 Okay, thank you to the organizers and to Annette and Mike for the introduction. And so I'll talk to you about some of our work looking not solely at the immune system but at colonization factors. And I think that many of the introductory slides that I normally use have taken out of this presentation because they've already been given, but I want to remind you of the data that we've already seen mostly yesterday and then Maria's work as well. We're talking about the succession of organisms that colonize the infant and how we assemble our microbiomes and what are the molecular mechanisms by which we assemble our microbiomes. And that's really what our lab is interested in is trying to understand on an experimental level, on a molecular level, how microbes colonize us and subsequently how they confer benefits in terms of immunologic and neurologic health. And so one introductory slide only just to remind you about this notion of dysbiosis or alterations. And so this is just another way of looking at the data that we've already seen. This is a healthy gut in people looking across many regions of the gut and highlighting the importance or the prominence of bactoideides and pharmacides in a healthy individual. But then we see that the landscape is quite different in patients with IBD, lowering of the bactoideides and pharmacides and an increase in mainly the proteobacteria, which are minor species in humans. And just by way of setting the context for the types of experiments that we do, I think that this implies there's no cause or effect relationships here, but that this implies that there are bad players and good players and perhaps health and disease is in the balance of the proportions of these organisms. And so once again, we don't really do a lot of metagenomics, and so we don't look at community profiles. But the approach that we've taken is to use model organisms, to use organisms that we can genetically manipulate, associate into mice, and then look at outcomes and then really once again get this molecular relationship between the bug and the host to try and understand how the microbiome impacts us. And so the organism, one of the organisms that we work with is Bactoides fragilis. So this is an organism that's been studied for many, many years, initially by people like Sidney Feingold, who studied many of these anorhose, many of the Bactoides species. And Bactoides, as you know, is the most common genus in most humans in the gastrointestinal tract. It's a very unique organism in many ways. And so people like Dennis Casper and Laura Comstock have shown that Bactoides expresses multiple capsular polysaccharides within each genome, and so this is once again very rare for organisms because most bacteria will express one capsular polysaccharide per genome. There are obviously different serotypes, but here we have multiple capsular polysaccharides, two of which have a very unique structure in that they're positively charged and negatively charged within each subunit. So there's one subunit of these very large molecules. And the molecule that we focused on is polysaccharide A, PSA, because it was previously shown by Dennis and others that PSA induces CD4 positive T cell proliferation. And so we became interested in how that impacts the host during colonization and symbiosis. And so many years ago, we sort of took a leap of faith at the time and wondered whether or not this organism and this particular molecule would be protective in models of inflammatory bowel disease. And we chose IBD because of its proximity to the microbiota. This is the TNBS model of colitis. We've tested several other models as well, where you see an acute weight loss in mice after induction of disease. But if the animals were fed PSA orally, you can see that they're ameliorated from the weight loss. This is the colitis that ensues in TNBS treatment. But oral feeding of these animals with PSA shows pretty much an unremarkable intestine. And we can do the treatment both prophylactically and therapeutically and get essentially the same results. And the idea is that someday PSA hopefully will be a therapeutic for inflammatory bowel disease. And so that's the disease. And so we've looked at the immune response. And here, once again, you can see that there is an increase in T817 cells, these IL-17 producing C4 positive T cells that we've already heard about in the TNBS in the colitic animals, but then highly reduced in animals that were treated with the polysaccharide. And so we wondered what the mechanism was. And there are multiple ways that one can imagine T817 cells, which are pro-inflammatory, could be reduced in number or inactivated. But it appears that PSA doesn't cripple parts of the immune system or the promo-climatic immune system. It actually activates other arms of the immune system, specifically regulatory T cells. And we've already, once again, heard a little bit about these as well. So regulatory T cells are marked by a transcription factor, FOX-P3. And here we use CD25 as another marker. And as you can see, both healthy animals and animals with colitis have the same relative proportions in the intestine of these regulatory T cells. But if we treat animals, even though they were treated with the colitic agent, and if we administer PSA orally, you can see the proportional increase in these FOX-P3 positive cells. There's also a numerical increase. And PSA also increases the transcription of FOX-P3. And so what this implies is that on a cell intrinsic basis, there's an increased regulatory activity. In