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129 Cards in this Set
- Front
- Back
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Microbiology Intro I
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-microbiology is the study of microscopic living organisms and their effect on other forms of life. Also includes virology and molecular biology
-Candidiasis is yeast that causes oral thrust -Over the years microbiologist have gone from the theory of spontaneous generation to the germ theory of disease |
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Disproving Spontaneous Generation
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1) The first guy to observe microorganisms was Leeuwenhoek who developed a strong lens due to the curvature. He was influenced by microscope making Robert Hooke
2) The theory of spontaneous generation where life forms from inanimate matter was introduced by Theodore Schwann 3) Pasteur was one of the first to disprove spontaneous generation. He put contents in a flask and molded to tops to be sealed or open. The open containers were the only ones contaminated. He also developed pasteurization to prevent spoilage, using heat I4) John Tyndall also disproved SG by using tyndallization. Filtered air without dust would not contaminate organic material |
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Introducing the Germ Theory of Disease
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1) Lister - Noticed that a wound infection was caused by microorganisms so he developed disinfection to remove most of the organisms causing infection. He used phenol to disinfect. Proved miamsa (smelly air) did not cause disease
2) Robert Koch wanted to prove that organisms cause disease. To purify the specific pathogen he was agar which was great because it solifies to a gelatinous matrix that contains bacteria nutrients and bacteria don't move on it. Therefore, when you streak bacteria on the surface the colonies stay in one place. Came up with Koch's Postulates -Germ theory of disease states specific microorganisms could infect humans to cause disease |
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Koch's Postulates
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1) Microorganisms are found in disease but not healthy animals
2) Microorganisms grow in pure culture from diseased animals 3) Cultured microorganisms produce disease in healthy animals 4) You can reisolate the disease microorganisms from the infected animals |
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Movies
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1) Leeuwenhook - Silver/copper frame to hold lens. Lens with large curvature to get better magnification.
-Hooke published micrographia about organisms and this inspired Leewen. Leewun first to see protizoa and bacteria (from teeth), first to publish these results. Also made infusion culture of microorganisms. Became the father of microbiology 2) Lister - First to perform surgery under anti-septic conditions. Microorganisms cause organic mater to ferment and rot (putrefication) published by Pasteur. Used phenol to disinfect instruments and physicians had to wear gloves. Stopped using porous material in manufacturing surgical instruments. |
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Organization of Microbial World
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-science of classifying species is taxonomy
-used to think that eukaryotes comes from prokaryotes but this not true. Domain model shows that a origin divided to produce prokaryotic bacteria on one end, and another molecule that split into eukaryotic organisms and archaea -3 major domains break it into 3 major phylogenetic trees. They are bacteria (procaryotes), eucarya (eukaryotes), and archaea -In this class prokarya synonymous with true bacteria -Archae come from extreme temperaturs, they are not bacteria |
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Comparing Eukaryotes and Prokaryotes
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-Carl Woese used genetic analysis of the 16S rRNA to determine the 3 domains. The 16S rRNA is a part of the 50S (large) ribosomal structure that contains RNA and proteins. Found that variable region of archaea extremely different from bacteria that gave them their own domain
-Margulis coined endosymbiotic theory to explain presence of bacteria like mitochondria in eukaryotic cells. Said that Pro's working together over years evolved into interdependent Eu's. |
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Comparing Eu's and Pro's
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1) Organelles - Eu's have many types of organelles while Pro's have no true organelle except some with glycogen or membrane extesnions
-Eu's are very compartimentalized while Pro's have no membrane in their cytoplasm 2) Sterols - Eu's synthesize sterols while Pro's don't 3) Ribosome - Eu's have a 80S ribosome (mitochondria has 70S) while Pro's have 70s 4) Outer Wall - Eu's have no cell wall or structure with cellulose while Pro's have a peptidoglycan cell wall 5) Transcription - Eu's have exon/intron (intervening sequence), splicing, with trans/trans in separate compartments. Pro's have everything in the same compartment, no exon/intron or splicing, and trans/trans happen at same time, it is COLINEAR |
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Comparing Pro's and Viruses
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-viruses cannot be grouped into the domain system because they don't have 16S RNA
-virus affects Eu's while a bacteriophage infects Pro's -Pro's have DNA and RNA while viruses have one or the other -Pros have lipids, viruses do not, instead they have a protein coated envelope -Pros have a energy metabolism and trans/trans while viruses do not -Pros autonomously replicate by cell division while viruses reply on a host |
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Size Matters
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-the surface area/volume ratio determines who big a cell can get and the size
-If the ratio is too low then the demand on biosynthesis and metabolism is too high, can't get enough nutrients -If the ratio is low then it also affects rate of cell division |
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Video Lecture 2
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1) Bacteria Shape - rod shaped bacilli, spherical cocci, helical shaped vibrio (curved), spirilla (thick helical) and spirochettes (thin and flexible), rarely cubic
-shape confers selective advantage in environment. Large surface/volume ratio to take up lots of nutrients for high metabolism. Use filiments and stalks to increase ratio -Fast swimmer is a rod, length to width important. Use helical shape to corkscrew through vicious medium. -Used to protect against prediction by being too large, small, or akward shape to be devoured -Shape is maintained by the cell wall peptidoglycan. Shape of wall is determined by how it is deposited which is determined by cytoskeleton. Cytoskeleton made by tubulin (construction of septum and poles) actin localize peptidoglycan synthesis in lateral wall of rod |
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Bacterial Classification 1
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-Developed by Linnaeus, it uses a binomial system of nomenclature to identify the genius and species in the name
Step 1) Use Gram stain to separate all bacteria (except acid-fast) into either gram positive or gram negative. After you stain with crystal violet and treat with iodine the violet is fixed to the gram positive. Washing with alcohol removes the stain from gram negative. Adding safranin adds a red counter-stain to the gram negative 2) Use the acid-fast stain for these bacteria like Mycobacterium Tuberculosis (leprosy). Add carbolfuschin (makes them red) and heat to get them red. Wash with alcohol to remove from non acid-fast. Counter stain with methylene blue |
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Bacterial Classification 2
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2) individual bacteria can be distinguished by their shape and color
Fusiform - Bulge in the middle Square - Grow in sheets 3) Bacteria can be classified by their colony morphology 1) Shape - circular, rhizoid, irregular, filamentous, spindle 2) Margin 3) Elevation 4) Size 5) Texture - smooth or rough 6) Appearence - Glistening or dull 7) Pigmentation 8) Optical Property - opaque, translucent, transparent -some colonies like S. Mutans change shape depending on their growth conditions -A aggressive periodontitis bacteria, A.A. has a star inner star shaped morphology 4) Bacteria can be classified based on their metabolism, called biotype Ex: A.A use catalase to turn perioxide to oxygen and this appears as yellow bubbles -Gram- Bacteriodes make melanin and this appears as black spots -whether or not bacteria use fermentation to make lactic acid -what kind of condition bacteria gorw in |
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Bacteria Classification 3
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5) Serotype - based on immunospecific reactions (test using ELISA, immunoflourescence, immunoblotting, any antigen-antibody reaction)
6) Genotype - You can use the 16S rRNA sequence to distinguish microorganisms into classes. -You can use PCR, northern blot (rna), southern blot (dna), and genomic sequencing which is cheaper recently |
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Bacteria Classification 4
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6) Phenotype - Use methods that rely on the physical attributes of the bacterium.
