Hello. My name is Ralph Isberg. I’m a professor in the Department of Molecular Biology and Microbiology at Tufts University School of Medicine. In addition, I’m an investigator of the Howard Hughes Medical Institute. In this talk, I would like to discuss some general principles on infectious diseases which I’ve learned over the years working in my lab as well as interacting with many of my colleagues. In the second talk, we’ll amplify on some of the topics which were discussed in this particular lecture. Now, most of you, growing up, have been warned about things called “germs”, which of course are bacteria, viruses, and parasites which can cause disease. And, over the years, your mothers warned you to wash well, wash your face, brush you teeth, wash your hands… to get rid of these germs because, of course, getting rid of all microorganisms from bodily sites, at least that was our prejudice, would protect us from infectious diseases. However, recently it’s become clear that microbes can not only be our enemies, they can also be our friends. And through… at each of the sites within hosts, including the human, and here we have a typical female, what you will notice is that microorganisms can colonize normally, without causing disease, in various sites within the human. These include oral flora, intestinal flora, flora on the skin at various sites… and these organisms we live with without having any apparent disease. So the questions is, why is it that some organisms, when they colonize hosts such as us, will cause disease while other ones will simply colonize us and continue to live there without, apparently, having any effect on our health. In addition, many of these microorganisms which colonize us, and which do not cause disease, are also health-promoting, and that’s a very active area of research which I won’t be discussing today. So now, let’s look at this view, now, from a typical contrast between an organism which causes disease and organisms which don’t. Now, in this slide we have sites where hosts have microorganisms that colonize them and there is no apparent disease. Now, we contrast this with an organism which can cause disease, Staphylococcus aureus. Staphylococcus aureus can colonize all these sites that normal flora can colonize. However, when colonization in these various sites occurs with Staphylococcus aureus, often the organism can cause disease. Some of these diseases can be extremely serious and can be lethal. So unlike normal flora, when Staphylococcus aureus colonizes these various sites which we’re used to seeing with normal flora, the organism can cause disease. This includes organisms growing within oral flora. Normally we have bacteria which will grow in our throats and in our mouths, but Staph. aureus can cause disease in these sites as well, and what I’ve shown here is a picture of Staphylococcus aureus within a culture from sputum, in which you see throat cells which are surrounded by this organism which is a Gram-positive organism, in blue, around host cells, which are stained with red. This organism is causing disease in the throat. In addition, the organism very commonly causes disease in skin sites, and here what we see is a picture of an abscess where, although we have normal flora which can colonize the skin, Staph. aureus, under various conditions, can cause disease, particularly when there’s breaches in the skin which allow the organism to grow in this particular site. In addition, the organism can enter into other tissue sites, for instance, the eye. As you see with this young lady right here, you see a rather serious case of pinkeye which is caused by Staphylococcus aureus. Again, this cannot be normal flora when the organism is growing within the eye… the milieu of the eye. We do obviously have organisms in our tears and in and around our eyelashes, but these are not causing disease, whereas Staph. aureus, when it starts to enter into this particular site, it will cause disease. And finally, this organism has the ability to enter into sites where we’re relatively free of normal flora, and that includes the lungs, where the organism can cause fulminant pneumonia. Now, it doesn’t stop right here. Staph. aureus is a relatively common disease in this country, but, in addition, infectious diseases can be extremely dramatic, and one of the most dramatic diseases are organisms which cause watery diarrhea. The most famous in human kind are outbreaks of cholera, and this is a picture of a cholera case where there was a lethal disease of Indian cholera in the 19th century. This organism, as you can see, looks relatively benign and, as a matter of fact, probably doesn’t look any different from many non-pathogens. It’s a curved rod with what is a polar flagella, which allows the organism to move throughout a marine milieu. The organism normally colonizes in marine sites and then when it’s ingested, when the water supplies are highly contaminated, it causes a disease which is a watery diarrhea. This