fact, we measured this in vitro. I won't show the data for the sake of time. But each regulatory T cell is more suppressive if it came from an animal with PSA. And so following our work, several other groups have shown very similar results. In fact, extended some of the results in other systems. And these include single organisms like fecalobacterium presnitsia. This is a wonderful story that this organism, and I think many of you already know this, was isolated from patients or was depleted in patients with inflammatory bowel disease, signifying that the absence of the organism may be a risk factor for disease, and then subsequently shown to have anti-inflammatory effects. Something very similar recently from Hiroshitaka's lab showed that an organism called bifidobacterium brevet induces interleukin-10 and anti-inflammatory cytokine, whereas another probiotic doesn't. And then this was recently published just last year. And then once again, the very famous story from Kenya Honda, both in mice and in humans, that a consortium of Clostridia induced regulatory T cells in the colon and expand their proportions, and also protect against inflammatory bowel disease. And another story from Andrew McPherson showing that altered shadeless flora can induce IT regs or inducible T regs, here marked by Helios expression. So many examples, once again, both individual organisms as well as consortia that induce regulatory T cells. And later today, we'll hear about a wonderful story from Wendy Garrett looking at the molecular mechanisms by which some of these organisms may be inducing regulatory T cells. So this notion of regulatory T cell induction appears to be quite broad in several organisms. So going back to PSAs, then we characterized what the signaling pathways were, and I'm just going to show you just a preliminary or just some data from that. And so we've worked out the entire signal transduction cascade. But here, TLR2 is clearly very important because this is the data I showed you, so this is colitis scores and wild-tap mice untreated and then treated with PSA. If we do the same thing in TLR2, knockout animals, we see no protection. In wild-tap animals, PSA induces interleukin-10. This anti-inflammatory cytokine that I told you about suppresses IL-17. We see no such phenotype in TLR2, knockout animals. And when we look at regulatory T cell proportions, those are not increased in TLR2 knockouts. So TLR2 is clearly important in PSA signaling. And in work I'm not going to talk about, we know that TLR2, both on the dendritic cell and the T cell, is important for these anti-inflammatory effects. And so to summarize the potential of PSA as therapeutic for inflammatory bowel disease, I think the way that this mechanism is working is that PSA is secreted by outer membrane vesicles from Bacteria Spigil. So we've shown that PSA is packaged into these vesicles, taken up by dendritic cells. And in a very unique process presented by MHC class 2 to naive T cells, induces the differentiation of these regulatory T cells in their expansion. Regulatory T cells then produce a cytokine called interleukin-10, which then ameliorates intestinal inflammation. And Lloyd Casper has also shown that the same process, oral feeding of PSA, is protective in models of MS and ameliorate information in the central nervous system. So perhaps there are effects beyond just colitis as well. And so then we got really interested in how this organism colonizes. And in fact, how the bacteriities colonize. And once again, many of the talks have alluded to this notion of who's there and what happens during perturbation. And so we approach this from a, once again, a very different angle of using model organisms to understand what people like David Rellman and many, many others have shown, is that there's a unique pattern in mammals for the speciation of organisms. And so even those in the back row can see that this pattern among humans and other animals is quite similar. So there's some specificity, so some factors that are mediating that colonization event and selecting for particular organisms which can inhabit the gut. Whereas all the other organisms in the ecosystem and terrestrial and aquatic environments are not permanent residents of our intestines. There's clearly specificity in what organisms colonize us and there's stability. And once again, we've heard about this concept as well. And here also is data from David showing in either I'll file up and more particularly in bacteriities is these are three subjects and these are the different organisms in those subjects. And you can see that before antibiotic treatment, there's a particular pattern. These blue bars designate a timeframe where they're treated with Cipro and you can see that there's a decrease in most organisms. But very quickly after the cessation of the antibiotic, those organisms come back. So clearly these organisms are stable on a global level and once again, multiple other studies have shown this and we became really interested in what are the mechanisms that mediate this. And so a very talented student in the laboratory, Melanie Lee took on this very ambitious project of trying to assemble a microbiome from scratch and the way she started doing this is working with organisms that we can culture and genetically manipulate and adding them sequentially to mice and looking at colonization. And so initially if we take a mouse and say this is a germ-free animal and we give it an inoculum of bacterias for jillus, we see a very nice colonization of this organism