-This can be achieved by a differential or selective growth media. For example you can select for bacterial products (enzymes/pigment) or metabolism of sugars (fermentation) -Using automated phenotypic identification you can put bacteria in media and depending on the results you can classify |
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Bacterial Classification Flow Chart
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-Start big and work your way down to more specific
-Start with Gram stain, then move onto shape and then sugar metabolism (anaerobe, aerobic), and then the biotype, serotype, and phenotype -Using a phylogenic tree you can get genetic relatedness based on GENOTYPE. The length of the line indicates how related they are |
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Types of Media
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-media is used to select bacteria based on phenotype, biochemical, and metabolitic difference
1) Nonselective Media - A media that supports many types of bacteria. Use it for isolating unknown bacteria 2) Selective media is used to eliminate a large number of bacteria at a populated site. It contains inhibitory agents that select against irrelevant bacteria. 3) Differential Media - Example of biochemical identification where you select for a certain bacteria based on its pigmentation, extracellular enzymes, or metabolism. Example is lactose to fermenting/nonfermenting bacteria |
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Bacteria Structure
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-all have a 70s ribosome
-all have inclusion bodies - cytoplasmic aggregate of proteins -all have a cytoplasmic membrane with a phospholipid bilayer -The cell wall between species is different. While Gram + have a thick peptidoglycan layer, Gram - have a thinner layer, followed by a periplasmic space (gel like with proteins/enzymes) and a outer membrane -Within the cell wall different species have extensions like flagellum and pilus/fimbriae -Outside the cell wall can be a capsule/glycocalyx |
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Mycoplasma vs. Mycobacterium
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Mycoplasma - Does not have a PG layer, only a cytoplasmic membrane
Mycobacterium - Acid-fast bacteria that cause tuberculosis. Have a intracytoplasmic membrane within the cell that contains the nucleus. Then around the cell there is a plasma membrane, a complex cell wall with a PG, and a enclosing membrane around the cell |
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Bacteria Cytoplasmic Membrane
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-typical phospholipid bilayer with proteins embedded
-metabolic activities that would occur in eukaryotic organelles must occur in the bacteria cytoplasm |
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Cell Wall Structure
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-made of a glycan backbone, tetrapeptide side chain, and a cross-bridge between the side chains
-While the glycan backbone remains constant, the tetrapeptides and the cross-bridge differ between species 1) Backbone is a repeated dissacharide chain with N-acetylglucosamine and N-acetylmuramic acid which form B-1,4 covalent linkage around the cell 2) The tetrapeptide is connected to the NAMA. It consists of a L-alamine, D-glutamic acid, 3 variable but usually D,L-diaminopimelic acid (DAP), and D-alanine -some oral bacteria have lanthionine at 3 instead of DAG 3) Cross-bridge (not every NAMA, but most)- The AA at position 3 of NAMA is connected to AA at position 4 of NAMA. Usually the amino group of DAP is connected to the carboxyl group of D-alanine. -Lysine precursor DAP is unique because it have make 3 bonds, 2 of which are peptide bonds, and is perfect for this role. -one peptidoglycan layer is one giant PG molecule, and cells usually have 40 or so |
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Peptidoglycan Properties
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1) PG is slightly porous so needed molecules can penetrate through
2) G+ PG is thicker and harder to penetrate because of the additional layers, plus G- is not as highly crosslinked 3) The PG helps the bacteria exist in different environments because it can withstand the turgor pressure from inside and outside so it doesn't plasmolysis (shrink) in hypertonic conditions (salt) or cytolysis (explode) in hypotonic conditions -boiling bacteria in sodium sulfate where you only keep the PG keeps cell as round sacs of PG and maintain cell shape when flattened 3) Peptidoglycan is anchored to the cell by interactions with the cytoplasmic membrane |
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Peptidoglycan Distruction
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-Lysozyme cleaves the B1-4 link between NAGA and NAMA
-THe results is a disaccharide crosslinked to another disaccharide instead of a uniform sheet, this is susceptible to lysis -Lysozyme is actually more effective on G- because they have less PG |
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LTA
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1) Lipoteichoic Acid (LTA) - These are Gram+ only amphiphilic polymers connected to cytoplasmic membrane by a glycolipid anchor. They are repeating with a glycolipid end (phobic) as part of the cytoplasmic membrane and a repeating polyglycerophosphate end (philic) that runs through PG layer
-Usually glycerol + phosphate or ribitol + phosphate as the polyglycerophosphate -LTA is used for adherence, anti-phagocytotic, and as a antigen |
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LPS
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The G- outer membrane is asymetrical due to outer leaflet LPA while inner leaflet like inner membrane
2) Lipopolysaccharide (LPS) - Only in G-, amphiphilic, and the lipid A is part of the outer membrane. Made of lipid A, core, and a O-polysaccharide a) Lipid A - Embedded in the outer membrane. It is a NAGA disaccharide with phosphates attached and a fatty acid tail. It can function as a toxic endotoxin (not secreted) that induces inflammation b) Core - A few monosaccharide chain attached to lipid A through 2-keto-3-deoxyoctulosonic acid (KDO). The core has 2 typical sugars, KDO and hexose c) O-polysaccharide - Binds to the core as a unique tetraglycan and then repeats several times. It is also known as O-antigen because it is variable between species, allowing us to target a Ab for serotyping -The LPS O-polysaccharide is negatively charged non-covalently cross-bridged by divalent cations so it keeps out hypophobic particles. Allows hydrophillic entry of a particular size through porins which are only in the outer membrane only. They are 3 subunits and protect against antibiotics |
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Acid Fast Membrane
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1) Cytoplasmic membrane with phospho-inositol
2) PG layer while manno-phopho-inositol repeat running through it 3) Mycolic acid arabinogalactan, and lipoarabinomannan create a waxy hydrophobic layer on the bacterium. prevents G+ crystal violet stain penetration. Mycolic acid and aribano-galactan is used to inhibit phagolysosome -Due to the mycolic acid these are slow to grow and take up nutrients |
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Extracellular Polysaccharide (capsule/glycocalyx)
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-The capsule is outside the membrane and attached to the PG, the OM, the LPS, or LTA
-Known as K-antigen because the chemical composition varies so it's good for serotyping -It is a highly branched polysaccharide, many times the bacteria area -It is anti-phagocytic, a energy reserve, used for adherence, a toxin -Some bacteria can have a capsule present in good or bad times, some just under extreme conditions |
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Flagella
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-a long and thick structure made out of flagellin it is used for bacteria motility and chemotaxis
-absent in acid fast -it is anchored into the membrane as a motor protein, and one directional rotation is movement and the other is chemotaxtic tumbling -polar flagella has a single flagella at the end, axial (Spirilla) has multiple on the end and can spin, and peritrichous have flagella all over which rotate and work in unison -flagella is known as H-antigen can the protein can be used for serotyping |
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Pili
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-A short and thin cell extension, made from protein pillin and used for adherence, coaggregation, and conjugation
-absent in acid-fast -The common pilli help bacteria bind together. The corn cob is a pili found in plaque -The sex pili is longer and used for conjugation |
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Peptidoglycan Podcast
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-have wide pores allowing proteins to diffuse
-targeting PG by mutation or antibiotic results in cell lysis -periplasmic space |
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Bacteria Metabolism