can be dramatically seen in a hospital… in hospitals where cases of cholera are treated, it’s commonly treated by patients being put on cots called cholera cots, which is a dramatic demonstration of this disease… where patients are placed on the cholera cots and this watery diarrhea, which is called a rice water diarrhea, simply flows out of these patients into these buckets. Fortunately, there’s a relatively simple therapy for this disease, if the therapy can be instituted in these patients early enough, and it’s called oral rehydration therapy… this can also be given intravenously. These bottles all are intravenous bottles which this patient took in during the course of his treatment for cholera. So, it’s a very dramatic disease which causes a large amount of watery diarrhea, and it colonized in a site, the intestine, where we’re used to having a large amount of normal flora. So, the real question here is, what distinguishes cholera from the normal flora which can be found in our intestine? Now, as our final example of an infectious disease, I’ll take you to one of the most common ones in the world, which afflicts about a billion individuals. And that is tuberculosis. Now, this organism, now, is different from normal flora in that it colonizes areas of the host where we don’t normally see normal flora, and that’s in the lungs, which are relatively free of bacteria. It’s an organism which is very similar to a number of soil organisms… it’s an extremely hearty organism… and when we stain it in slides of the organism, and this is an example of a sputum sample where you can see cells that are coughed up by this individual, the blue cells are cells from the sputum and then the red cells you see are these acid-fast organisms, which is a type of stain which is very harsh and will permeabilize most organisms and prevent them from taking up stains, but in the case of this organism, which is called Mycobacterium tuberculosis, the stain is taken up very well and we can see this disease as it occurs. Now, when fulminant tuberculosis occurs, it has very dramatic effects on the lung and, from an example of such a disease… we can see in the next slide an autopsy from a patient who died of tuberculosis, and this person has cavities in the lung, which you can see right here. This was caused by the tuberculosis. This is not the normal morphology of the lung after this type of disease. Alright, so now what you’ve seen are some very dramatic examples. The organisms which cause these diseases cannot possibly be normal flora, because if they were normal flora they wouldn’t cause the effects that they do. And it’s clear that our normal flora don’t normally cause these types of disease. So the question is, what is it that distinguishes a pathogen from a non-pathogen? So, when you look at it, they appear very similar. In the case of both [a pathogen and a non-pathogen], there’s a series of steps that must occur in order for the organisms to actually be present at our various bodily sites. The first thing that must occur is that the organism must encounter us. So for instance, in the intestine, it’s believed that our normal flora are acquired from our mothers, so the encounter that occurs is the movement of intestinal flora from the mother into the child. And then, the organism must enter into the appropriate site. In the case of normal flora from the intestine from the mother, it would enter into the intestine of the child. And then, once within the intestine, this normal flora would multiply and then grow within that particular site. And then it might spread throughout the intestine in sites which are appropriate for replication of the normal flora. All these things occur both in pathogens and non-pathogens, but there’s something very distinct about what occurs when an infectious disease happens, and that’s the following. In the case of an infectious disease, all the things that you see a non-pathogen promote are also promoted by the pathogen, but in addition, there’s a very important step that’s added on, and that is that the pathogen causes damage to the host. This, again, is dramatically seen in this view of the Middle Ages, in which bubonic plague has infected these two individuals, and you can see these cysts, called buboes, which are packed full of this pathogenic organism called Yersinia pestis. So, this is clearly not normal flora. The organism is found growing in lymph nodes, and it’s causing this distinct damage which can be easily seen by both professionals (MDs) as well as by the rest of us. Alright, so, what is damage? And how does it occur in the host? And how does it distinguish a non-pathogen from a pathogen? So, for the rest of the talk, I’ll talk about some aspects of pathogens which are able to promote damage, and this is a kaleidoscopic view, I’m not gonna go through everything that pathogens promote in order to cause damage, but I’ll go through a few select favorite topics of mine which are good examples of how