because germ-free animals are amenable to colonization by almost anything. But if then we, a few days later, come back with bacterias for goddess, a very highly related species, different from bacterias for jillus, you see that there's really no competition between these two organisms. We can do this with bacterias for jillus and bacterias data at Omicron. Once again, see no difference and this makes sense because many of us are colonized by multiple bacterias species. We can reverse the orders of colonization. There are really no effects there. But the surprise came when we took an animal and mono-associated with bacterias for jillus and then challenged that same animal with the isogenic strain, the same exact strain of bacterias for jillus just marked by an antibiotic-resistant gene. And now in a germ-free or at least in a mono-associated mouse, we can colonize or challenge with bacterias for jillus and this organism does not colonize. And this was, once again, very surprising to us. Initially we thought it was the antibiotic-resistant gene so we swapped those around. We've done this by other methods as well. Every single time bacterias for jillus went challenged to a bacterias for jillus and mono-colonized animal does not colonize. The same thing with bacterias for goddess and now we've shown this with four different bacterias species that there's competition within an organism but not between organisms. We don't see the same effect with E. coli. We don't see this with enterococcus fecalis. There's something specific about the bacterias that mediate this phenotype. And once again in data I'm not going to show you. If we take the animal that's mono-associated with either of these bacterias and then give an antibiotic at the time of challenge to which the initial strain is sensitive but the challenge strain is resistant, we can actually displace the initial strain and get the challenge strain to colonize. And I think what this suggested to us that there's some limiting nutrients or some limiting space that the initial organism occupies and then excludes or resists the colonization by the challenge strain. And so what we did instead of taking a larger metagenomic approach is we took an experimental mix approach and I think Fred stole a little bit of my thunder yesterday by introducing this concept and so this is what we do in the laboratories when we have a problem, we design experiments to tackle this. And the way Melanie devises this very ingenious screen is that she took genome fragments from bacterias fragilis and transfected them into bacterias for goddess. Remember these two organisms do not compete with each other. And so imagine if there was a locus or a piece of DNA that conferred bacterias for gillus specific colonization, phenotype perhaps here expressed in red, that we'd have some clones that contain this element whereas most of the clones of bacterias for goddess do not contain this element so they act like bacterias for goddess. And then we took germ-free animals and mono-associated them with bacterias for goddess and then challenged them with pools of these clones that contain fragments of bacterias for gillus. And so imagine most of these in green are bacterias for goddess and as I showed you before they will get cleared over time because they cannot compete with the indigenous strains but if there was one isolate shown in red that contained a bacterias for gillus colonizing factor then it should be able to dual-associate this animal and then after time we can isolate that organism and sequence the genes and see what we find. So she did this for 2,100 clones all in mice and what we discovered were two clones out of an entire pool, the rest were all cleared. Both of those clones contain the same genetic region and that's shown here and then when we sequence this to our surprise maybe not to our surprise is that the genes contain polysaccharide utilization loci and so they have these characteristics C-sustee homologs and I'll talk a little bit about those as well they have regulatory elements up front so the sigma factor and anti-sigma factor and we've shown that they control expression of these genes and we started to characterize this and so once again we work with organisms that we can genetically manipulate so we can make mutants in these organisms and then test colonization factors and so here what you're looking at is day 30 of the exact same graph I showed you earlier so the initial strain with the challenge strain once again by day 30 everything is cleared so you can see very clear phenotypes but if we take animals and monosocate them with the sustee homolog which we call CCFC or the sustee homolog CCFD that we lose this phenotype so if the initial strain does not have this factor then now the challenge strain can colonize CCFE does not have a phenotype and I think we know why that is because there's redundancy in that gene and if we knock out the entire operon we see that there's a loss of function and classical microbiology we can take the mutant and then complement the mutant on a plasmid with the entire operon and once again restore this colonization resistance phenotype so we can do this with bacterias fagilis we did the exact same thing with bacterias vulgotis and saw a similar phenotype where bacterias vulgotis will compete against itself but if you make a mutant of bacterias vulgotis it can no longer exclude challenge by the wild type strain and so as I mentioned these polysaccharid utilization loci have been studied for many years one of the pioneers of