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1) Carbon Needs
a) Autotrophs - Can grow with only carbon dioxide present, the majority b) Heterotrophs - Require a organic source of carbon, all of the oral bacteria. Can grow in CO2 but not use it alone 2) Oxygen Utilization 1) Obligate Aerobes - Require oxygen for growth 2) Obligate Anaerobes - Cannot grow with oxygen present 3) Faculative - Grow with or without oxygen -anaerobes can't grow in oxygen because they are missing Superoxide Dismutase. SOD gets rid of oxygen radicals which others cause DNA problems 4) Bacteria that require CO2 for growth use a process called heterotrophic CO2 |
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Bacteria Temperature Requirements
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Mesophiles - only grow between 20-40 celsius. The ones in our body
Thermophiles - Grow between 50-60 and used for PCR Psychotrophiles - Grow between 10-20 and found in the artics -thermophiles and psychorophiles mainly belong to archae |
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Bacteria Transport
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1) Simple Diffusion - Penetrate membrane without help. Passive
2) Facilitated Diffusion - Substrate helped by a transmembrane protein. Passive 3) Active Transport - Couples transport to ATP to either move in molecules against their gradient or phorphorylate sugars so they cannot leave because bacteria membrane and sugar are now negatively charged -examples are proton symport, shock sensitive, sodium cotransport, and phosphotransferase system |
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Phosphotransferase System (PTS)
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-uses carrier proteins and enzymes to phosphorylate sugars as they enter so they cannot leave
Glucose: 1) Enzymes 2 is a transmembrane protein that bring glucose to the inner cytoplasm surface 2) Enzyme 1 transfer a phosphate from PEP to HPr, which transfers it to Enzyme III 3) As glucose enters the cell enzyme cell phosphorylates the glucose to make G6P -One set of PTS enzymes for every sugar Sucrose Transport -a little different. Sucrose goes through a porin in a G- and meets up with enzymes II (sucrose) which does the work of glucoses enzyme III and enzyme II. -Sucrose enzyme 2 will take sucrose across the cytoplasmic membrane and transfer a P from HPr to make Sucrose-6-Phosphate (uses same Enzyme 1 and HPr for all sugars) |
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Bacteria Glycolysis
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-oxidizes sugars to produce usable energy through catabolic pathways and intermediates for biosynthesis through anabolic pathways
-Glycolysis is diverse among species, doesn't always complete, the pathways are interconnected, and tries to oxidize the initial substrate to the simpliest chemical form (carbon dioxide) |
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Embden-Meyerhof-Parnas (EMP) Pathway
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-uses substrate level phosphorylation and 2 ATP to make 4 ATP (2+), 2 moles of NADH, and 2 3-carbon products
-Not an isolated reaction, works with other reactions which are coupled to it. An example is PPP Glucose = > G-6-P (-ATP) => F-6-P (Phosphofructokinase - PFK) => Fructose-1,6-diphosphate (-ATP) => 2 moles of triose-3-phosphate => 1,3-diphosphoglycerate (+NADH) => 3-phosphoglycerate (+ATP => 2-phosphoglycerate => PEP => Pyruvate (+ATP) |
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Pentose Phosphate Pathway (PPP)
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-uses 1 ATP, make 2 ATP for a net gain of 1 ATP. Also produces 1 NADH and 2 NADPH2
-At pentose-5-phosphate the pathway can continue to supply intermediates for nucleic acids, or it can feed back into glycolysis. If it feeds back into glycolysis there is only 1 3-C product so that's why it yields just 1 ATP G-6-P => 6-phosphogluconolactone (+NADPH) => 6-phosphogluconate => pentose-5-phosphate (+NADPH)...and this point it splits a) Yields acetyl phosphate and triose-3-phosphate which continues with glycolysis b) erythrose-4-phosphate => nucleic acids |
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Pyruvate Metabolism
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-for anaerobes they proceed with fermentation to produce carbon dioxide, acids, alcohols, and NAD. Most bacteria have a single characteristic pathway. No ATP is made
-This yield 2 ATP and is a example of substrate level phosphorylation -for aerobes they continue with respiration to make ATP by catabolism of pyruvate through the TCA cycle. This yield 38 ATP and is oxidative phosphorylation 1) Pyruvate (3C) loses a carbon dioxide to become acetyl-CoA (2C) 2) PEP gains a CO2 via biotin and PEP carboxylase to become OAA (4C) 3) The acetyl-CoA and OAA combine to make citrate and begin TCA -Through this part of the cycle no ATP is made, only NADH, and then in TCA more NADH is made and CO2 is released. -the carboyhydrate metabolisms in bacteria (EMP, PPP, TCA) are not a complete cycle -facilitative can do either depend on what is present -Since there are many different products of bacteria fermentation metabolism we can use this to classify bacteria -Some bacteria like Streptococcus are homolactic, only make lactic acid, some are heterolactic |
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Electron Transport Chain and Protein Synthesis
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-The chemiosmotic hypothesis by Peter Mitchell reports that ETC is coupled with ATP synthesis
-Mitochondria membrane proteins (riboflavin) oxidize NADH and pass electrons down the chain until the final electron acceptor which is oxygen. As the electrons are passed H+ is pumped through a proton gradient to the outside -As H+ rush back into the cell this drives ATP synthetase which couple ADP to Pi to make ATP |
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Reductive vs. Oxidative Arms of Bacteria Carbohydrate Metabolism
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-For facultative bacteria glycolysis goes by fermentation if oxygen unavailable, but to switch from respiration to fermentation a change occurs in the TCA. This change reverses the direction of the upper half or reductive arm to make TCA 2 branches
-When oxygen is plentiful the oxidative arm of TCA is functional which produces a-ketoglutarate and then succinylCoA via a-ketoglutarate oxidase -When oxygen is low, a-KG oxidase turns off as does the oxidative arm, and the reductive arm turns on to yield succinyl CoA -Instead of going from OAA => a-KG => succinylCoA => OAA, the reductive arm turns on to go from OAA to succinylCoA -Oxidative arm yields NADH2 while reductive arm yields NAD |
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Heterotrophic CO2 Fixation
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-requires atmospheric CO2 for ATP synthesis
-lower half of TCA (oxidative arm) is missing 1) Pyruvate is converted to acetic acid as in the fermentation pathway 2) Atmospheric CO2 (really HCO3) and ADP is used with PEP carboxylkinase to fix PEP from the EMP pathway to make OAA and ATP 3) A second mole of ATP is made by converting pyruvate to acetyl phosphate and ultimately acetate -Basically a modified TCA cycle where the main focus is taking PEP, ADP, and atmospheric CO2 (HCO3) and using PEP carboxylkinase to make ATP and OAA (recycles NAD through reductive arm). Additionally, pyruvate further metabolized to make ATP. Get 2 ATP and NAD in total Aerobic and Heterotrophic PEP decarboxylation are very different. -For aerobes/faculative this part of the cycle nets no ATP production, only NADH, and then in TCA more NADH is made and CO2 is released. This is VERY different than heterotrophic CO2 fixation because these organisms cannot go through oxidative phosphorylation, so this part with PEP and pyruvate, although it looks similar, actually has different enzymes and atmospheric CO2 to produce 2 moles of ATP for usage |
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E. coli O157:H7
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-causes famous diahrea
O157: LPS o-polysaccharide o-antigen recognized by AG 157 H7: Has a flagellum -Contaminated hamburger -It is a enterohemoagic E-hec strain -Produces large quantities of toxins to that damage intestine/kidney -Produces viratoxin and shigalatoxin |
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Horizontal (lateral) Gene Transfer
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-process where a microorganisms get genetic material from another microorganism that is not its offspring (or even same species sometimes)
-allows the receiving organism to evade host defense, acquire resistance to antimicrobial agents, and form a new species (salmonella and e. coli is just inversion of half chromosome) -the mobile genetic elements that pass between bacteria are extrachromosomal plasmids, transposons, and bacteriophages |
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Nonmobile and Mobile Genetic Elements
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1) Nonmobile - Chromosome - supercoiled
