---start--- microbial invasiveness schifferle this is an elective course...allegedly for 20 people but we have too many people, so... this course is organized so that each tuesday...(he didn't finish the sentence) we'll do a voyage of microbe through host, from point of view of microbe and host. you will study that. course requires participation and preparation. we need 5 groups of 8 people. each group essentially takes one day. each time is a topic and these have been chosen to describe the voyage a microbe takes through a host. first colonization on the surface, then minimal invasion, then we discuss about papers, the typical case where a microbe invades the whole body and causes septicemia, etc. sometimes the toxin is more important so we'll discuss that too...and host responses to that...another session will have microbial adaptations to host defenses- here we'll discuss an important issue - the capacity of microbes to stay in a host due to antigenic variation. last day,we'll have a bitch of a day. this course is not really a clinical course but a bridge b/w last year and clinical information. he invites a clinician to come participate in the last lecture. practically... for today, pick up some of those handouts he has here then, for each time, one group of 8 people will have one day to prepare for. there will be essentially three to four papers to prepare. you have to read the paper,and present the topic to the class. you can describe the paper, you can do more reading and do more, you can ask questions of the class, there are many ways, so be creative. one year, two students went up together and one defended and one attacked- it was a debate format. what are these papers? each time, one is more clinically relevant- maybe a case, sometimes something else, whatever. one will describe or present all the issues dealing with the pathophysiology of the problem, and one will be a more complex type of paper dealing with more specialized issues. this gives you some perspective of what's being done at the research level and which may impact what you do in practice in the future wrt vaccines, therapeutic factors,etc. antibiotics don't work so well now,so we're looking for new targets on microbes, new virulence factors to get rid of,etc. so it's going to require participation and preparation, and a lot of it. at the end of this hour we have to form five groups. equal sized. each group has to pick a day to present their presentation. Dr. Littman is here too. question: who answers the questions that are presented? well, in addition to the papers he gives us, he also gives a set of questions for each paper. try to answer them. you do not have to present a q and a format, but try to answer the questions to guide your preparation. if you need help, go see dr. schifferle. better safe than sorry. regarding time - if you have groups of 7 or 8 people, you have to time it so that we can use these twice 45 minutes...you may want to speak 8 minutes each or 7 minutes each and leave time at the end to summarize. another good thing is that the group that's preparing the session each time, the others also essentially look at it and prepare some questions for those people. grading: done on participation and presentation. participation is showing up and asking questions as well as doing presentation. if we have more than 25 students in this class,he is going to be forced to have an additional exam, a take home exam, given at the end of the last session with one week to prepare it. this is new. books: third edition ImmunoBiology; Mim's Pathogenesis of Infectious Disease; Bacterial Pathogenesis; Pathogenesis of Bacterial Infections in Animals. also Carter's from last year. -- VOYAGE OF THE MICROBE THROUGH THE HOST Virulence factors and host defense reponses (see handout) first, we'll define essentially Koch's postulate,we'll use it to define what a pathogen is. maybe you know the two fathers of microbiology - Pasteur and Koch. Louis Pasteur died too soon to get a Nobel prize, but Koch got one in 1905 for his work with vibrio cholera. postulates: microbe should be present in all clinical cases you have to isolate the pathogen in pure culture isolated organism should reproduce the disease (not always easy with slow viruses, or prions, or whatever) molecular version of koch's postulates: phenotype or property should be associated with pathogenic strain specific inactivation of gene associated with trait should lead to measurable loss of pathogenic virulence this is the knockout concept - if you KO the gene for the virulence trait, you should lose disease. restoration of the gene should result in restoration of virulence/pathogenicity now, this is good if the phenotype you study is due only to one gene. but often more than one gene is involved,so there is still a lot of work to do. same thing in immunology - take host, KO gene, look what happens. they do this in mice, mainly. that's how you answer specific questions. there is a lot of redundancy not only in virulence factors/traits,but also in host gene products, host responses to virulence factors, etc. if we make this voyage, we begin - the first thingyou need for disease is an encounter b/w host and pathogen. 