this is Abigail Salyers who really figured out much of the biochemistry of this and the way these systems work in a generalized fashion is that Sus C is a poor Sus D is an outer membrane lipoprotein and they take complex sugars channel them through, break them down channel them through Sus C and then the organism uses it for nutrients and so these operons are widely dispersed in the bacterias so data out of Omicron has 88 of them Fragilis has 36 of these and what we've shown is that the CCF homologs those genes that we isolated are very, very unique on many characteristic levels and they're only found once in each of these genomes if you look at their homology and so once again as I mentioned these genes have been studied before but they've been studied in the context of foraging and so this is a very nice work by Eric Martens and Jeff Gordon where they knocked out a sigma factor that controls expression of five of these polysaccharid utilization loci and if they colonize mice on a rich diet they see no competition but if they switch to a restricted diet they can see that the wild type can colonize but now the mutant cannot and so that's where this concept of foraging comes from is if the diet is restricted that the bacteria then use host mucus, likely host mucus as an energy source because they can't use them the more abundant dietary fibers and so once again you get this colonization phenotype once again only upon switching to this restricted diet and then there's a defect in vertical transmission I know this is hard to see but this is this 2008 cell-hosted micro paper if you want to look at this defect in vertical transmission in fact we don't see a defect in vertical transmission I'll get to that in a second but the operand that we're studying is clearly different from this phenotype and Justin Sonnenberg also showed something similar where if he took two organisms in this case bacteria stayed out of Omicron bacteria, Kake, put them on a rich diet and you see that they can both colonize to different levels but if you switch them to a saccharide inulin that Kake prefers and data doesn't then you can see that only during this restricted diet you see this competition and in some cases you do not see competition and so we became interested in this concept of long-term colonization on a rich diet and so the way we approach this is gavaging animals with laboratory grown bacteria is artificial so we looked at horizontal transmission so we took two different groups of animals one colonized with wild type one colonized with the mutant and then co-housed these animals and looked for transfer of the wild type to the mutant colonized and vice versa so here you're looking at the wild type animals that were after co-housing for 14 days so these animals were initially colonized with the wild type bacteria, you can see that here but if we then co-housed them with the mutant bacteria the mutant never transfers into a mouse that has already been established with the wild type but when we do the converse and the initial organism is the mutant then the wild type can very efficiently transfer over and in fact every animal can now colonize so even during horizontal transmission there's a defect for the CCF genes where are these genes expressed so they're expressed in very very low abundance in culture and various degrees throughout the intestine but most highly associated with the ascending colon and so Melanie became interested in what's going on in the ascending colon and started looking down at the colonization of these organisms in intestinal crypts and so here you're looking at images of a confocal micrograph of a whole mount microscopy looking at one particular crypt but of course we've looked at many of them and when you look in the center of the script only wild type bacteriose fragilis can colonize this mouse so once again these are germ free animals and we're looking in the intestinal crypts only in the ascending colon whereas if we mono-associate the mouse with the mutant even though the luminal contents are identical none of these bacteria wind up in the intestinal crypt here's the control for germ free animals and another way of looking at this is through two-photomicroscopy so this is a crypt so the dark area is the void space of a crypt and as you can see only the wild type bacteria can penetrate into the script the mutant is at the epithelial surface but never really gets into the crypt and we can quantify hundreds of different crypts and you can see here we're looking at the distance from the epithelium and the wild type penetrates further into these crypts than the mutant and so what we conclude is that this particular clade of polysaccharid utilization on the side the CCF are required for colonization of these crypts and so finally what does this mean in terms of resilience or stability and so here what we did is we took SPF mice and we used a protocol by Thad Stappenbeck to be able to colonize these SPF mice with bacteroides so we can initially treat them with an antibiotic to displace the indigenous microbes and then colonize them with either bacteria for Jo's wild type or the CCF mutant and here you can see very, very similar levels of colonization but if we then challenge that those animals with ciprofloxacens are very similar to the experiment that David did in humans that you can see that the wild type bacteria is maintained but the mutant bacteria is greatly reduced and that's much more sensitive to ciprofloxacens and in fact what we've done now with this experiment is we've