2) Mobile -Plasmid (conjugative plasmid)- dsDNA that is a circular and linear array of genes (one after the other in sequence) and caries a set number of genes and controls its copy # in the cell Toxinogenic Factor - Codes or toxin that affects humans, a surface antigen usually Metabolic Factor - Codes for a enzyme in involved in catabolism like a fermentation gene Bacteriogenic - Produce toxins that affects other bacteria. Usually results in a clearing zone, due to competition of bacteria who live in similar environment and compete for resources. Exp: Membrane hole puncher colicin |
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Transposons
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-linear dnDNA that is incredibly mobile and able to excise itself from chromosome in one location and move to another or even to a plasmid
-It has inverted repeats at the ends that allow it to remove and insert itself -Uses repeat to search for a direct repeat site -Discovered by Barbara McClintock |
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Bacteriophage
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-Held within a virus the nuclear material can be DNA, RNA, ss, or ds
-The nucleic acid is in the head which is surrounded by a protein coat (capsomere) that self assembled (nucleocapside) -The tail is a hollow sheath and ends in a baseplate with attached tail fibers are feet that bind to the cell -Classified based on nucleic acid, presence of a tail, and size of capsomere (6 types) dsDNA - contractile trail, long tail non-contractile, short tail non-contractile ssDNA - filamentous, no tail large capsomere ssRNA - no tail, small capsomere |
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Bacteriophage Infection
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1) Method - Uses tail fiber to bind the bacteria pili, LPS (o-antigenic), or another cell region. Injects nuclear material in the cell
2) Growth - In the eclipse phase you don't see bacteriophage cause still taking over machinery. Then a exponential increase which levels off when bacteria supply is low. This is a intracellular growth curve -One-step growth curve (extracellular): |
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Transformation
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-naked dsDNA picked up from environment
-major experiment done by Griffith. He used S-strain pneumococcus (streptococc)i that killed and R-strain that did not. When S-strain boiled first it did not kill. However, when boiled S-strain and live R-strain added, it killed. Obviously genetic material was transferred from S to R, he called this a transforming principle 1) Bacteria sharing DNA release a competence factor 2) Receiving bacteria has cell surface receptor that binds competence factor and tells bacteria to turn on transformation genes 3) Bacteria produce autolysin which cleaves the membrane exposing DNA-binding proteins and nuclease 4) The DNA is bound and one strand degraded by nuclease 5) ssDNA associates with competence-specific proteins and enters the cell where it replacements a endogenote DNA (host) with the donor |
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Conjugation
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-conjugative plasmid or transposon for genes required to have bacteria love making
-Cells with the F-plasmid produce the sex pili needed to mate -The F+ cells mate with F- and transfer F-plasmid so they become F+ -Sometimes F-plasmid incorporated into chromosome and then cell is a Hfr, super male -During mating a F- can receive the Hfr F-plasmid and some of it's own genetic material to become a F'. -During Hfr mating the gene transfer starts right next to F-plasmid and ends at F-plasmid. Therefore, depending on when sex ends different combo of genes can be transferred. This information was used to map E.Coli chromosome -major contribution made by the Lederbergs and Tatum |
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Transduction
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-bacteriophage gene transfer
-After a bacteriophage injects its genetic material it can continue in 2 paths 1) Temperate phage make bacteria enter enter lysogeny where they incorporate their material into the chromosome, this is called a prophage. The material will replicate with the cell 2) Virulent phage take over the bacteria machine and make new phages that release, killing the bacteria, and infect a new host. This is putitng the bacteria in a lytic cycle Temperature phages can be triggered into virulence -The lytic cycle sometimes leads to bacteria genetic info being incorporated into the virus leading to new strands |
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Control of Bacteria Metabolism
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1) Biochemical synthesis does not take place until the substrate is used up
2) If multiple substrates exist then the one more quickly metabolized (and supporting faster growth) is used first |
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Mechanisms of Control - Enzyme Control
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1) Feedback Inhibition - A metabolite has an affect on a earlier enzymes. Turn anabolic/catabolic pathways on or off
-Often the enzyme has a substrate binding site and a allosteric site that closes the substrate binding site a) Cumulative - multiple products can culumlative enhance, turn off, or have different effects on the pathway b) Sequential - Products can turn off certain portions or branches of the pathway but not the whole thing c) Feedforward - Earlier product can actually turn on or inhibit a later enzymes -In glycolysis F-1,6-diP turns on lactate dehydrogenase and G-6-P turns on pyruvate kinase, and triose-3-phosphate turns off pyruvate formate lyase. Switches cell from heterolactic to homolactic fermentation |
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Transcriptional Control
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-used to regulate gene expression
1) Local - control of a single operon like the lac operon. Have a cluster of genes participating in lactose transport and metabolism and this is constitutive (always expressed) 2) Global - control multiple opersons. -An example of this is catabolite repression where rapidly metabolized substrate glucose suppressed the synthesis of a few gene products |
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Lac Operon
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-This is a negatively controlled operson where the genes are induced
-lactose pathway converts lactose to glucose/galactose, and galactose to G-6-P -The 3 genes in order at Lac Z (B-galactosidase to cleave lactose into 2 sugars), Lac Y (galactoside permease to bring lactose into the cell), and Lac A (B-galactoside transcetylase converts G to G6P) -If no lactose present the promoter has a LacI repressor blocking transcription because LacI gene constitutively on -Once lactose given it will bind the repressor and change conf so it doesn't bind promoter and this induces transcription |
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Arabinose Operon
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-example of positive control system
-If arabinose not needed to has a repressor bind to the operator -When arabonose is available it binds and activates the repessor to help transcrription |
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Catabolite Repression 1
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1) Bacteria uses the substrate metabolized quickest first. This decision is called catabolite repression. When given glucose and lactose the bacteria will use on the glucose first because it's ready to be converted. Once glucose is gone, then the bacteria will upregulate lac operon genes and make lactose through this method (measure B-galactosidase)
2) If the bacteria are given only lactose there will be a lag phase while they metabolize it, then a log phase as they grow exponentially. If glucose is added they stop using the lactose and use the glucose. However, if glucose and cAMP added while growing on lactose, the continue to use the lactose |
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Catabolite Repression 2
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-it turns out that in this example of catabolite repression, the cAMP controls level of transcription
-When glucose is active the PTS uses enzyme 3 to transfer phosphate to glucose to make G-6-P, no cAMP made. -However, when glucose is absent the enzyme III will phosphorylate and activate an associated adenylate cyclase which makes cAMP -the cAMP binds to a protien called catabolite gene activator protein (CAP) and the cAMP-CAP complex promotes binding of RNA Polymerase to many operson promoters for global transcription -This works because lactose enters the cell trhough a proton symport mechanism and doesn't use PTD |
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Antimicrobial Agents
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-many antibiotics are produced by microorganisms that use them to ward of competeting microorganisms
