MICR 141 Cheet Sheet Exam 2 PDF

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S. pneumoniae Background: Gram (+), capsulated. Colonizes the nasopharynx then lower airways. Into bloodstream is meningitis. Leading cause of community acquired pneumonia. In hospital, its aureus. Under 6 are more likely to get it and elderly. Fluid accumulation in the lungs due to fluid and cell influx. Neutrophils(PMNs) release neutrophil serine proteases and ROS to kill it. Capsule protects from ROS. Contained in granules. 90 different bacteria serotypes so difficult to combat. Colonization mediated by binding of bacteria on epithelial cells by surface protein adhesins. Can be prevented from entering respiratory tract by being enveloped in mucus and removed from the airways by action of ciliated epithelial cells. Bacteria prevent being enveloped by producing secretory IgA protease and pneumolysin. Regular IgA traps the bacteria, the protease prevents this interaction. Pneumolysin binds cholesterol in the host cell membrane and creates pores, but this activity can be destroyed by ciliated epithelial cells and phagocytic cells. PLY (similar to LLO) activates the classic complement pathway. Then cytokines IL-1 and TNF-a are produced by leukocytes, leading to migration of inflammatory cells to the site, leading to damage. S. pneumo Phosphorycholine present in the cell wall binds to receptors expressed on endothelial cells, leukocytes, platelets - then bacteria can enter the cells and protected by opsonization and phagocytosis (spread of disease). Primary Paper: HXA3 promotes recruitment of neutrophils in the airways, which leads to tissue damage. PLY deficient bacteria was impaired in triggering neutrophil transepithelial migration. Data indicates that PLY is an important factor in promoting HXA3 dependent neutrophil recruitment across pulmonary epithelium. PLY induces PMN migration across pulmonary epithelial cells, and PLY-dependent PMN migration requires pore-formation and epithelial 12-LOX activity. Diverse pore forming toxins induce PMN migration. Decreased PMN recruitment leads to higher bacterial burden. Strong response leads to edema, lung damage, etc. Favorable is a good timing and degree of PMN recruitment orchestrated. Purified PLY activates Phospholipase A in endothelial cells which release arachidonic acid, suggesting eicosanoid signaling may be involved. The final step of PMN entry into the airways during infection is transmigration across the lung epithelium, which is associated with disruption of the mucosal barrier function and spread of the bacteria into the bloodstream. Additional Variables: PLY into another bacteria, other virulence factors, another serotype. Time of incubation, different host cell type, PLY inhibitor. Pore formation is not just something PLY can do. PLY must be able to bind and form pores to induce PMN migration. Figures: fMLP is a leukocyte chemoattractant (+) control. PLY required for PMN migration. ILY, SLO, PFO, a-toxin, are pore forming toxins which induce PMN transmigration as well. PLY requires the ability to form pores to induce PMN migration. PLY CBS lacks the cholesterol binding site. PLYEP is a mutant locked in early prepore formation. PLYLP is locked in prepore formation. 12-LOX is required for PLY induced PMN transmigration. Ci-Di-Cy and Baicalein are 12-LOX inhibitors. PLY requires pore formation and the 12-LOX pathway to induce robust PMN migration in vivo. HXA3 drives pro-inflammatory PMN migration. It’s a bioactive lipid, and bacterial infection induces apical secretion of HXA3 by the epithelium. It is derived from membrane phospholipids and processed by the enzyme phospholipase A2 to generate arachidonic acid. Arachidonic acid can make 60 dif chemoattractants or biomolecules. 12-LOX the enzyme, yields HXA3 from arachidonic acid. The inhibitors of 12-LOX are written above, which blocks HXA3. Chemoattractants recruit WBCs to different sites of infection. Lipid chemoattractant is HXA3. Tight Junctions Connect host cells together. HXA3 serves as several cues for PMNs to migrate the epithelium to try to clear the infection. Infection: Bacteria comes down to the lungs, adhesin to specific receptors, bind to epithelium, and start accumulating. Basolateral chemoattractants are released from bottom of epithelial cells (basolateral). PMNs sense this and transendothelial migrate. Follow the chemoattractant by 3D transition (ambeoid approach). HXA3 is released apically. High affinity for HXA3 so follow it. They also follow pericyte (ICAM-1) expression. Some epithelial cell receptors called JAM-C that bind to the neutrophil receptor ligand (JAM-L) which promote attachment to epithelium and help neutrophils affiliate with site of inflammation and infection. Strep pneumo realizes it needs to