1. contact! encounter requirements: travel (host and/or microbe), environmental conditions (vaccination status, etc), transmission (aerosol, fecal/oral, venereal, vector-driven, transplacental/vertical) for example anthrax, carpet makers can get from wool used to make carpets, which is coming from afghanistan or other areas, and the wool contains the spores. aerosol is easiest way to spread disease. venereal disease is harder to transmit (brain power can avoid it...). aerosol can't be avoided. fecal/oral is second easiest way. in a building, you can count 1000 bacteria per cubic meter. since we as humans essentially take 6L air/min in our lungs, we take 6 bacteria per minute into our airways - 10,000 per day. most get stuck in upper airway and then get swallowed or coughed up or whatever. when someone sneezes...lots of bacteria come out of the person. 10,000 droplets, most filled with viruses. depending on size of droplet, they may remain in air and get breathed in by other people. foot and mouth dz virus agent went through the channel from france to england via these droplets,carried by the wind. so distance may not help. 2. productive interaction microbial adhesion to host surface molecules. requirements: microbial ligand - appropriate host/surface - receptor. bacteria attach to skin, mms, conjunctiva, mouth, etc. bacteria utilize sugar and create acid around our teeth resulting in dental caries. the prophylactic use of fluoride in our toothpaste and water makes teeth more resistant to low pH. some molecules (ligands) are expressed only once the bacteria is signalled it is in the right place. viruses already have a ligand on the surface, usually. we'll talk about this later. bacteria like neisseria gonorrhea or moraxella bovis will turn on or off the expression of their pili. so you need to have this thing turn on at the right time. additionally, you need the host to have the receptor for the ligand. this is influenced by host's genetic constitution. ETEC with the K88 fimbriae bind only to specific piglet's receptors. some are susceptible, some are not. age can be a factor - susceptibility may be regulated by variable expression of a receptor in the host, or sometimes when an animal gets older the receptor moves to a different location - so if you have a receptor changeto a soluble receptor it carries microbe away so it can't colonize a surface. say you have the right ligand, the right receptor, the right host... 3. potential progressions -colonization of host surface - local multiplication. this requires appropriate nutrients and resistance to hostile environmental factors. microbe has to outcompete competitors. antibiotic treatment may remove competitive organisms and make your problem worse. if salmonella is there but poorly colonized, and you tx with abx, you can eliminate competition w/o eliminating salmonella, and make a problem.... also microbe can have a protease or something that prevents host defense from working. microbe can circumvent host defense as in gut, host uses IgA, but bacteria may have IgA protease. 4. invasion of epithelial barrier - requires ligand/receptor interaction to be successful. this has to be more than just binding and colonizing on the surface- it has to do or to initiate something, crosstalk b/w microbe and host. usuallywhat happens is that the microbe, if bacterium like salmonella, ETEC, etc, it initiates crosstalk by contact b/w host cell and bacterium, bacteria organize a secretion pathway which allows them to inject some form of toxin into the host to deregulate host biochemical machinery - subverting signal mechanism transduction so host cell thinks it should allow the microbe in. viruses do this too. this is microbe induced entry 5. intracellular survival is the next step. once all we've discussed so far is the epithelial surface.mammalian hosts are covered with epithelial cells on all surfaces. once microbe goes there it can stay there or continue the voyage - cross the apical area of the cell to the basal area of the cell , cross into the subepithelial tissue. well,this is new. something happens there. immediately there is an inflammatory response. rubor,dolor, calor, tumor. you have these invading microbes, cytokines are expressed by host cells... but what happens before that? some microbes can interact directly with mphages by lectinophagocytosis. they have some sugar on their surface which binds a receptor on the mphage and they enter the mphage - but these are special cases and may not induce cytokine response. more typically, the first things that occur are --- what's in the