-penicillin (b-lactam), streptomycin, quinoline Antibiotic Characterists 1) Bactericidal - kills 2) Bacteriostatic - inhibits growth 3) Broad Spectrum - effects G+ and G- -antibiotics tested on bacteria by the Kirby-Bauer method where bacteria colonies added to a filter and placed with antibiotics. Clearing zone indicates sensitivity. How effective antibiotics are is tested by minimal inhibitory concentration where a diluted antibiotic is added to equivalent # of bacteria in culture. Eventually you see what it takes to kill (minimum) -The goal is a antibiotic that kills bacteria without harming our cells so you look for bacteria specific structures like the cell wall and ribosome |
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Classes of Antibiotics
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1) Inhibition of Cell Wall Biosynthesis
2) Disruption of Cell membrane 3) Inhibtion of nucleic acid biosynthesis 4) Inhibition of protein biosynthesis |
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Inhibition of Cell Wall Biosynthesis - Cytoplasm Stage
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1) Cytoplasm Stage - Use D-cycloserine (broad spectrum)
-for normal PG production 1) UDP-NAMA adds L-alanine, D-glutamate, and meso-DAP 2) Before D-alanine can be added an enzyme creates a dipeptide D-alanyl-D-alanine and then this adds onto DAP 3) You can use with a d-alanine dipeptide added onto DAP. After this UDP-NAGA added via B1-4 bond 4) D-cycloserine looks like D-alanine so it will bind the enzyme and block PG synthesis |
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Inhibition of Cell Wall Biosynthesis - Membrane Stage
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2) Membrane Stage - Vancomycin, bacitracin, penicillin
For normal PG incorporation 1) After the NAMA/NAGA crossbridge made the molecule is attached to a undecaprenyl phosphate carrier lipid and brought to the surface 2) To be recycled the carrier lipid must lose a phosphate to be recycled back to the cell 3) Outside the PM a transpeptidase forms the crossbridge between adjacent NAMA by cleaving terminal-5 D-alanine and adding 4-D-alanine to 3-DAP How drugs work.. 1) Vanomycin binds D-alanine dipeptide making the molecule too large to exit the cell. Only works on G+ 2) Bacitracin prevents recycling on the lipid carrier because it blocks phosphate cleavage 3) Penicillin blocks the function of transpeptidase so the crossbridge does not occur |
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Penicillin
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-Discovered by Sir Alexander Flemming and now a family of antibiotics
-structural analog of D-alanyl-D-alanine -The β-lactam moiety of penicillin binds to the enzyme (DD-transpeptidase) that links the peptidoglycan molecules in bacteria. The enzymes that hydrolyze the peptidoglycan cross-links continue to function, which weakens the cell wall of the bacterium (in other words, the antibiotic causes cytolysis or death due to osmotic pressure). In addition, the build-up of peptidoglycan precursors triggers the activation of bacterial cell wall hydrolases and autolysins, which further digest the bacteria's existing peptidoglycan. -B-lactam antibiotics have different R groups which change potency to allow it to affect G+/G- |
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Cell Wall Antiobiotics Additional
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1) They are bactericial, kill bacteria
2) They are only affective on growing bacteria, so bacteria suspended or in arrest it doesn't work on 3) They are very effective because they target a bacteria specific structure 4) Work better against G+ cause have more PG and G- has outer membrane which is a barrier. However, ampicillin uses its charge to be more affective against G-. 5) Others are cephalosporins and cephamycins (have thiazine rings) and 6-amino penicillanic acid which has a thiazolidine ring |
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Antibiotics that disrupt the cell membrane
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1) Polymyxins - A polypeptide antibiotic it alters the permeability by insertion of positive self into the cell membrane via interaction with negative phospholipids to cause leakage. Specific for bacteria membrane. Won't affect humans because we have less phospholipids
2) Polyenes - Nystatin and AmpB are specific for eukaryotes (yeast and fungi) because they alter permeabilty of membranes by interacting with sterols |
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Antibiotics that Inhibit Nucleic Acid Biosynthesis
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1) Quinolones - They inhibit DNA replication by binding DNA Gyrase which is needed to unwind helical bacteria DNA
-Includes nalidixic acid(stop growth), ciprofloxacin, norflaxacin, and novobiocin(kill). Cipro and Nor are flourinated (block enolase in EMP) 2) Metronidazole - Inhibit DNA replication by increasing the cells free radicals which disrupt DNA. Very effective against anerobes 3) Rifamycins (kill)- inhibits transcription by binding RNA polymerase 4) Sulfonamides - They inhibit tetrahydrofolic acid synthesis so no folic acid (vitamin B) is made. Without B9 nucleic acid synthesis stops (stop growth). Won't affect humans because we ingest our B9 |
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Antibiotics that Inhibit Nucleic Acid Biosynthesis
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Normal Bacteria Protein Synthesis
1) mRNA binds to the 30S ribosome (rRNA + protein) 2) The 30S initiation complex forms with the tRNA 3) The 50S joins the 30S to make the 70S initiation complex 4) Through peptide bond formation protein is made How antibiotics fuck it up 30S 1) Kanamycin - A aminoglycoside that prevents formation of the 30S/mRNA complex by binding 30S proteins 2) Tetracyclin (static)- Binds a 30S protein to prevent tRNA binding 3) Streptomycin - A aminoglycoside that binds a 30S protein blocking tRNA joining the 30S initiation complex 50S - all bacteriostatic and broad spectrum 4) Erythromycin (a macrolide), chloramphenicol, clindamycin, and lincomycin bind the 50S ribosome preventing growing chain going from A to P so no protein made |
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Tetracyclin
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-synthetic antibiotic with 4 R-groups that can be changed to improve the mechanisms of tetracyclin and prevent resistance
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Inhibition of Acid Fast Bacteria
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-in mycobacterium tuberculosis because they are waxy and slow growing usual antibiotics can be a problem
-We use small, charged Ab to penetrate and inhibit mycolic acid biosynthesis and carbohydrate metabolism -Isoniazid and ethionamide block mycolic acid production while ethambutol blocks carbohydrate metabolism. Another one is pyrazinamide |
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Antibiotic Podcast - Mouth Study
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-oral antibiotic macrolides (ethromycin blocks 50S protein biosynthesis)
-Found that oral bacteria streptococci became macrolide resistant after a long enough treatment. Need a correct treatment length or else resistance explodes |
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Resistance overview
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-resistence genes evolved years ago probably may the microorganisms that produced the antibiotics, or from housekeeping genes, or by spontaneous mutation
-The resistance gene can decrease cell permeability, alter the antiobiotic or antibiotic target site, or encode a pump that pushes the antibiotic out of the cell -The spread of resistant genes is due to our use killing off the nonresistant organisms, and those surviving passing on the resistance gene using conjugative plasmid or conjugative transposon. Transduction not really used -E. Coli is a reservoir of gene transfer |
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Resistant Genes that Alter Permeability
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1) The G+ PG layer not great at preventing the antibiotic
2) The G- outer membrane more efficient at restriction to antibiotic, but Ab gets in through porin 3) Acid-fast have an even better membrane and their porin is more restricted |
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Mutations that alter the target site of an antibiotic
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-this is not a new gene. It's usually a mutation that doesn't affect protein function just changes it enough that the antibiotic won't bind
1) A mutated transpeptidase won't recognize penicillin 2) Vancomycin won't recognize a mutated PG layer 3) Spontaneous mutation of ribosomal proteins 4) Ribosomal protection - A protein protects the 30S from tetracyclin. This is not a mutation but a new protein 5) To avoid erythromycin or any 50S antibiotic, the rRNA can develop a new signature by methylation 6) Mutated DNA gyrase won't be recognized by quinolones |
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Tetracyclin Resistance