leave since environment is not hospitable and endothelial cells undergoing stress. Escape lungs and enter the blood supply, which can cause sepsis and meningitis. PLY: Has 4 distinct domains. Each domain works together to undergo a distinct conformational change. First step of PLY interaction is host cell monomer binding. PLY released through unknown mechanism. A lot is contained in cell wall of the bacteria. Once you have monomers released, they can bind to the host cell. PLY belongs to CDC family which means they bind to cholesterol. No binding if first incubated with cholesterol. LLO has same 4 domains as PLY, and Strep O. After monomer binding, it now transitions to a prepore formation. You have an oligomerization step on the membrane of host cells, and starting to form pores. This step allows PLY to undergo a conformational change and it is now in a more energetically favorable state, to undergo pore formation (final step). This is when alpha helices are inserted into the membrane of host cells, and pop out the tissues of host cell membranes to create pores. Leads to efflux o host cell ions, and activation of host cell immune responses. This pore formation leads to release of HXA3 in epithelial cells. It is involved in vasoconstriction, calcium release, and chemoattractant. Netosis to PMNs at high dose. Don’t know if PMN underwent netosis because HXA3 or PLY. PLY induces netosis in neutrophils. Pyroptosis in macrophages. Necroptosis in epithelial cells. NSPs: Present in neutrophil granules and kill during degranulation. Neutrophil elastase degrades E-cadherin, Cathepsin G degrades occludin and VE Cadherin (necessary for tight junctions). Proteinase 3 promotes VE-cadherin stability. NSPs also hurt hose cells as well. -Remember, PLY induces 12-LOX dependent PMN migration, so PMN migration is dependent on 12-LOX, which makes HXA3, which is the PMN chemoattractant. With deficient 12-LOX signaling, no PMN migration if you give it PLY. Neutrophil Transepithelial Migration Assay: Measure toxin concentration. Sometimes, same PLY won’t yield same pore results because losing activity of certain aliquots. Propidium Iodide, is impermeable to intact cells, which shows pore formation. ____________________________________________________________________________________________________________________________________________________________________

Bordetella Pertussis: Introduction: Gram (-), rod-shaped, non-motile. Attachment of organisms to ciliated epithelial cells is mediated by protein adhesins: pertactin, filamentous hemagglutinin, and fimbria. Systemic toxicity is produced primarily by pertussis toxin. Toxins: PTX, ACT, TCT, T3SS. This toxin inactivates the protein that controls adenylate cyclase activity, leading to an increase in cyclic adenosine monophosphate (cAMP) levels and a subsequent increase in respiratory secretions and mucus production, characteristic of the paroxysmal stage of pertussis. Only human reservoir. Infection initiated when aerosols inhaled and bacteria become attached to and proliferate on ciliated epithelial cells. CEC extruded in paroxysmal stage. Growth: Incubation period is the initial growth phase of bacteria in the host. Catarrhal phase: Mucous inflammation, runny nose, nasal congestion (peak of bacteria). Paroxysmal is a fit attack of intense coughing and high pitched cough. Convalescent is decline in coughing which can last weeks or months. Pathogenesis: Inhalation of aerosol droplets, then bacterial attachment to the ciliated airway epithelium and production of toxins (using all adhesins to bind to cells, then upregulate their toxin). Bacterial multiplication, influx of PMNs, damage to host cell epithelium by TCT and LPS. Mucus hypersecretion delays neutrophil influx. Effects on Immune Cells: Resident airway macrophages – important for neutrophil recruitment (PTX inhibition of protective anti-bacterial function). Neutrophils (PTX – inhibition of influx to airways, ACT- inhibition of phagocytosis and killing, FHA-specific antibodies inhibit phagocytosis of filamentous hemagglutinin antibodies). Dendritic cells (ACT and T3SS inhibit maturation and transport to lymph nodes. Affects DC ability to present antigens to adaptive immune response. Manipulates LPS/TLR4 and ACT increase IL-10 and IL-23 production, and generate a Th17 response. Suppress Th1 responses. PTX Toxin: AB5 type toxin. A is the catalytic portion. And 5 membrane binding/transport B subunits. To assemble, individual subunits are transported to the periplasm. The holotoxin (toxin with multiple subunits) is assembled in the periplasm. Toxin is secreted by Type IV SS. PTX does not require cell to cell contact. Shoots PTX wherever, can damage any host cell. Targets: Sialoglycoproteins, intoxicates any mammalian cell. Toxin binding it