Study Guide, Exam 2 2021

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MICR 470/670, Microbial Pathogenesis, Exam 2 Study Guide 6. Cell Adhesion and Signal Transduction What are the two basic components of plaque structures for both adherens junctions and focal contacts? Actin Cytoskeleton and anchor proteins The membrane receptor in a focal contact is an integrin. Describe the molecular structure of an integrin. Integrins are heterodimers of ꭤ and β subunits that bind Ca 2+ What are three proteins that bind directly to the cytoplasmic domains of integrins? Fibronectin, vitronectin, collagen, laminin, etc The membrane receptor in an adherens junction is a cadherin. Describe the molecular structure of an E-cadherin. Actin Cytoskeleton  Plaque  E-Cadherin  E-Cadherin  Plague  Actin Cytoskeleton Connect two cells by extracellular domains What extracellular molecule is bound by an E-cadherin? Cadherin Receptor What are three proteins that bind directly to the cytoplasmic domains of E-cadherin? Β -catenin, plakoglobin, and p120-catenin plaque proteins What are two molecules unique to focal contacts and absent from adherens junctions. Integrins and Talin What are two molecules unique to adherens junctions and absent from focal contacts? Cadherins and ꭤ/ β -catenin Describe three basic molecular mechanisms for regulating the formation of plaque proteins besides activation of their membrane receptors (i.e., don’t include integrin or cadherin activation). 1. PIP 2 binding – closed to open transition is induced by binding PIP 2 2. Synthesis of proteins such as vinculin, ꭤ-actinin, and actin 1

MICR 470/670, Microbial Pathogenesis, Exam 2 Study Guide 3. Phosphorylation & Dephosphorylation – protein kinases also regulate interactions of come plaque proteins by phosphorylation What structural protein in focal contacts can also activate transcription of genes and how does it do this? FAK has a signaling function by phosphorylating protein, thereby turning on transcription of some genes What structural protein in adherens junction can activate transcription of genes and how does it do this? free B-catenin and plakoglobin can translocate to the nucleus, where they bind transcription factors and activate specific genes One protein that Rho activates is PIP kinase. How does this activation influence the formation of a functional focal contact plaque? Increased levels of PIP 2 contribute to formation of focal contacts by binding Talin, vinculin, and ꭤ-actinin, which opens these proteins, allowing them to bind to each other and actin What is the molecular pathway leading from an activated Rho to cell contractility? Rho  Rho Kinase  Mysoin II Light Chain-Phosphorylation  Cell Contractility via actin 7. Bacterial Adhesins What are the two major classification of bacterial adhesins and where are the adhesin molecules located in each one? 1. Fimbrial adhesins are located at tip of a scaffold-like structure: pilus or fimbria 2. Afimbrial adhesins are anchored directly in the bacterial membrane or cell wall The E. coli P pilus is attached to the outer membrane and consists of several proteins. What is the role of the PapH protein? PapH is the anchor subunit of the P pilus an dis located in the outer membrane. PapH anchors the major pilus protein to the outer membrane. What is the role of the PapG protein? PapG protein is attached to the end of fibrillum. PapG is the adhesin subunit that binds host molecules. 2

MICR 470/670, Microbial Pathogenesis, Exam 2 Study Guide What is the role of the PapD protein? PapD protein is a chaperone, which tranfers P pili subunits from the cytoplasmic membrane to the outer membrane. What does the P pilus bind to? P pili bind to glycolipids on host cells lining upper urinary tract, allowing E. coli adherence. What protein makes the rod (not the base) of the type IV pilus of Neisseria gonorrhoeae ? PilE rod = Pilin protein What are the two different adhesin proteins on the type IV pilus and what do they bind to? 1. PilE Adhesin binds to CD147 and β 2 adrenergic receptor in the brain. 2. PilC adhesin binds glycoprotein CD46. What role does PilE play in N. meningitidis pathogenesis? PilE adhesin is responsible for the binding of bacteria in the brains microvasculature endothelial cells and is mediated by Type IV pili. The type IV pilus binds to host receptors. What are two additional activities of the type IV pilus of Neisseria gonorrhoeae ? 1. Extracellular Neisseria DNA can be entangled in retracting Neisseria type IV pili and taken in the cell 2. Type IV pili retract in order to make close contact between the bacteria and a host cell Describe the assembly of E. coli Curli pili. CsgA protein or curlin is a curli subunit. Curlin protein polymerization occurs on a surface bound nucleator (CsgB protein). Secreted curlin undergoes self assembly to form a stable curli structure. What proteins are required for proper assembly of the Afa-I adhesin of E. coli ? Periplasmic chaperone/usher protein and anchor in outer membrane with adhesins attached 3