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-normally tetracyclin is brought into the cell by a transport protein
-in ribosome protection a protein binds the 30S so tetracyclin cannot bind -in tetracyclin efflux a cytoplasmic membrane protein (tetB) pumps tetracycline out of the cell Regulation: A repressor protein usually binds the tetB promoter. The inducer is tetracyclin which binds the repressor so it leaves the promoter. TetB is then active to remove the inducer |
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Penicillin Resistance
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-The gene (LamB) encodes B-lactamase/penicillinase which cleaves the B-lactam ring enough so that tetrapeptidase doesn't recognize it as a structual analog of D-alanyl-D-alanine
-This is NOT a mutated gene but a newly acquired gene |
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Chloramphenicol Resistance
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-Modifies the antibiotic so it no longer recognizes its substrate
-Chloramphenicol transacetylase changes the structure by adding a O-acetyl group so it no longer recognize the 50s ribosome |
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Resistance Conclusion
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-conjugation the most likely method for resistance transfer
-Transduction rare but used in Staphylococcus and Salmonella transfer of B-lactamase gene |
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Immune System Overview
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-protects against bacteria, fungi, virus, and parasites
-pathogens can live either extracellular (blood, lymph, interstitial space) or intracellular (phagosome, cytoplasm) |
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Routes of Infection
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1) Mucosal Surface - Airway, GI tract, or reproductive tract
2) External Epithelia - external surface, wound, insect bites -the immune rxn that protects these routes of invasion are all different |
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Types of Immune Response
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Immune system broken down into
1) Innate - Absolutely need this to survive 2) Acquired - Further has humoral (involves B-cell and antibodies) and cell-mediated (APC and T-cells) |
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Innate Immunity 1
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-present at birth and in place prior to a exposure to act rapidly
-They are non-specific and repeated exposure produces the same response 1) Physical Barrier/Physiologic - our epithelial cells held by tight junctions, air and fluid flow, mucus movement by cilia -We also have salivary enzymes, stomach enzymes and a low pH, and antibacterial peptides throughout a body -Our natural microbiological flora helps as well 3) Phagocytic Barrier - Immune cells in our innate system like macrophages (secrete cytokines to stimulate inflammation) and neutrophils (first on the scene) and NK cells (lyse infected cells) have PRR (pattern recognition receptor) that bind bacteria PAMP (pathogen associated molecular pattern) like LPS to induce phagocytosis -By endocytosis bacteria are brought in phagosome which merge with lyosome to make phagolyosome and has degredative molecules -phagocytosis also releases molecules to recruit more cells |
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Innate Immunity 2
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3) Inflammatory Barrier - As phagocytosis occurs macrophages produce a chemokine IL-8 (cxcl8) that triggers the endothelial cells to change surface expression
-They upregulate E-selectin which will loosely bind leukocyte siayl-lewis causing it to roll. Leukocytes always floating around -Eventually the leukocyte will bind the endothelial cell ICAM-1 strongly with its LFA-1 (integrin) and move through the tissue -The leukocyte will tmove its way through the basement membrane and to the site of infection via its IL-8 receptor which follows the chemokine path 4) Complement - 9 proteins and 3 pathways lead to a) Release of peptide mediators of inflammation b) Opsonization of pathogen (can involve antibody - adaptive immunity) c) Membrane attack complex that lysis cells |
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Acquired Immunity
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1) Inducible - It is not always active, turned on (reason behind vaccine)
2) Specific - The defense is specific against the pathogen infecting our body 3) Memory - While innate immunity always responds the same, adaptive remembers seeing a pathogen and the second time it responds quicker and more effective 4) Diversity - Millions of different pathogens it can bind 5) Non-reactive to self -classic experiment: when a antigen is introduce the response (serum ab conc) takes a few days and is at a minimal level. During the second exposure the response is super quick and the Ab produced are extremely high |
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Antigen 1
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-any molecule that can bind specifically to a antibody or t-cell receptor
1) Inorganic molecules are bad antigens, organic proteins are the best since they are diverse and not repetitive 2) Size is an issue 3) Charge is an issue 4) Foreigness - Want something that humans don't have. Bacteria specific LPS is a great example 5) Mode of Administration 6) Genetics of the Host - We all respond differently to the same antigen Making a antigen - one protein can provide many different antigens (epitope) 1) B-cell can recognize a specific part of a native protein 2) T-cell receptor needs the epitope chopped up and presented in a certain way |
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Antigen 2
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-our receptors recognize virus sialidase and hemagglutinin
-normally the body only sees infectious agent and tumor cells are antigen. If the body sees self organ or a innocous substance it can lead to allery and autoimmunity |
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Antibodies
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-antibodies are the basis of humoral immunity
-antibodies are made of 2 heavy chains connected by a disulfide bond and 2 light chains -Each antibody has different heavy and light chains, but on a specific antibody the two of each class are identical -The light chains are bound to the heavy by a disulfide bond -The part that sticks into the membrane is the C-terminus and the part that binds is the N-terminus -antibodies are flexible -When cleaved by papain the result is 2 Fab (light chain and variable part of heavy) and 1 Fc (2 heavy chain constant region bounded by disulfide bond) |
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Classes of Antibodies
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-5 classes of AB each with their own function and structural differences
1) IgM - Can be a monomoer of form a J-chain pentamer. On naive antibody 2) IgD 3) IgG - Most populous, in fetal circulation 4) IgA - can be a monomer or dimer. Dimer helps in transport across the epithelium 5) IgE - Important in mast cell allergic response and eosinophil destruction of parasites |
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Antibody Structure
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-the light and heavy chains have stable constant regions between them and then variable regions where the Ag binding occurs
-it takes a combination of the light and heavy chain variable region at the N-terminal to bind the antigen -Ab:Ag binding occur through electrostatic, hydrogen bonding, van der waals, and hydrophobic forces -The rest of the light and heavy chain is the constant region, and gives the antibody its biologic effector function -light chain has 2 constant regions, k and lamba, while the heavy chain has the 5 discussed before |
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Physical States of Antibodies
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1) Integrated into the B cell plasma membrane as a antigen receptor
-has a transmembrane and cytoplasmic domain -interacts with Iga and Igb to transduce a signal 2) Associated with cell surface Fc receptors -Example is mast cells which has a Fce associated with IgE (binds constant region) 3) Secreted in soluble form |
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Effector Function of Ab
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-B-cells are the ONLY cells that have antibody as a membrane receptor
-B-cell effectors called plasma cells secrete antibody ONLY 1) Antibody binds the antigen to block pathogen process or toxin 2) Antibody binds pathogen which enables phagocytosis by a leukocyte 3) Antibody uses help of serum protein complements to carry out function |
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Effector Function of Ab
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1) IgG - It helps fix complement and assist in opsonization. Has lots of cells with Fc receptors.