mediated by B oligomer. Key effect is ADP-ribosylation of the alpha subunit of heterotrimeric G proteins (G alpha) locks the alpha subunits into an inactive state (GDP bound form). Our cell use G proteins to function in a lot of different ways, and responsible for mediating different signaling cascades to trigger immune responses. The bacteria is able to modify these G proteins an put them in the inactive state. The net effect of this is increase cAMP levels. Translocation: PTX binds to its target cell, PTX enters via receptor mediated endocytosis, and enters the host cell. It undergoes a series of steps, called retrograde transport, to the host endosomal compartment. It follows a system of cellular organelles in order to get to the cytoplasmic reticulum. It is endocytosed into a vesicle and it is not interacting with the cytosol. PTX continues retrograde transport through the golgi apparatus. PTX eventually reaches the E.R. and remember, up until this point, no interaction with cytosol. The E.R. is the only subcellular compartment that contains ATP, besides the cytoplasm. ATP binding to PTX destabilized PTX and disulphide isomerases may reduce PTX disulphide bonds. ATP makes the A subunit less stable with the B oligomer which means a greater chance of separation. This allows the A subunit to escape the vesicle environment, and enter the cytoplasm. It can then now mediate toxic effects in the cell. How PTX it affects cell function: The A subunit translocates into the cytosol and stays associated with exosomes and traffics to the cytoplasmic membrane. It migrates towards the cytoplasmic membrane and this is where ADP ribosylation occurs. The A subunit ADP ribosylates the subunit of G proteins. There are a lot of G proteins, there are multiple subunits of these G proteins and this specific toxin, ADP-ribosylate, adds a ribosyl group, to the G alpha subunit. Upon ADP ribosylation, it leaves the heteromeric complex, because it is leaving in an inactive state, it is no longer able to function normally. This leads to increased cAMP levels in the cell. If you have PTX that is able to be released from the E.R., it will ADP ribosylate the G alpha subunit, and this will lead to increased cAMP levels, which is an important part of pathogenesis. Increased cAMP levels dysregulate the host immune response. Brefeldin A disassembles the Golgi and protects host cells from PTX damage. The golgi is an important step in retrograde transport and if the golgi is disassembled, then its difficult for PTX to enter the E.R. If not, then the toxin is stuck in endosomes and the endosomes don’t go to the E.R., they go to the golgi. If you treat host cells with Brefeldin A, you are able to protect them from the toxin’s function in terms of ADP-ribosylation, because it can’t leave the vesicles and its stuck in the endosomes. Adhesins: PTX disrupts ciliary movement by creating ciliary stasis s it can more efficiently colonize our airways. Ciliary movement is constantly trying to brush away toxins, but with stasis, which is caused by adhesins, the epithelial cells are no longer able to move efficiently. Primary Paper: Type I IFNs able to modulate immune response, but detrimental in a number of ways.However, this response may impair adaptive immune responses to some infections. Weaken host resistance to infection. Include IFN-a and IFN-B. Decrease in IFN-y, and IL-17. Less proliferation and increase in cell death. Less recruitment of neutrophils, asIFNs mediate this affect. Th17 has TF, RORC3 and expresses IL-17A,F,22,21,26. Results: See a significant Th17 cell population. And a lot of cells expressing IL-17A so this is important because it serves as a clue that they may be involved in mediating the immune response to B. pertussis infection. IFN-y produced from Th1 cells, which is very low. No influx of IL-17 cells until 15dpi. You first see a higher population of Th1 cells, where no resolution of disease. Then a massive influx of Th17 cells. Th17 cells important for resolving infection. More robust results in lungs and not blood since infection taking place in the lungs. Notice the bracket. IL-17 mRNA expression is significantly upregulated in lung tissue at 5, 10, 15 dpi and important because they can assess where IL-17 is coming from. Increase at 15dpi means influx of Th17 cells. Pulmonary inflammation at 15dpi. More intense staining means inflammation more severe. PTX is important for inducing a significant Th17 response in the blood. Mutants lacking PTX, this trend disappears and no massive influx. Remember: Type I IFNs suppress Th17 differentiation and IL-17 responses. In vivo blocking of iFN-a produced by pDCs during infection with the bacterium resulted in early incrase of Th17 frequency, inflammation, and reduced bacterial loads in the airways.