pathogens cause damage, as well as it’s a good setup for the second talk in this particular series. So, damage is… if you look on Wiki… if you look on Wikidictionary on the web… it causes physical harm, so as to impair normal function. And that’s our definition for what pathogens are able to promote. When they cause damage they impair normal function, and that’s a good way to think about the various topics that I’ll be discussing. So, when we think of impairing normal function, what do we usually think of? Well, there’s three things that imply impair of normal function. The first one is there’s some obvious tissue pathology. That was most dramatically seen in the case of the host which had tuberculosis, in which Mycobacterium tuberculosis caused these cavitous lesions within the lung. That’s very dramatic tissue pathology which could be seen on autopsy. The second is loss of organ function, and that was dramatically seen in the slide in which I showed the young lady in which her eye was infected, and which she is no longer able to see effectively out of that eye. Fortunately for her, this is a temporary condition which could be treatable with antibiotics. And then finally, there’s growth in normally sterile sites. So this is something that pathogens are able to promote that non-pathogens aren’t, in that they’re able to grow in sites where non-pathogens don’t normally grow. An example of this is the pneumonia which is caused by Staph. aureus or by Mycobacterium tuberculosis, which was shown in the previous slide. Clearly, organisms don’t normally grow in the lung, or they certainly don’t grow to levels that we see in those particular cases. We saw dramatic X-rays which were full of organisms within the lung. And so, pathogens which are able to cause disease in the lungs, and cause pneumonia, have the capability… the genetic capability to cause disease in these sites. And so, what are the aspects of the genetic capability of these organisms? And so, what they are able to do is, obviously, promote colonization in novel sites, and they way they do that is by promoting damage in these particular sites, which then allow a niche for colonization. An example of this is meningitis. So, organisms which cause meningitis first invade into the bloodstream, and they’re able to survive in the bloodstream through the eliciting of a capsule which protects the organism from phagocytosis. These organisms are able to get into the meninges eventually, and within the meninges, where they drain from the blood supply, they cause damage. And the damage in the meninges, then, causes this disease which allows the organisms to colonize in this site. And a common organism which causes meningitis is the meningococcus, which causes damage to the meninges and then the organism is able to grow in this damaged site, which is protected from the immune response because of the necrotic damage that occurs in that particular site. So, promoting colonization in novel places is an important aspect of damage. The second thing is that these… the organisms have the ability to antagonize host defenses. Their ability to antagonize host defense means that they can grow to levels that are higher than non-pathogens, because normally non-pathogens are kept in check by the immune response, but pathogens, on the other hand, have a series of proteins which they make, and sometimes carbohydrates, which protect against the ability of the host defenses to attack them. An example of that is in the case of Staphylococcus aureus, again this is a favorite organism for these kinds of examples. Normally, pathogens, when they invade tissues, will induce an adaptive immune response in which antibody is directed against the organism. This antibody then can act to opsonize these organisms and facilitate phagocytosis, and also act to direct the complement cascade to cause pore formation. What Staph. aureus does in order to bypass this particular step is it has a protein on its surface called protein A, and what protein A does is its able to bind the Fc portion of immunoglobulin and turn immunoglobulin on its head, so the active portion of immunoglobulin, the antibody combining region of immunoglobulin, is no longer facing the organism and the opsonic capability of the antibody can no longer work. And furthermore, the complement cascade is no longer able to work on Staphylococcus as well, because the antibody is in the wrong direction. And then finally, what damage is able to do is it facilitates spread. So, once the organism causes some damage, this allows it to go into different sites within the host. And this can be seen, dramatically here, in this case of this skin disease called impetigo caused by Streptococcus. The organism has spread throughout the skin, and the organism causes damage, which allows the organism to colonize at these different sites. Once the damage to tissue occurs, it allows a place for the organism to invade into these tissues and establish disease. Alright, so it’s