2) IgA - Used for mucosal immunity, lots of cells have Fc receptors. Defend when we inhale and found in alimentary canal 3) IgM - Helps fix complement and a B-cell Ag receptor. No cells have Fc. It is found in the heart and blood 4) IgD - B-cell Ag receptor, no Fc 5) IgE - Helps with basophil and mast degranulation, and helps eosinophils attack parasites. The Fc receptor is on these cells. It is in the connective tissue and helps defend against wound |
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Effector Function of Ab - Ag:Ab Interaction
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1) Antibody bind to virus capsid proteins to prevent cell attachment. In HIV they bind to GP41 and GP120 which use CD4 to gain T-cell entry
2) Toxins function to bind cell and other part determines function. Our antibodies can bind toxins to prevent their cell attachment 3) Our cells have receptor for bacteria attachments (pili). Antibodies can bind these and prevent cell attachment |
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Mucosal Immunity
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-there are direct interactions between mucosal epithelia and lymphoid tissues: diffuse lymphoid tissue and organized like Peyer's patch, lymphoid follicles, tonsils
-M-cells in peyer's patch, adenoids, and tonsils are there for specialized antigen uptake mechanism -mucosal immunity is mediated by secretory IgA which forms a dimer -Plasma cells makes the IgA monomers and J chain from separate gene. The 2 IgA bind to the J-chain to form the dimer -To get from the submucosa to the lumen the J-chain of the IgA binds the epithelial poly-Ig receptor which takes it through the epithelial cell to the lumen -A portion of poly-Ig stats with IgA dimer to make the secretory component and stabilize the dimer |
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Antibody Secretion
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-For a B-cell to change antibody classes and secretes it involves interaction with helper T-cells
-B-cell can go from IgD and IgM to any other one -During secretion IgA dimer can have 3 functions as it goes from the submucosa, to the lamina propria, to the lumen 1) Bind toxins in the lumen 2) Bind and neutralize antigens in the epithelial cell endosome 3) While being secreted it can export toxins from lamina propria to lumen |
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Dentistry and Antibodies
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-Periodontitis involves IgG mediated inflammation while caries/decay involves IgA
-Oral and nasal vaccine involves IgA -Intramuscular and IP vaccine involves IgG |
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Antibody Effector Function 2: Interact with Accessory Cells (1)
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-involves Fc receptors which cells use to bind antibodies. When antibody binds bacteria and Fc then it triggers phagocytosis
-Fc can both activate and inhibit a pathway -Fce => secretion of granules, Fcg => phagocytosis, Fca => phagocytosis -Macrophages, neutrophils, and dendritic cells are the major phagocytotic cells -phagocytosis is a good mix of innate and adaptive immunity cause leukocytes ability to uptake pathogens is increased a lot by antibody opsonization |
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Antibody Effector Function 2: Opsonization and Phagocytosis
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1) IgG bind microbe surface, thereby opsonizing it
2) The antibody constant region binds the cell Fcg receptor 3) Phagocytosis is activated as it the lysosome enzyme process 4) The phagosome and lysosome combine and bacteria dies in phagolysosome Popular PAMP and PRRs 1) G- LPS is bound by TLR, CD14, LBP on phagocytotic cell 2) Mannose-rich glycans from microbes is bound by the macrophage mannose receptor and the plasma mannose-binding protein |
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Antibody Effector Function 2: ADCC
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-another function of antibody in cell interaction is antibody-dependent cell-mediated cytotoxicity
-Good for killing infected cells, tumors, and parasites 1) Infected Cell -infected cell express virus/bacteria protein on cell membrane -IgG binds to these "flag" protein -Fcg on NK cell recognize and bind antibody -Binding crosslinks the Fc receptor and trigger NK cell to kill target by releasing granules which cause apoptosis 2) Killing a Parasite -IgE is bound to the parasite surface -Eosinophil binds the Fce and releases granules with enzymes and major basic protein to injure the parasite |
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Mast Cells
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-have lots of Fce receptor on their surface
-Will become coats with IgE bound to the Fce -When Ag binds the IgE the Fce and IgE dimerize to become active -This causes eosinophils to release the granule content which in small number is helpful but in large number causes allergies Histamine - causes vascular dilation (leak), bronchoconstriction, and intestinal hypermotility Cytokines - cause inflammation Enzymes - Cause tissue damage |
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Allergies
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-normal reaction to allergens is mediated by IgG, but allergic person uses IgE
-First intro to allergen produces little result, subsequent can produce anaphylaxis -To cause anaphylaxis, molecule that causes IgE activation gets into the blood stream -Type 1 allergy is immediate hypersensitivity |
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IgE and ADCC Killing of Helminths
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-during worm invasion mast cells can produce cytokines (IL-5) to promote other inflammatory cells recruitment and activation like eosinophils
-Eosinophils have Fc receptor for IgG and IgE. They will bind parasite covered w/IgG and IgE and release major basic protein -Takes a number of eosinophils to kill a parasite |
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Complement System
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-can be part of both innate immunity and adaptive immunity
-involves soluble complement proteins C1-C9 and membrane receptors -C1-C9 are usually inactive plasma proteins but are activated by sequential proteolysis to yield proteolytic enzymes and inflammatory molecules -The products of this cascade become covalently attached to a microbe or antibody attached to a microbe (membrane pore/phagocytosis) -The pathway is regulated by proteins found on host cells but not microbial -the results are pathogen killing (membrane pore), opsonization and phagocytosis, and recruitment of inflammatory cell |
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Complement 3 Pathways
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1) Classical - Dependent on antibody so acquired immunity
2) MB-Lectin - Host protein (MBL) recognizes carbohydrate on the pathogen surface. Innate immunity 3) Alternative - Occurs through spontaneous cleavage of C3 which binds a bacteria specific structure like LPS |
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Classical Pathway
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1) Ab binds to microbe membrane. Either IgG or IgM
2) C1 binds to the Ab constant region (Only 1 Fc per Ab). Since IgM pentamer has 5 Fc it is more likely to trigger complement 3) C1 conformational change allows it to bind and cleave C4 to activate it. During cleavage one part stays near the cell (enzymatic) and other released for other role 4) C4 binds and cleaves C2 to activate it 5) C2 binds and cleaves C3 to activate it 6) After this it is in seqeuntial order. Where C5-C9 are activated and activate the next molecule through enzymatic cleavage 7) C5-C9 form a membrane attack complex that puts a pore in the pathogen membrane causing osmotic lysis |
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Complement other proteins
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-Some released products like C3a, C4a, and C5a lead to inflammation by acting on blood vessles
-leads to increase vascular permeability, Ig and leukocyte/lymphocyte extravasation by more EC surface molecules, and increase leukocyte microbicidal activity -C3b and C5a increase opsonization by binding the bacteria and a phagocyte cell receptor. Work in conjunction with a Ab binding both bacteria and phagocyte |
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Passive Immunity
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-child not exposed to pathogen until birth but has a line of defense because the mother passes her IgG to the fetus and through breast milk later on
-Differs from active immunity in which we are injected with a antigen (vaccine) Bad Side: If Mom has autoimmune disease like Graves she will pass these IgG to the fetus which will suffer Graves until the IgG leave |
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Corresponding the Antibody and the Type of Immunity
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1) Extracellular Pathogen in blood, lymph, interstititial - Use antibodies, complement, phagocytosis, and neutralization
2) Extracellular Pathogen on Skin - Use antibodies (IgA) and antimicrobial peptides 3) Intracellular Cytoplasmic Pathogen - Use Tc cells and NK 4) Intracellular Vesicular Pathogen - Use Th-cell, NK-cell to activate the macrophage in which pathogen lies |
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B-Cell Differentiation
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Primary Lymphoid Organ - Where lymphocytes differentiate from stem cells. B-cell in the BM and t-cells in the thymus. A t-cell will start differentiating in the BM and move to the thymus
Secondary Lymphoid Organ - Where mature B and T cells go to become activated by their specific antigen -B,T,NK cells differentiate from the lymphoid pregenitor. All other immune cells come from the myeloid progenitor |
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Cell Surface Markers
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-immune cells are distinguished by their expression of cell surface markers called CD (Cluster of Differentiation) - phenotypic markers
B-Cell: Produce antibody (humoral) and have MHC II, CD19, and CD21 T-Helper: Stimulate B-cell activation (humoral) and activate macrophages by secreting cytokines (cell-mediated) have CD3 and CD4 T-Cytolytic: Kill virus and tumor-infected cells and reject allograpfts (cell-mediated). Have CD3 and CD8 |
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B-Cell Differentiation II
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-the B-cell leaves the marrow as a resting mature cell and is covered in either IgM or IgD Ab bound to PM as receptors. 2 methods of activation
1) T-Dependent (majority-for all proteins): B-cell must bind Antigen and the T-helper cell. T-helper cell will then secrete cytokines that further trigger the B-cell 2) T-independent: A antigen alone activated B-cell by binding to the PM bound antibody receptor. The antigen has a repetitive epitope so activation cross talks |
