factors for example pentrexin 3 ah SNP single nucleotide polymorphism is known to be high risk factor for invasive aspergillosis same like masp that’s a serine protease on the other hand in Mouse model it’s already been shown that SPD knockout mice or c1q fcn2 contractions are highly susceptible to invasive aspergillosis
So the question is whether we can use these components for therapeutic therapy of IPA for example it’s already been shown in mice model if you add SPD internationally or pentroxine 3 intravenously there is increased survival of IPA mice so that leads to the question whether they are having any therapeutic benefits
Most interestingly what we did was we took this mutation ah this resistant mutant and here you see its resistant to oriconazole but when you add SPD it doesn’t matter it behaves like reference a sensitive isolate so that means SPD is also active against Azure resistant isolates so now we have
Done rna-seq analysis to see what’s the mechanism of action so there was lot of emphasis on secondary metabolite what we see is that SPD is shutting down all secondary metabolite formation in aspergillus fumigators so it is not only shutting down secondary metabolites it is also decreasing many more Pathways
So probably we are going to present that data in aam Triple A M next year and just today morning I got data from Netherlands it shows that there is a single nucleated polymorphism in SPD which makes patients more susceptible to invasive aspergillosis so that’s about the host side so proteomic from fungal aspect
So here we try to extract surface proteins from dominance pores and during germination we saw that there was overlapping protein in both conditions but interestingly what happens is if you give this extract to the neutrophils then there is less production of il-8 or less phagocytosis so that means fungus is trying to
Counteract the defense mechanism of the host system so we know I think today morning there was a talk about metaloprotease and alkaline protease so what we found was there is metaloprotease and alkaline protease associated with fumigators which are trying to counteract this humoral immune factors so all together
Host is trying to kill fungus but fungus is also trying to counteract the host system so with that I hope that proteomic I convinced you that proteomic provides a platform to understand host fungal interplay because we see the reality here because I’m not against genetics and genomics but genetics and genomics
We don’t see the reality because for example there are 9 900 genes but when you see proteins you see only 5000 proteins so it gives the question whether we have to simultaneously look into proteomics or not so with that I’m ending my talk and these are my acknowledgments thank you [Applause]
Thank you very much vishu and thank you to raise the debate between genomic and atomic because you have something to answer because I think all together now only 5000 proteins have been identified out of 9900 genes yes so what is the rest okay no I’m not I’m not saying it is uh one
Is important one is not important I think parallely one has to look into yeah as I said they are they are different sides of the same coin but it is a really good point is what is happening with the the other four thousand or more you know if we see more
Gene content in other fumig artists what is happening to those are they redundant are they are they are they junk that’s right um what is it um and and so on and so forth but I really are really in enjoyed that and and um what was interesting was the SPD SPD
Shuts down secondary metabolic metabolism in aspergillus is that do you like it yes correctly so that’s what we are observing it is diminishing or down regulating all the transcripts of secondary mental Alliance in thank you yes right now we have time I just I found that fascinating from at
Least one point of view it’s been years I played with compliment and I love somebody getting back to compliment work yes it’s massively important and it has a central role and I feel so uncomfortable when we look at in vitro models where complement is ignored because it’s complicated and it’s
Difficult to add your serum gets inactivated and you know what do you do and your bugs die and um you just leave it out and then you do massively fine proteinomics or other analyzes on a sort of half a model I really like that it’s done properly
But same applies to the genomic and looking at the gene expression side of things that you know you have to have the whole model there to to get anything just like you have to have the right phase and the right right you know of side of of the fungus itself yeah that’s
What I’m I’m a bit worried about some of the research I’m not I’m not commenting on anything for example most of the in vitro Studies have been done using fetal cough serum Which is far away from Human serum okay so we it’s depleted uh in uh
I mean it is it is heat inactivated fetal cough serum what we use wherein you shut down all complement system and it’s slow in immunoglobulin so we are ignoring many things there so and the other side of the story is that that we’ve we’ve learned in the past
Already that of course human body protects itself from its own complement by Massive array of regulators and some microbes can exploit those regulators and use them for their own protection a Candida known for for that for Factor H and and that sort of stuff so I think it’s really important to realize that