clear now that the organisms must make different factors that allow these different aspects to occur, and these factors are, for the most part, not shared by non-pathogens, and facilitate the ability to cause damage. Okay, so now what causes it? So, damage is caused by organisms by a variety of factors which the organism is able to produce. These include secreted toxins, which the organism will use in order to impair host cells as well as misregulate them, as well as some organisms are able to cause damage by growing within host cells. Again, most non-pathogens don’t do this, but some pathogens have the ability to grow within host cells and bypass normal killing mechanisms of these host cells. The second way in which damage can occur is by the host response, and we’re most familiar with the host response causing damage when there is inflammation. And so, in presence of inflammation, there is damage which is induced in response to the organism, which we see the damage that can occur in hosts. I won’t talk very much about this latter aspect of disease, and we’ll focus primarily on microorganisms and how they’re able to promote damage. The most dramatic form of damage that’s elicited by pathogens are secreted exotoxins, and these secreted exotoxins take two forms. The first form I’ll discuss are membrane-active cytotoxins. These toxins perform two different duties. The first duty is to bypass the immune response, and the second duty they perform is to acquire important nutrients for the organism to grow within host tissues. These are really interesting proteins, which obviously can insert into membranes, but they are secreted and are soluble before they attack membranes of the host. And, the best characterized of these are the hemolysins. These are multimeric proteins which are secreted by a number of organisms, and what I’ve shown here is one of the first crystal structures of these hemolysins, which is one of the hemolysins that’s elicited by Staphylococcus aureus. And you can see this beautiful multimeric structure in which there is a pore which the protein is able to make, and then at the base at the pore what you can see is that there is a beta-sheet structure which is inserted into the membrane of target cells. The way this protein works is it’s secreted from hosts in a conformation in which the protein is soluble. This can be seen on number 1 in this particular slide. As you can see in conformation number 1, the protein which is secreted by Staph. aureus is perfectly soluble, and then when the protein hits hosts tissues, the protein then undergoes a conformational change in which this beta-strand structure is then inserted into the membrane and then forms a pore in the host membrane. This, then, solves a very important problem for the organism. It’s able to produce proteins which act on the membrane of host cells, but able to also be soluble at the same time. That is facilitated by the fact that the beta-strands, which allow the protein to be inserted into the host cell membrane, are hidden in the initial conformation which is secreted by the pathogen. Membrane active toxins play a number of roles in promoting growth of pathogens, and perhaps the most important role they play is in sequestering nutrients for the pathogen. One important nutrient is iron. Now, it may be surprising to most people that tissues are relatively low in iron. The reason they’re so low in iron is that the host takes iron and binds it in a number of iron binding proteins. The most familiar one for you is probably hemoglobin, which is found within red blood cells. Now, the name hemolysin conjures up this idea that these toxins are able to lyse red blood cells and, indeed, many of these hemolysin-type proteins are able to lyse red blood cells, and this ends up being a very important first step in sequestering iron for a number of pathogens. This first step occurs and the red blood cell is lysed open, and then hemoglobin is released. And then the host can either directly… the pathogen can either directly bind to the hemoglobin or it can make proteins which will degrade the hemoglobin and then allow sequestration of iron through a number of different paths. Now, to illustrate how important iron sequestration is for pathogens, most pathogens actually have a number of different pathways in order to take up iron. This can be seen right here. This is a view of a Gram-negative bacterium, E. coli, which has a variety of different iron sequestration paths that allow iron to be taken up from host cells. Here you see, actually, four different paths which the organism is able to use. Several of them involve the use of hemoglobin to be taken up, or degraded hemoglobin, which can allow iron to be directly taken up into the microbial cell. So, this is a very important aspect of the pathogenesis of disease. The organism makes these toxins in order to facilitate iron sequestration, and to illustrate the importance of this particular aspect of infectious