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B-Cell Differentiation III
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event 1/2 events must occur at approximately the same time. Signal 1 w/o signal 2 leads to anergy
1) Antigen binds the B-cell antibody leading to signal transduction 2) T-helper receptor binds the b-cell MHC II whiles T-cell CD40L binds B-cell CD40. This releases cytokines from the T-cell that bind the B-cell cytokine receptor 3) After stimulation the B-cell will proliferate in a process called clonal expansion, and this leads to 4 fates a) Turns into a plasma cell secreting original antibody (IgM) b) Through isotype switching it becomes a plasma cell that secretes a different antibody. This may be dictated by the T-cell. The Fab remains the same but the Fc changes c) Becomes a memory B-cell that keeps Ab as receptors and continues to circulate d) Undergoes affinity maturation |
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How is Ag Specificity Determined
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Selective Hypothesis: A single B-cell in specific for 1 Ag. Specificity is determined before a pathogen is seen. However, since you have millions of different B-cells, by chance you are covered
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T-Cell Mouse Experiment
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-example of t-cell mediated immunity and skin graft rejection
-First exposure to a skin graft leads to delayed rejection while second exposure leads to quicker rejection -First exposure by a different mouse of same strain as first mouse still is a delayed response -Shows that skin graft rejection is done by acquired immune response -The T-cells are recognizing MHC proteins on the surface of the donor tissue |
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MHC
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-Heterodimeric glycoprotein of 2 noncovalently associated polypeptides
-The 2 polypeptides form a 3D groove that acts as a peptide binding site Class I: 3 subunit alpha chain that is variable between MHC 1. Has a B2-microglobulin that does not vary. Expressed on all nucleated cells so cytotoxic T-cell can induce killing if virally infected Class II: an alpha and beta highly variable polypeptide of 2 subunits each. The beta 1 and alpha 1 make the peptide-binding clef. -Expressed on only B-cells, macrophages, dendritic cells, and active T-cells. On these cells so T-helper can induce immune response -Cells that express MHC II can also express MHC I, but not vice versa |
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MHC Genes
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-Genes that make MHC in humans is called human lymphocyte antigen gene HLA. All clustered on chromosome 6
-Each person receives 2 copies of each MHC gene, one from each parent -The HLA genes vary in polymorphism but are generally very polymorphic (except B2) |
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T-Cell Development
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-T-cell receptor gene rearrangement and development occurs in the thymus
-Receptor specificity is antigen INDEPENDENT -Process of negative and positive selection makes sure that the receptor doesn't bind self too strongly or too lightly or else it is eliminated -The interaction between self MHC and T-cell receptor should be sufficient |
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T-Cell Receptor Development
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1) Cells that leave thymus are CD3, CD4, and CD8 negative
2) Gene rearrangement leads all T-cells to express CD3, CD4, CD8 positive to become double-positive 3) After positive/negative selection the T-cell leaves the thymus are CD3+/CD4+ or CD3+/CD8+ 4) If the T-cell is autoreaction to undergoes apoptosis (more than 95%) 5) The alpha/beta receptor T-cell is critical for immunity, the gamma/delta is not |
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T-Cell DIfferentiation
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CD8+ - Cytotoxic T-cells Interact with cells that have MHC I and induce virally infected cells, tumor cells, and transplant cells to kill themselves via apoptosis
CD4+: T-helper cell receptors bind cells with MHC II and assist by binding and secreting cytokines for cellular and humoral immunity. The different Th cells produce different cytokines Th1: Activate macrophages infected with bacteria to fuse with lyososome Th2: Activate B-cells to become effector plasma cells and secrete Ab T-reg: Prevent activation of self-reactive cells T-17: Secrete IL-17 which is involved in bacterial infection immunity |
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Cytotoxic T-cells
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-To become active the cytotoxic T-cell must recognize a foreign peptide bound to SELF MHC. Can't just be self MHC and can't be a foreign MHC
-T-cells recognize peptides from denature proteins associated with self MHC molecules (unlike B-cells that recognize full protein) -The cell which a T-cell recognizes is called a antigen presenting cell (can be any cell). T-cells require this cell to capture the antigen, process it into peptides, and display it on a MHC - |
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Professional Antigen Presenting Cells
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-Cells that express MHC II on their surface (B-cell, dendritic, macrophage)
-APC are constantly taking up proteins, breaking them to peptides, sending them to the rER, and bringing them to the surface with MHC -If it's a self peptide then the T-cell won't recognize and the peptide will disocciate. If foreign peptide then T-cell will recognize |
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T-Cell Receptor
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-Composed of 2 polypeptide chains (alpha and beta) which are glycosylated, disulfide bonded, and have transmembrane region
-There are constant and variable regions -Activated T-cell does not secrete receptor, always on the T-cell surface -T-cell receptors associate with CD3 to help amplify signal transduction -T-cell needs to engage a specific peptide bound to a specific MHC, and the strength of this interaction causes signal transduction -T-cell receptor looks like 1/2 of a antibody Fab |
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T-Cell Activation
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-T-cell activation requires 2 signal
1) Receptor binding the specific peptide:MHC complex 2) Binding of T-cell accessory CD4/CD8 to APC co-stimulatory molecules and sometimes cytokines -Without this co-stimulatory binding anergy occurs -these signals lead to signal transduction, cell division first, and effector function later -T-receptor to MHC:peptide leads to IL-2 cytokine autocrine expression which triggers T-cell growth |
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Cytotoxic T-cell function
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Activation: To become activated the Tc requires Th stimulation. Stimulation Th secrete cytokines like IL-2 which activates the CD8+ T-cell
Function: Once activate the Tc will look for infected cell, bind them, and induce apoptosis. Can affect many cells -Tc contacts target and releases granules with perforin and granzymes to poke holes in the membrane -Tcell recognize: 1) Viral proteins on the surface of virally infected cells 2) Cancer cells produce unique proteins |
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Helper T-Cell Function
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-Th stimulation produces IL-2 required for their growth
-Will differentiate into a subset of Th and produce a unique set of cytokines Th1: Helps in cell-mediated immunity to activate cells of the immune system like macrophages, and neutrophils. Induces fusion of bacteria and phagosome. -Produces IFN-y to help B-cell become plasma so it can secrete opsonizing antibodies Th2: Function in humoral mediated immunity to activate B-cells to produce antibodies. -IL-5 activates eosinophil to fight parasites based on IgE that B-cell secreted due to Th2 |
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Immunity Against Intracellular Microbial Infections
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-bacteria want to be phagocytized and hide in phagosomes or even better hide in the cytoplasm
-Th1 activates the phagolysosome through IFN-y cytoskine secretion (TH1 binds MHC2:peptide on infected APC and secrete IFN-y) -Main difference is that TH doesn't lead to cell death, just bacteria death with while Tc kills the cell |
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Tuberculosis
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-this mechanism is for any cell that gets into the cytoplasm
-Because you can't kill the cell you form granulomas -Giant multinucleated infected cells surrounded by T-cells which block them off |
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Hypersensitivity
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1) Immediate - Cause immediate reaction due to IgE. Causes by metals, poison ivy, haptens
2) Delayed Response IV - The antigen will be taken by a langerhaan to lymph node and activate a Th1 which releases cytokines that act on the vascular endothelium to allow recruitment of phagocytotic cells -Caused by proteins! -responsible for look of lepers and latex sensitivity |
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Effector Function of TH2
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-responsible for humoral immunity based on Ab
-When TH2 binds APC with MHC2:peptide 3 major functions 1) IL-4 production leads to B-cell activation to produce neutralizing IgG antibodies and IgE antibodies that cause mast cell degranulation 2) IL-5 cause eosinophils to become activated and attack a parasite 3) IL-10 and IL-4 suppress macrophage activation -When Th2 directly binds a B-cell it not only makes it differentiate into a plasma cell, but it also determines the class of Ab produced |
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Transplant Rejection
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syngenic - transplant to twin
allogenic - transplant to same species xenogenic - transplant to different species For dental transplant 1) Can use autograph from own body bone 2) Can use allographs from cadavour with allogro 3) Can use xenograpfts with Bioss from cows |
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Transplant Rejection 2
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1) Acute Rejection - T-cells recognize a foreign MHC on graft and attack it
2) Hyperacute - Person has pre-existing antibody from a blood transfusion which leads to a quicker response 3) Chronic Rejection - takes a while, rare -to prevent rejection we take immunosuppresdents which usually inhibit IL-2 production so there is no T-cell activation. Can also take anti-inflammatory steroids |