That it’s the bigger picture is far more complicated that’s true and uh yeah we we look at so many chemical compounds again I’m not against anti-fungal drugs anti-fungal drugs are targeting only one uh enzyme whereas if you look at SPD um I have not given you the complete picture it is
Down regulating all see there are four forming proteins so cider4 is important for iron acquisition for fungus so for fungus to germinate iron is essential okay so it is attacking multiple Pathways so first I think we have to look into what is the normal condition to combat any fungal or any microbial
Pathogen then probably it will give or lead you to a better idea how to tackle it with and so vishu do you think that in the near future we will have to screen our patients for the SPD polymorphism in order to to evaluate the sensitivity to susceptibility to aspergillus that will
Be very fantastic because uh uh that’s why that’s what I got the mail this morning at 11 there is one snip particularly with the probability value of 0.05 which is quite interesting uh showing the risk factor for IPA so so perhaps yeah but 10 years ago we had the nice paper from
Bush I don’t remember his uh and collaborators and they showed that tlr for polymorphism could be Associated to the susceptibility in um allergenic transplant patients for aspergillus but 10 years after we don’t do that so so tlr is a receptor which is expressed depending on the presence or absence of
Any microbial threat whereas SPD is a constituently present in the lung because it’s the purpose of SPD is something structural so probably it is more stronger candidate to look for than all tlr or any other components because tlr is a I mean it it’s a highly expressed or low expressed depending on the
Presence or whether there is any microbe or not so that’s that same thing holds good for cytokines also because I know some people are interested in cytokines but what time you will be looking at cytokines and you mentioned susceptibility to invasive aspergillosis but regarding chronic aspergillosis
Or abpa what do you think we see that uh in C because uh the proteomic analysis what we did was with the combination of colonization CPA and IPA so it is absent that means it is absent in CPA condition also so ABP I don’t know okay thank you very much we have five
Minutes and I learned during the last meeting in who that now we can it’s nice to have one minutes stretching moving from our our chairs Etc so if you want what you want during one or two minutes you can upload you can do everything but now yeah yeah
It in the different International uh meeting I I saw that so no I have to be there yeah yeah so just just stretch and not moving from uh you can stay here we are going to to move to the next presentation you made such a rich program yeah yeah
It has to be five days not two days so right so no no no it’s not a break no no just foreign okay so we are going to move to the next talk so please we have the coffee break in uh in half an hour thank you so now it’s my pleasure uh
To introduce myself Institute and NECA hospital she’s a medical Mycologist and doing very nice research on candida in the team of Christopher Master Institute thank you Elizabeth thank you Jean Pierre and thank you Robert for the kind of invitation in this wonderful meeting here and thank you for giving me the
Opportunity to talk about genome evolution of candidate and I am going to focus my talk today on a specific question that is how the evolution of considered genomes enable the acquisition of phenotypes important for candida pathogenesis uh among all considered species responsible for infection considered because it’s probably the most relevant
Species for the suffering this equation because candidate because is uh deployed as deployed genome with a complex whole production and also because consider albicans evolves uh all the their the life cycle in an unique overall the human being And recently we show that consider albicans colonize or almost all LC individual but who is very low quantity and with very variable quantity you know is also a major opportunistic pathogen responsible for superficial infection but also for invasive infection in immunocompromise patients and also it’s associated with eye mortality
Therefore in Human Condition because it’s comforted with very different environment different niches with stress full environment which require a significant genetic adaptations uh this is a short reminder that the Genome of Continental because is deployed and with a pair of chromosome chromosome one two chromosome seven and chromosome are but it is very
Important to keep in mind that the Genome of candidal because has a high level of heterozygosy with an average of one heterozygous position every uh every uh area 200 base pair in comparison in human there are there is one heterozygous position only every two kilo per square there are a lot of There are a lot of mechanisms driving genome changes from nucleotide to wall chromosome levels and the Genome of Continental becomes is able to tolerate a rearrangement such as an employee and loss of heterozygosy loh and this loh events can cover short regions of a large segmental part of chromosome or even or chromosome
And just to to remember using uh using data from a wall genome sequencing of multiple uh clinical isolate we demonstrated and we confirmed that the population structure of candidate because is organized organized in different genetic cluster Gathering strengths genetic genetically very close and also we confirm that the reproduction is predominantly clonal with