disease, pathogens often have several different ways in order to sequester iron once it’s freed from a tissue site. The second way in which membrane active toxins work is to kill or misregulate immune cells, and the most important immune cell which these toxins are able to kill or misregulate are phagocytes. So, phagocytes are the first defense against pathogen attack, and what I’ve shown you here is a movie of chemotactic phagocytes, which are moving on a substrate of particles. Hopefully you can see these particles, there’s one right here in the center of the screen. The phagocyte moves toward these particles and then engulfs it, and what you can see is that the particle will then be internalized by the phagocyte and then will disappear, and then eventually will be degraded by the phagocyte. So clearly, this is something that pathogens don’t want to occur, they don’t want to be phagocytized, and as I’ll go into in a few slides, these phagocytes have a number of killing mechanisms which can destroy pathogens. So, rather than subject themselves to these phagocytes, many pathogens are able to kill these host cells, or they’re able to misregulate them. And then finally, in the case of some pathogens, but not all, some intracellular pathogens use membrane active toxins in order to lyse open the membrane in which the organism is phagocytized in the host cells. Some pathogens can grow within phagocytes, and they’re able to grow within phagocytes by breaking down the membrane in which they’re engulfed in. And membrane active toxins are able to break the membrane in which the organism is engulfed, and then the organism can grow free within the cytoplasm once that membrane has been destroyed, and allows the pathogen to grow within that particular site. I might add that this is a very small subset of pathogens, but this is a very important aspect of the disease process of these pathogens, in that this toxin is necessary to break through the membrane and allow growth. So just to summarize, there’s three main reasons that organisms will make membrane active toxins. To reiterate, they allow iron sequestration, a very important nutrient which is very difficult to sequester in host tissues, they allow them to kill or misregulate immune cells, in particular, phagocytic cells, and then, in the case of a subset of some pathogens, they’re able to facilitate growth of some intracellular pathogens. So, now what I’d like to do is to talk about another class of toxins. Now, these toxins do not necessarily have to be active against the host cell membrane, but instead, are translocated across the host cell membrane, and they target activities which are found within the host cell. Now, these proteins which can enter within host cells can target a number of different proteins within the host cell. Now, it’s very common for pathogen proteins to target regulatory proteins within the host cell, and it seems as through one of the favorite sites in which these organisms can encounter host cell proteins is the regulatory cascades which are promoted by GTP-binding proteins. Now, GTP-binding proteins regulate a number of processes in the host cells. These include membrane trafficking, it includes translation, it includes other aspects of the host response, which we’ll discuss on the next slide. These GTP-binding proteins regulate host processes by being in the ON position in the GTP-bound form or in the OFF position in the GDP-bound form. Clearly, by regulating the ratio of the GTP-bound form to the GDP-bound form, pathogens can regulate these host proteins and determine how they regulate host cell function. Typically, these GTP-binding proteins, in the ON position, will then signal to downstream targets, and these downstream targets include the following. They include host cell translation: there are GTP-binding proteins that regulate the elongation factors which allow translation to occur and there’s a number of translocated proteins which are elicited by pathogens which can regulate elongation factors and terminate translation in the host cells. The second is signaling from plasma membrane. This is most famously illustrated by trimeric G proteins, which regulate a number of processes in host cells, including signaling from hormones which come from the host cell and then control a variety of regulatory events within the host cells, as well as control secretion in the host cell. This is most dramatically seen in cholera, where the toxin is able to control trimeric G proteins from the host cell membrane. The next is cytoskeletal dynamics. There’s a number of small GTPases which control cytoskeletal dynamics, and these are favorite targets of pathogens, in particular a series of small GTPases called Rho family members, which a variety of pathogens are able to regulate by controlling the GTPase cycle of these small GTPases. And then finally, membrane trafficking. Membrane trafficking is regulated by small GTPases called Rab family members and, again, a number of