interstitium? all i can think of is electrolytes. oh, complement. right. it's in there. ok. in the reticuloendothelial system there are big gaps in the endothelium... in other tissues, there are smaller openings, like the kidney, which is more selective. but you have proteins like complement in the interstitium. what's the first thing that happens whenn complement interacts with bacteria? activation of complement pathway - C3b. this starts the process of going further til you get C3a and C5a. then the bacteria produce molecules which can have chemotactic activity.this is the beginning. you have to shout out these answers here....this is how the bacteria shout . then you have a host response starting by having PMNs accumulate at the site. studies have shown that after 30 seconds you can see PMNs accumulating once infxn starts. the PMNs come not only from blood but also from bone marrow. they go there and then you get your cytokines coming later on too. then you have some of these molecules which activate cells like mast cells,which are hiding all over like guardians in the submucosa,and they release histamine, serotonin, leukotrienes, other inflammatory substances. so essentially you have a lot of molecules all very early in the process. once you have this chemotaxis, and pmns arriving,then you have mphages arriving - the second wave. mphages and other immune cells will also produce cytokines. IL1 is made early, locally, causing positive feedback. also IL8 to increase chemotaxis. more slowly, others like IL6 for Ab response, acutephase proteins from liver, molecules like IL12 to activate NK cells, TH cells, etc. then you have a whole set of different cells involved. the whole set of things is going on in the inflammatory response. with epi cells wheninfected by viruses they also make interferon a and b. these are small molecules which react/are produced as soon as you have viral replication in the cell, b/c cell recognizes the viral double stranded RNA. these molecules also help initiate inflammatory process. think about phagocytosis too. how are the bacteria able to subvert, avoid, or circumvent these processes? if you have bacteria which resist complement, or viruses which do, or resist PMN products, the you have bugs doing a good job of surviving. virulence factors which make complement useless: capsules, glycoproteins. capsules may not inactivate chemotaxis,but the MAC isn't able to punch a hole into it. so no lysis takes place inhibition of phagocytic uptake - some molecules made by strep - streptolysin, etc - cause lysis of red cells on your agar plate, and also in the host they regulate surface molecules of host cells so that chemotaxis can't occur. adsorption - mycoplasma can just adsorb onto the surface of mphages and the mphages ignore them. it's easy to contaminate cell cultures with mycoplasma also can lyse the cell - pasteurella hemolytica makes leukotoxin, a hemolysin or leukotoxin or whatever you want to call it, which is a problem in shipping fever in cattle. can lyse host cells and cause problems. when you lyse PMNs or mphages, you liberate a whole set of molecules geared against the microbe, so that also can be bad for the microbe...bu you release lysozymes, cationic peptides, etc which can damage the host as well as the microbe. the respiratory burst releases radicals which have strong reducing power and can inactivate host enzymes and stuff. --break--- "bug of the day" lecture at the end is -- the first years they did it, he just chose one. last year, he gave an alternative for students to come with an idea. someone came with an idea - leptospirosis. it has relevance to humans and animals because it is an important zoonosis. the key is the vaccines that are used to protect pets and livestock do not reflect the field strains, so are not protective. so it's a problem. also the signs of the field strians are different from what most people think... but if something new emerges before 10/13, maybe we could talk about that. chlamydia? for bug of the day, everyone prepares, each group has 3 papers or so, every person in the class has to speak. it's tense, it's active, it's fun, you'll see. ok. so we talked about inflammatory response, subepithelial invasion, microbial circumention of host defenses.... 