Diversification with Snips and aloe rash we should also that we can see gen flow events between genetic cluster but it is a raw situation now I would like to just focus my talk on a specific cluster the cluster number 13 because trends of the cluster are phenotypically difference of strengths
Of the other cluster these Trends are less fit less virulent uh the strength of clusters sir steel were called consider Africana because the first isolate were from Africa and the strength uh the strength of high capacity to colonize genetic tract and cause vaginal infection and genetically these Trends are highly
Similar with reduced heterozygous well it is very well very well it’s very well possible to see in this figure because you can see the density of heterozygous of heterozygosity along candidal because according to the cluster and we if we compare the density of sniff we can see that’s for cluster Thurston the the
Color is blue because the number of heterosis tea is very low and we demonstrated finally that we demonstrated finally that some genes uh important for virulence are this routine in clusters and these Trends are very important in the in the very last and it is played why we have a cluster 198
13 restriction we found an accumulation of homozygous non-stop due to nonsense mutations specifically in cluster thirstin and this is fixed is this in this cluster in this foreign therefore here you have an interesting and unique example of Niche restriction in candida population due to extensive loh with accumulation of deleterious
Mutation engines involved in the fitness of conditional because but in during infections and fiction strengths can also also undergo Um different gender variations and we know that loh has a frequency of loh events increase in reference to different stress such as antifungala is position and this is was just this was very well demonstrated by Alex Costa several years ago and she thought that after acquisition of mutations in ERG eleven
For Azul Target or takwan on codeine for transcription Factor regular regular with regulation of ethnic sperms you you obtain and You observe the full advantage of this mutation when the strength is in homologous State following loh events therefore loh is a major driver of Clinic of continent adaptations therefore and we just
Learned that the employee also can facilit facilitate adaptation to niches this is a new paper that showed that and when strength of candidal beacons were pressed in gastrointestinal tract we can see the emergence of strengths with the search chromosome 7. actually this Trends with the search chromosome 7 with a Tresemme of
Chromosome seven are more fit in competitive experiment experimentation in gastrointestinal colonization but this Trends have a low capacity to produce filamentous cells and it is a very different in very different phenotype actually on the chromosome 7 is located energy one energy one is a gen that encodes a negative regulator of
Filamentations and it was demonstration by the author that energy one inhibits filamentation of candidal because in a gen dependent fashions therefore when is trans chromosome sevens this strengths have an fitness advantage in gastrointestinal colonization model this is the first demonstration that the possibility that we can have a reversive
Reversible adaptation to different or snitch by again or loss of a nucleus and you know during infection genetic genetic change can occur in infection strength and often this genetic change are measurable and I would like for illustration to present a short case report of relapse of infections in immunocompromised patients in these
Patients this patient has had candidemia and with blood catheter and urine culture positive foreign and and 14 14 months later the patients as a relapse with multiple multifocal arthritis with culture positive for candidal beacons from HIPAA ankle and and if you look at the whole genome second season if you compare the whole
Genome second season of these three strands collected at the same time you can see different evolution of the Genome of each of his tribe for example you can see an increase of copy number variation with in IPA strength the presence of anuploidy in chromosome are and also in chromosome are you are and
Trismic chromosome one in the strength from ankle secondly you can see a large loh tract events in the chromosome 2 of the IPA strands and in the chromosome are on the chromosome are of the strength from me and interestingly this this case show that each strength have evolved and importantly each other
And it’s very interesting and if you look at the summary of all genomic alterations can be occurred over time and between the different and effective seats you can see that the the each strands have has developed a specific rearrangement and what are the significance of these differences and this rearrangement in
Terms of pathogenicity what are the significance of this rearrangement for the relapse of the infection it is not possible to proceed we will I did we will add some other experiment to a better understand the the rules the impact of this rear arrangement in the tissue and in the pathogenicity of the graphics
In conclusion the main mechanism responsible for evolution of genome during basketballism and affection are elohich and an employees loh Is frequent in conditional beacons with evidence for active diversification of coded albigon through short runs allowed during commercialism evidence of a large Ranch loh during Affliction in human and evidence also