pathogens are able to translocate proteins into host cells that regulate these Rab proteins. Alright, now, how is it that these pathogen proteins are able to control the GTPase cycle of these GTP-binding regulatory proteins. So, there’s a variety of strategies which they use. The first one which I’ll discuss are the bacterial transferases. So, translocated proteins which are moved from the cytoplasm of the bacterial cell into the host cell are able to enter into the host cytoplasm and then find their target, then transfer small molecules to the GTPase, and these small molecules… and these can be a variety of things… ADP-ribose is one small molecule that it commonly used to regulate these proteins, but also there is glucosylation of these proteins as well, which can lock these proteins in either form… these then will lock the protein in either a GTP- or a GDP-bound form. If the transferase locks the protein into the GTP-bound form it locks the protein into the on position, and then there is continued signaling through downstream targets. The consequences of this transferase can be very dramatic, and most dramatic was probably what I’ve shown you previously, which is in the case of Vibrio cholerae. In the case of Vibrio cholerae, what happens is there’s a trimeric GTPase which is regulated such that it’s turned on constitutively, so that now there is this watery diarrhea we see, where there is unregulated secretion of water and an inability of other cells, which are normally absorptive in the intestine, to take up water. So, here you see a disease which can be explained, in large part, by a single protein which is transferred into the host cell and causes this watery diarrhea. But as you might imagine, bacterial transferases are able to do other things as well. For instance, they’re able to lock the GTP-binding protein in the GDP-bound form and also cause it to be off. Now, in addition, there’s other ways in which these cycles can be regulated, and there are more and more proteins that are being discovered that regulate this cycle in a different fashion. And, one of them is to mimic, actually, a host protein. So, host proteins regulate this cycle by having proteins called guanine nucleotide exchange factors, which take the GDP-bound form of the protein and convert it to the GTP-bound form of the protein. Pathogens can overtake this cycle by secreting bacterial exchange factors into the host cell cytoplasm and then force exchange from the GDP-bound form into the GTP-bound form, and then force the protein into the on position. This in some ways mimics these transferases, but acts directly by mimicking a normal host protein. As a matter of fact, the crystal structures of these bacterial proteins, now that they’ve been determined, look very similar to the crystal structures of host proteins that regulate these same events. And finally, in addition, of course there are, very commonly, bacterial GTPase activating proteins. These, again, mimic proteins which are normally found in the host cells that regulate these processes, and these bacterial GTPase activating proteins, which now force the GTP-bound form of the protein to assume the GDP-bound form of the protein, now knocks this protein into the off position and prevents it from regulating downstream events, and we’ll see an example of this here. An example of this is in bubonic plague. The ability of Yersinia pestis to cause disease is in large part due to inactivation of phagocyte function. Phagocyte function requires the activity of cytoskeletal proteins, which are regulated by these small GTPases, and Yersinia pestis secretes a protein which is a GTPase which then locks these regulatory proteins into the GDP-bound form and prevents normal phagocyte function, with one of the consequences being, in Yersinia pestis, the formation of these buboes – unregulated growth of this organism in lymph nodes. Now, this raises a problem. How do these soluble bacterial proteins get across the host cell membrane? Now, its clear that these proteins are soluble: they’re soluble in the bacterium; they’re soluble within the host cell. So, somehow you have to cross at least two membranes, coming from the bacterium, across the bacterial envelope, perhaps into the extracellular milieu, and then taken up within the host cell. Well, there’s two primary strategies which pathogens use in order to get these proteins into the host cell. The first is this process known as the AB-type toxin, in which these proteins are assembled in two subunits. One, the A subunit, or the Activity subunit, which has its activity in the cytoplasm of host cells, and the B subunit, or the Binding subunit, is the subunit which allows the A subunit to bind the host cell and then translocate into the host cell cytoplasm. This is displayed on this particular slide, in which the AB toxin is secreted by the bacterium and then the toxin is bound by host cells… the specific receptors. The toxin is then internalized into an endocytic