Mycobacterium tuberculosis: Introduction: Rod-shaped, acid-fast, non-motile, slow growing. Respiratory pathogen, aerosolized droplets. Humans are only reservoir. 5 Type 7 SS. At the time of exposure, M. tuberculosis enters the respiratory airways, and infectious particles penetrate to the alveoli where they are phagocytosed by alveolar macrophages. Mtb prevents fusion of the phagosome with lysosomes by blocking the specific bridging molecule, EEA1. At the same time, the phagosome is able to fuse with other intracellular vesicles, permitting access to nutrients and facilitating intravacuole replication. In the presence of IFN-y, the infected macrophages are activated, leading to increased phagosome-lysosome fusion and intracellular killing. Alveolar macrophages with intracellular mycobacteria form the central core of a necrotic mass that is surrounded by a dense wall of macrophages, CD4, CD8, and NK T cells. This structure is called a granuloma, and it prevents further spread of bacteria. If a small antigenic burden is present at the time the macrophages are stimulated, the granuloma is small and the bacteria are destroyed with minimal tissue damage. If many bacteria are present, the large necrotic or caseous granulomas become encapsulated with fibrin that effectively protects the bacteria from macrophage killing. Virulence Factors: ESX 1-5 are required for virulence. Adhesins HBHA, PE_PGRS, and has a capsule. Pathogenesis: M. tuberculosis transmitted host-to-host via aerosolized droplets. Mtb is phagocytosed and begins replication in macrophages. Endosomal lysosomal fusion - slowly starts to grow in macrophages. Neutrophils and innate lymphocytes are recruited to the site of infection. They try to control the infection - b/c macrophages can’t destroy Mtb cause can’t fuse lysosom to endosomal compartment. 3 Outcomes: 1) T Cells and innate immunity can lead to control indefinitely 2) Caseating granuloma becomes increasingly necrotic, liquefies, and ruptures Bacteria here gets the upper hand - rupture of granuloma - then reactivation and dissemination happens again 3) Reactivation and dissemination in 10% of infected individuals Cell Wall: Supports cell growth, crucial for virulence, barrier for antibiotics. Layers: Mycolic acids, arabinogalactan, and PG. Secretion Systems: Cell wall also has ESX – Like Type 7 SS. Referred to as export systems. From outer to inner: Capsule - Outer lipids - Mycolic acids - Arabinogalactan - PG - Cytoplasmic Membrane – Cytoplasm. Within the cytoplasmic membrane, you have ESX membrane complexes, there are 5 different types of these. The mechanism of protein secretion across the envelope is perhaps one of the most prominent unanswered questions in the field. The envelope acts as a impermeability barrier. MOM=Mycolate Outer Membrane. Proteins traverse the cell wall via an unknown mechanism - scientists hypothesize that there is porins. T7SS export protein substrates from the cytosol across the cytosolic membrane (unclear how) ESX-1. Transports Esat6 and CFP10 across the cytosolic membrane. Required for virulence. Damages the phagosomal membrane while residing within phagocytes. Uses ESX-1 for extrusion from the phagosome of macrophages into the cytoplasm. Need this to be a successful Mtb pathogen. What would be a useful toxin or partner in crime for a ESX-1 Mtb mutant? - LLO - b/c LLO used to escape from the phagosome. Phagosomal permeabilization is ESX-1 dependent. This leads to activation of a macrophage IFN-B response. This response promotes Mtb infection. Secretion Assay: Take host cell, and load it with dye, CCF-4-AM. Enters host cell cytosol. Esterification Traps CCF-4 into host cell. Excitation at 409nm of CCF-4 generates emission at 535nm because FRET occurs. However, if you have Mtb in the cell, and it secretes beta-lactamase in the host cell cytosol, the presence of beta-lactamase cleaves the dye, CCF-4. Now the dye is modified. Excitation at 409nm of cleaved CCF-4 generates emission at 450nm because no FRET occurs (WT bacteria) Now, the light is blue (450-no FRET) instead of green (535nm). So active secretion is going on. Purpose of this assay is to measure the ability of a bacterium to secrete a substrate, like beta lactamases. If ESX1 present, then blue. W/o ESX-1, green If you infect mice unable to produce IFN, they actually survived. Permeabilization of the phagosome induces a Type I interferon response. This response is bad, because IFNs contribute to Mtb pathogenesis. ESX-1 wants to help permeabilization of the phagosome. Worse outcome for the host. ESX-2: Might be required for survival in dendritic cells ESX-3: Required for iron and zinc uptake. Prevents phagosomal maturation via the EsxH effector protein by disrupting host endosomal machinery from fusing into all these lysosome ESX-1 and ESX-4: Help coordinate conjugation between cells. Conjugation is a process by which genetic material is transferred from a donor cell to a recipient cell ESX-5: Promotes nutrient uptake essential for survival in phagocytes. Secretes a large number of proline-glutamate/proline-proline glutamate (PE/PPE) proteins which promote virulence Membrane phospholipids are processed into arachidonic acid. Arachidonic acid can become HXA from the enzyme 12-LOX from strep pneumo. Arachidonic acid → Prostaglandin G by enzyme COX (Cyclooxygenase rxn). Prostaglandin G → Prostaglandin H by enzyme COX (Peroxidase rxn). Then specific isomerases (enzymes), that are going to differentiate which molecules are made. Tissue specific isomerases take Prostaglandin H can then have additional levels of specificity via tissue specific isomerases Cyclooxygenase Inhibitors (COXi): Aspirin and Celecoxib are 2 inhibitors of COX - There is COX-1 and COX-2. Membrane lipid → Arachidonic acid (e. Phospholipase A ) → endoperoxides (COX-1 or COX-2). Endoperoxides (PGG2, PGH2) produces eicosanoids → Prostaglandin (PGE2, PGD2) via Isomerases. NSAIDS (aspirin/acetaminophen blocks both COX 1 and COX-2 enzyme activity). COX-2 inhibitors (Celecoxib), blocks COX-2 activity. Corticosteroids inhibit phospholipase A act...