7. intraphagocytic survival: now, some other microbes essentially will use the phagocytic cell as a little house, and will go in andlive their happily and will cause problems that way. have you heard of PRSS? also Dengue. these diseases it is thought that the viruses hide in mphages. also mphage function is disrupted. intraphagocytic survival -how do they survive in there? viruses survive in the cell - well, not the cell, but the DNA or RNA or whatever form they want to take, the nucleic acid takes some form, and gives information to host cell. if it isn't doing anything or provoking a reaction,it can hide there forever. think herpes. it is going to sit in the ganglion, until you stress out and expose yourself to UVlight, and you get vesicles on your lip. also pseudorabies is a herpesvirus in pigs...similar thing where it gets reactivated from a hiding state. HIV can hide also. for bacteria, the way microbes can essentially survive in a cell like mTB or salmonella typhimurium is to inhibit granulation- prevent fusion of phagosome and endocytic vacuole. many microbes go into host cells and then prevent formation of phagolysosome, by subverting the host system - they crosstalk with host molecules, sending signals to go into wrong compartment, they modify compartments to suit their needs. they go into post golgi exocytic vacuoles that look like they should be leaving, and prevent cell from realizing what is going on. they are sending signals - phosphatases, kinases, etc. a lot of these also interact like salmonella, with some kind of protein- changing the way GTP binding proteins interact with normal cells- proteins that are important for cytoskeletal movement. some bugs polymerize actin behind themselves to move around in the cell. you can have viruses moving cell to cell, and listeria and other bacteria moving cell to cell, by creating projections of the cell poking into another cell - so they can avoid going extracellular and risking complement or antibody attack. so, well, all these subversions cause inhibition of degranulation, diversion of lysosomal vacuoles to the cytoplasm (cell lysis), resistance to lysosomal products or acidification -- salmonella for example reacts to acidification by making a new set of gene products. resistance to O2 radicals, oxidative burst... also promotes intraphagocytic survival. so does resistance to NO (produced by mphages), and escape into the RER or cytoplasm from the phagosome - the cytoplasm is very nutrient rich. 8. local or directed dissemination this requires spreading factors or enzymes - once the microbe has been able to survive up to this point, it needs to be able to spread. remember streptococci had an enzyme called galluronidase (?) there are enzymes to degrade collagen - collagenases are produced by psuedomonase...there are also elastases,etc. some fibriae in salmonella and other bugs are known to bind plasminogen and activate it to plasmin, which allows the host to essentially to solublize coagulates.so it binds an anticoagulant of the host, activates it,and uses it for its own nefarious purposes. viruses use the host machinery because they don't have their own. but other pathogens do it just to be mean :) no, seriously, all pathogens use host mechanisms for their own purposes,or if they are new pathogens can cause huge problems b/c they haven't adapted very well and then they will kill the host very quickly - as with ebola or HIV which in the end kills. also the spreading not necessarily around where the entry site was, but also the tissue tropism of the pathogen will sometimes determine the route of spreading the microbe or toxin. some follow nerves - rabies, herpes, clostridial toxin (tetanus - tetanospasmin, tetanolysin) another way more typical of how spreading will occur,mainly if microbial agent will now spread in the host, what's typical in interstitium that makes large compartment proteins go through and get taken up again? lymphatics. you have to imagine this big filtering system. that's where the bacteria will go. frequently mphages pick up the bug, and the microbe uses the mphage like a taxi and goes around to where it has to go. lymphatic system is typical site of early immune response.you have all these activated cells there, you have other cells...in some diseases, you have abscess formation in LNs. Bubonic plague (yersinia pestis), brucellosis, rickettsia, etc. also the place where you have the interaction - think back to immunology - T and B cells. these interactions occur most efficiently in the LN. one of these books has information in it that says if you think about the # of T cells you have to interact with APCs, there aren't that many,but there are so many places where an infection can start...it's like only a few T cells can recognize a few epitopes...for efficiency these interactions have to occur at the LN where there is a funnel effect. if it is too diffuse, there is little change of having the right T cell encounter the right epitope. the LN is the place to be for T cell action :) so...now the microbes can be stopped, or they can get past you again. you get primary bacteremia or viremia if they get past the LN. then they get to reticuloendothelial organs. some microbes can escape lymphatic system when they come through gut and go directly to portal vein via subendothelial blood system. so in liver, the kuypffer cells work to grab pathogens...but they do not do any antigen presentation. the dendritic cells/langerhans cells are the best APC's but they are not in the liver. ---end---