compartment and then, from this endocytic compartment, the A subunit is able to translocate into the host cell cytoplasm. Once within the host cell cytoplasm, this A subunit is able to see targets. This, indeed, is how cholera toxin works, through this AB toxin-type pathway, in order for the A subunit to find its target and misregulate host cells. There’s another strategy, in which some organisms have specialized secretion systems. So, instead of eliciting toxins in this fashion, the organism directly docks with host cells, deposits these translocated substrates into the host cell, and then they’re found within the host cell cytoplasm. These specialized secretion systems, which I’ll discuss some more in the next talk, have a variety of functions which can misregulate host cells in fashions which I described previously. Now, the one which is one of my most favorite ones is this protein called YopE, which is a GTPase activating protein which causes the effects in bubonic plague, and which we’ll discuss in detail in the next talk. And, the effects of this particular protein is to interfere with phagocytosis, so that a phagocyte which encounters the microorganism that has the YopE protein has its phagocytosis inactivated by the GTPase activating protein activity. Now, what is it that translocated proteins like to target? And what is it that toxins like to target in general? And a favorite target of secreted toxins and translocated substrates are phagocytes, and the reasons for this are obvious, and what I’ve shown here is a movie of a phagocyte engulfing particles. What you can see is a very dramatic event occurs in which the particle is internalized. And, these are extremely effective host cells which are able to internalize particles. This can be seen also in this slide, where a phagocyte is challenged with a number of particles. As you can see, rapidly, these particles are bound by the phagocyte and then internalized. In general, this is not good for microorganisms, because the phagocyte has a number of killing mechanisms which prevent the microorganisms from growing. When phagocytosis occurs, or binding of the phagocyte to a pathogen occurs, what happens is the phagocyte will elicit a number of killing mechanisms which will act to destroy the microorganism. So it’s clear that toxins, the secreted proteins which pathogens often elicit, are targeting phagocytes because these particular host cells are a first line of defense against the pathogen. Phagocytes have a number of strategies that allow them to kill microorganisms and I’ll go over a few of these right now. This is not… again, this is not a comprehensive list of the strategies, but these are the ones that I think are the most important or the most interesting. So, the first one is there are oxidative killing mechanisms in which pathogens are targeted by host macrophages and neutrophils. Host phagocytes have a complex called the NAPDH oxidase complex, which assembles on the host cell complex in response to binding of a microorganism to the surface of the phagocyte. Most famously, this complex is assembled on neutrophils, which are short-lived phagocytes which normally circulate in the host cell… circulate within the circulatory system of the host and then can migrate into host tissues in response to pathogens. In response to a microorganism, the NAPDH oxidase complex will assemble on the membrane, and then its assembly on the membrane has the consequence that molecular oxygen is converted in oxygen free radicals, and the oxygen free radicals are converted into other molecules which are highly antimicrobial. Clearly, this is something that microorganisms want to avoid, and microorganisms which make secreted toxins are often able to inactivate this particular complex, or they have substances on the surface… that are placed on these pathogens which prevent the phagocyte from actually even recognizing the microorganism and initiating this cascade. The second killing by which phagocytes are able to target pathogens are small antimicrobial peptides, and these small antimicrobial peptides, for the most part, are found in membrane-bound compartments and they are released either into the extracellular milieu in response to pathogens or they are released in a compartment called the phagosome, which engulfs the microorganism shortly after uptake in a fashion which is similar to the movies which I showed previously. So, these small cationic peptides act on microbes by inserting directly in the membrane, and they can occur and kill through two different models… it’s still controversial in the literature how these small antimicrobial peptides kill, but one model is that they form pores similar to what you see with the hemolysin toxins, which pathogens use against phagocytes, and here the phagocyte would be basically turning this mechanism against the pathogen to form pores in the envelope of the pathogen. But there’s other models… I call it the carpet bombing model, in which these