taken wild type associated mice and given them a very high concentration of cipro for a very long period of time we can clear the luminal bacteria but if we look in the crypts of these mice on antibiotic treatment the bacteria are still in these reservoirs they're still in the crypts and so it looks like those bacteria in the crypts are resistant to the antibiotic and another predibration that we use was such a bacterial rodentium infection the paradigm for colonization is the same and then we introduce the cirodentium infection and as you can see in the dark blue bars the wild type bacteria are maintained but here we get even a more dramatic phenotype for the mutant it's completely cleared from the ecosystem and you can never recover this organism again once again the CCF genes are required for resilience and in the black bars are the citrofactor so all in all it looks like these bacteria have evolved mechanisms to stably associate with the host and to populate specific regions of the gut in a way that we think is using polysaccharides and glycans based on homology and we were really interested in understanding what those glycans are and so to conclude I'm gonna actually take a quote from Ralph Fredder proposed 30 years ago and what Ralph did was he took mice and he looked at transit times of various organisms and then used mathematical modeling to come up with this concept that and I'm just gonna read this is that most indigenous organisms in the gut are controlled by substrate competition they're competing with each other for nutrients particularly nutrients and that some species are better than others in acquiring these nutrients and that the population level of those species is controlled by the concentrations of a few limiting substrates and I think this is very important because this nicely explains our data for how organisms will compete against each other but not other organisms because they're using very, very few of these substrates but different substrates across different species and what we've done I think is extended that to this notion that there are populations of cells perhaps microbial stem cells if you will that allow persistent occupation of these satriple niches and once there's a perturbation to the system some sort of environmental stress that disrupts the luminal bacteria that these reservoirs still exist and these reservoirs can be used to repopulate the gut and if our bacteria that only live in the male and colon I would evolve this mechanism to ensure my long-term colonization and perhaps this is one of those mechanisms at least in bacteriities and so I'd like to acknowledge the people who did the work so the PSA work was done by a very talented postdoc June Round who now has her own lab at the University of Utah as I mentioned Melanie worked on the Cryptocupancy project with help from a new graduate student who's dragging the lab as well as SOVA these are the other members of the lab our collaborators and I'd really like to acknowledge Klaus Leu who takes these wonderful images for us and the colon this is an actual crypt of a mouse colon and so we drew in the bacteria but you can see the beautiful architecture here and he was instrumental in us understanding the mechanisms by which the CCF genes are working and of course the funding agencies we've had two grants from NIDDK and one from GM as well as funding from the CCFA and so then in terms of questions and gaps and a lot of this really dovetails off some of the concepts that we've already heard is maybe on a more teleological level how do we assemble the microbiota? Do we choose our own microbiota or do they select us? Have they over the millennia evolved mechanisms to associate with hosts in a very specific way? And I think that if we can understand those molecular mechanisms then it can really understand microbial secession and so once again we've heard a lot about that and maybe we've heard a little bit less about biogeography and so there are different organisms and different regions of the gut I think we know this quite well harder to sample in humans but I think the mechanisms are coming online soon easier and mice but yet still not that many people are doing this I'd like to remind you that there's a different biogeography if you go longitudinally versus cross-sectionally I think that's also important as well and these molecular mechanisms may really help us understand that and the other important question is can we somehow exploit this colonization or phenotype to help us resist pathogens? So there's a lot of history going back many years from Dwayne Savage and Charles Conway talking about colonization resistance in bacteria and if we can somehow engineer organisms to compete against pathogens if pathogens use similar systems then we could perhaps target pathogenic infections by making better probiotics or targeted designer probiotics and ultimately is there a way to exploit the system to correct dysbiosis and so once again we've heard about this concept over and over and if we can understand what those sugars are or understand what expresses those sugars then perhaps we can pharmacologically induce the substrates for these organisms and then once again correct dysbiosis by promoting colonization and then the needs I think some of these were already mentioned yesterday but I think that they're worth repeating because they're quite important is this incredible heterogeneity in organisms or in mice and their microbiota from different vendors and so we know this from the work of Dan Lipman and