That loh can be associated with fitness advantage and recently we learned that measure the measure and freons of an employee unconscious pathogenicity through changing in the expressions of virulence factors and the interaction Reserves what next it’s your volunteer I think that the future longitudinal studies should assess the evolution
During infection of the gen’s changes to uh to better understand the real contribution of this rare Arrangements in the pathogenicity of the contagious uh just to finish I would like to thank the unit at astitute Pastor that is specifically genre that people work in specifically in
System in the unit thank you for your attention [Applause] thank you do you have some questions about this evolution of Canada because um I always do thank you sorry um yeah my question is about polyploidy yes and we have a patient who has become colonized has a CMC Syndrome has become
Colonized with the panasal resistant candidal begins and we’ve used one drug after another um latest one I brexit van gerb and with every drug now we’ve been recycling them for her management and she responds to all of the drugs for two weeks and then just stops whatever drug you
Throw at her ambosome abrexa and we the only explanation that comes to mind is that there’s something like polyploidy happening and it’s just the the fungus is producing so much Target protein or something and then when we grow it on the plate for a couple of generations
It doesn’t look like it’s resistant to anything because it goes to you probably we haven’t tested that but it’s just single patient but could it happen that quickly and would that or what do you think what’s going on yes it’s all the question because we we don’t know exactly the
Significance of this rare Arrangements but these Arrangements finally uh are very frequent and it’s possible that these Arrangements allows the persistence and the relapse of the infection for example is is a multifocal uh arthritis is not usual in during candidiasis these patients uh has a polyartritis and it’s possible
That after the strength is located in the hip and in the several rearrangements occur occurred and after we have another infection and it’s very and it’s possible that this rear Arrangement and this Trends carrying this world are lost and it’s very important to to understand better understand the contribution of this rearrangement
Because it’s probably very important for us that the infection and to why not to find a prophylactic antifungalow prophetic yeah in her case she has a syndrome she will always get oral and other candida infections over and over again so we just have to play with it this
Okay thank you very much yes one more question so after the debate on proteomic and genomic aspergillus and candida what is the most interesting uh uh hi thanks for your great talk so I’m just wondering how stable is an employee what do you think how stable
An employee is not stable yeah but then and it’s very important thing because uh it’s uh it’s uh things important in the fact of the adaptation is reversible because you can gain an apology and you have an adaptation because you have a search chromosome Activity with different expressions and we can lost this anopology and it’s I think that is mean very important it’s very major driver of evolution fast rapid Evolution during infection in human If it’s unstable how can you catch this an employee and you get the good sequence yes if it’s unstable so when you capture it and you do the extraction when you sequence them yes okay you easily catch the unemployed stage yes but I I catch uh I like I catch an
Employee but there are no place is stable in in the in the in the culture yes it’s a culture yes it’s stable yes in the culture but if you pass this trends in animal models in the gut of animal model you can loss okay yeah because I think that’s exactly
The question that uh let’s say that the candida has become has four chromosomes or three eight eight sorry no sorry I didn’t mean the chromosomes but copies of it to you know been polyploid yes um and that has happened because of the anti-fungal pressure in the patient
You take the isolate the fungus out of the patient grow it happily on Sab agar with no antifungals just nice food why would it carry on with that polyploidy and how quickly does it go back because it probably why would it bother yes probably to be polyploid yes so it
Probably goes back to euploidy so exactly how do you how if I wanted to show that my patient’s isolate does this switching from euploidy to polypodium back and back and forth how can I show it because genetically it just looks like there is one and a half fungal cell
How do you measure it no yes we will uh measure this this fragrance an idea about that is yeah technically it is unstable how can you catch it aneuploidy happens for a reason and you know we kind of say oh well it’s due to pressure to antifungal drug stress but
What triggers the aneuploidy and we don’t know so it’s it’s that that you’ve got to look for what is the mechanism that causes the the the ploidy to essentially turn on that say you know whether your patient’s got it or not but we just don’t know what it is
Yeah so that would be pre-growing the isolate or when taking the isolate from the patient having a bit of azol in the plate and keeping it sort of teased patient yeah I I wonder if you um you could do the reverse put some antifungal drugs on
Your culture yeah yeah to try if you get the unemployed the polypriotic yes you will answer for us okay so thank you very much and now we have a coffee break and thank you for this excellent session on evolution okay foreign Beautiful images thank you thank you Foreign Please