proteins will destabilize the envelope of the microbial cell and cause death to the microbial cell. And, one of the most recently discovered, and I think most fascinating, strategies in which phagocytes are able to target pathogens are in the case of neutrophils. Again, neutrophils circulate within the host bloodstream and can migrate into tissues in response to microorganisms. What happens in this particular process is neutrophils, in response to a pathogen signal, will form something called a net, and what the net is, actually, is a response to the microorganism that results in death of the neutrophil and then eliciting of DNA and histones in the extracellular regions, and the DNA and histones which are released by the neutrophil are actually antimicrobial. This can be seen in these various steps. Here we see a neutrophil which is exposed to a pathogen. The neutrophil starts to decay. The red is the nucleus; the green is a cytoplasmic marker. You can see that, in response to pathogen signals, the neutrophil nucleus begins to expand and then eventually the neutrophil begins to explode, and then releases DNA and histones into the extracellular medium. And then these components, actually, are interestingly quite antimicrobial. The reason why they’re antimicrobial is still a very, very active… it’s a very, very active form of research currently. Now, in the next slide what I’ll show you is a movie of this actually occurring. This is actually one of my favorite movies in the whole pathogenesis literature. So, this is an example of what was shown on the previous slide, of a neutrophil responding to a pathogen signal. In this particular slide, the neutrophil responds to the pathogen signal, and then the nucleus will expand and then eventually the cell will explode. So, here you see the nucleus in blue. The cell is becoming degraded. It’s undergoing necrotic death, and then in response to this death what you see… you’ll see an explosion of the cell, and when it explodes it will turn red, through now allowing a fluorescent marker to become exposed to the cell cytoplasm. Alright, then the final thing that I’d like to discuss will be talked about in detail in the next talk, and that is the killing mechanism called nitric oxide. So, nitric oxide is thought of as being a very late antipathogen response by the host. Usually, it only occurs when the host has difficulty in clearing out a pathogen, so that if an organism begins to grow within tissues and neutrophils are unable to clear the microorganism, then there’s a secondary response that occurs, in which host cells which are high in producing nitric oxide come to try to save the tissue from the organism. And the primary host protein which protein which produces nitric oxide is Inducible Nitric Oxide Synthase, or INOS. This protein is found in a lot of different cells. It’s very low in neutrophils, but it’s high in macrophages. And, it’s simply… it’s a biosynthetic enzyme. It takes arginine and molecular oxygen, and then coverts the arginine and the gaseous oxygen to citrulline and NO, and then the NO has a variety of regulatory properties within the host cell, but it’s also an important antimicrobial molecule. And, when it’s produced, the nitric oxide then produces other reactive nitrogen intermediates, and it’s believed that a combination of these intermediates, perhaps not NO directly, but the intermediates that are made in host tissues, act to kill the microorganism. Alright, so this summarizes this talk. What I’ve hoped to tell you about is… and convince you… is that what distinguishes a non-pathogen from a pathogen is the fact that it undergoes a series of steps which non-pathogens undergo which involve colonization of tissues. But then, in addition, it has this added step, which is the central aspect of the pathogenesis of disease… is the ability to cause damage. The reason why it causes damage is not a mistake, but is actually important for the pathogen, because the ability of the pathogen to cause this damage allows the organism to colonize and cause disease. In addition, the damage that occurs now facilitates the organism to spread into other hosts, because it’s able to efficiently colonize within the patient. And finally, the other aspect of damage that’s important is it gives the organism a selective advantage over the normal flora which are in hosts. By causing damage, the organism is able to now establish colonization in niches within the host, which will allow it to efficiently establish what we see as the disease process, but which these pathogens see as the colonization process. Alright, well, I’d like to thank you for taking in this particular talk. In the next talk, I’d like to add a little more color to these particular aspects of infectious diseases. In addition, I’d like to modify some of the ideas which I’ve brought forth to you. I’ve given you a very simple model for how disease occurs, and then in the next talk I’m gonna add some complexity to the issue.