others that the microbiota is very, very different in different from different commercial vendors and this really affects our experiments and so if you look across the literature for let's say Treg development in the gut you see very different numbers from different facilities and different countries and I think that having some sort of standardization would really help with that and perhaps we can even think about humanizing these mice and having a cohort of mice that could be disseminated to the community and so that we could all be doing experiments on the same plane and then something that I think is quite important but often overlooked is whether or not our germ-free mice are the same and so we clearly know that there's genetic drift in different colonies and even our germ-free animals whether how rigorously we've tested them may have genotypic differences in fact we know this because if you look at microsatellite mapping of a black six mouse from different vendors the microsatellites are different so there has been genetic drift all black six mice are not the same so a central repository I think would be terrific and finally just to reiterate some of the concepts that Maria talked about but perhaps more mice because they're feasible is longitudinal multi-generation studies so looking at not just the lifespan of one organism but the ability of that organism to transfer its microbiota to its offspring on several generations but including perturbations that are affecting our lifestyle such as diet and antibiotics we've become quite interested in environmental antimicrobials I think many of us already know that there are very potent antimicrobials in all parts of our lifestyle and we're ingesting these antimicrobials and perhaps they can have an effect on our microbiome and our ability to pass that microbiome onto our offspring I think this might be a nice way at least in mice to test cause and effect relationships and get away from associations and understand the cause and effect relationships that could be mediating the increase in allergic and autoimmune as well as behavioral disorders so those are my thoughts and I'd stop there and I think we have a minute for questions Sarcas thanks for a wonderful talk as always is there a quick question from anybody? Right in the center Great talk Sarcas can you comment on the strain specificity? So this question of what keeps the same bug from coming in where it already may or may not be and the question of dynamics of different species coming in I think you've hit on something that could be an important part of that so do different strains of fragilis show the phenotype or what's the how far do you have to go before you see this? Yeah we've never looked at strains but those can be important as well and so we've looked at we work with Bacteria fragilis 9343 and we've looked at the genome of 638R as well as the other sequence Bacteria fragilis there are some differences in the CCF loci they may mediate colonization differences but we've actually never done the experiment Sarcas what do you think is going on in the crypts in the differential colonization is that something like substrate availability? Yeah so I think that the homology to these SUS systems and to the polysaccharide utilization loci suggests that there is a limited glycan perhaps a host glycan most likely a host glycan that is specific to the crypts and in fact when we look at a field of a monocolonized animal not every crypt is occupied by bacteria and so even though there's only one organism it doesn't get into every crypt so I think that perhaps what this argues is that not all the crypts are the same and so whether the bacteria induces its substrate or is there the entire time the bacteria have figured out which crypts to occupy and which ones not to so there might be some developmental biology that we can understand through these organisms and I think ultimately once again the way this is going to work is that that limited substrate most likely a glycan is utilized by a specific CCF and if it's utilized by an initial strain then the challenge range can't compete but two different species are not using that same glycan so therefore they're not competing against each other One more quick question So you've shown really elegantly that this works for one strain or one species how many different species substrate combinations do you think there are there are thousands of different bacterial species in the gut I mean is it possible each one of these has its own separate substrate that it's working on? Yeah so we don't know but in terms of so there may be many other mechanisms for colonization that don't involve polysaccharide utilization loci and so the answer is we don't know but when we look at the CCF genes and what types of organisms we can find them in they're only in intestinal bacteriities they're not in bacteriities or even bacteroidities that are not part of the mammalian gut so if you look in other ecosystems they don't have these CCF genes and so I think that this is one colonization mechanism I think it's a colonization mechanism that's conserved in bacteriities but things like clostridium and proteobacteria probably use different mechanisms maybe entirely different mechanisms and once again those are not competing against each other in a way that we can measure Sorry thanks again for a wonderful talk and please Gene Chang from the University of Chicago the title of his talk is Ground Zero the impact of the gut microbiome on host epithelial functions and responses