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MICR 470/670, Microbial Pathogenesis, Exam 2 Study Guide What does Helicobacter pylori BabA adhesin bind to and how does this adherence contribute to stomach epithelial cell carcinogensis? BabA adhesin binds to sialyl-Lewis x/a antigens and the Lewis b blood group antigen expressed on stomach epithelial cells and red blood cells Bordetella pertussis filamentous hemagglutinin (FHA) contains an "RGD" motif that mediates binding to a leukocyte integrin. What integrin does it bind to and what are two effects of this binding? FHA binding to macrophage CR3 (complement receptor; a β 2 integrin) causes internalization of bacteria into macrophage without triggering oxidative bursts so they survive. Also activates host signal pathway that upregulated expression of CR3 Bordetella pertussis FHA and PT adhere to respiratory epithelial cells. What do they bind to and what effect does this have on the respiratory epithelial cells? PT and FHA adhere to lactose-containing glycolipids on respiratory epithelial cells and interfere with normal ciliary movement. Describe three major steps that occur when Neisseria uses its type IV and Opa adhesins to bind to mucosal epithelial cells. 1. Anchorage is a relatively weak binding mediated by type IV pili (PilE) 2. Tight Adherence - PilE retracts to allow a tight secondary adherence using Opa protein binding to CEACAM1, CEACAM5, and CEACAM6. 3. Invasion and transcytosis – tight binding results in internalization How does Yersinia use YadA and Invasin to bind  1-integrins on epithelial cells and what is the outcome of this binding? YadA is an adhesin which binds fibronectin, which acts as a bridge to bind to the β 1- integrin that binds fibronectin. Invasin is a yersinia adhesin which binds to β 1-integrin. Together they promote internalization of bacteria into epithelial cells. What does Streptococcus Protein F1 bind to and what effect does this have on epithelial cells? Protein F1 binds fibronectin in soluble and immobilized forms, this helps promote internalization of the bacteria into host epithelial cells. Describe the function of the three domains, W, M, and C of Protein F1. M (– for membrane) and C (– for cytoplasmic charged amino acids) sequences temporarily hold the protein in the cytoplasmic membrane. W (– for wall) sequence is proteolytically cleaved between the T and G resulting in a permanent anchor. 4

MICR 470/670, Microbial Pathogenesis, Exam 2 Study Guide Mycobacterium species have a FAP adhesin, which mimics  1-integrins. What does this adhesin allow these bacteria to do? This integrin mimic is used to mediate adherence to the host ECM in respiratory epithelia. What is the effect of deleting the cna gene on all diseases caused by Staphylococcus ? S. aureus cells lacking can have reduced capacity to bind to collagen type II in cartilage and diminished capacity to cause septic arthritis but still cause other kinds of infections What is the effect on pyelonephritis caused by E. coli when they lack the P pili adhesin protein? PapG mutant that form P pili lacking PapG tip adhesin can infect bladder but have lost the capacity to infect kidneys. What is the effect of deleting the gene encoding Invasin on Yersinia pathogenesis? Mutant yersinia lacking invasion do colonize luminal surfaces of the intestine but do not transverse the epithelium. Invasin is needed for penetration. What is the effect of deleting the gene encoding YadA on Yersinia pathogenesis? Mutant yersinia lacking YadA adhesin show decreased binding to luminal surfaces and reduced colonization of the intestine. YadA is needed for intestinal colonization. What is the effect on disease caused by Salmonella when they have double mutations in both the lefC and invA genes? Causes a strong attenuation of virulence and inability to colonize animals when bacteria are given orally. What stage of the Salmonella pathogenesis is affected when both lefC and invA genes are deleted? Intestinal Colonization 8. Bacterial Pathogenesis via Cell Adhesion Describe three characteristics of bacterial lectin adhesins. 1. often involved in initial adhesion stage 2. sometimes results in tissue tropism to a specific host tissue when bound to host surfaces or ECM 5

MICR 470/670, Microbial Pathogenesis, Exam 2 Study Guide 3. can occur with ECM components Describe four major aspects of attachment and effacement lesions. 1. Intimin binds very tightly to Tir 2. intimin binding TIR causes polymerization of F-actin, dramatically altering host cell shape. 3. Adherence of EPEC to epithelial cells results in effacement of microvilli 4. Reorganizing actin cytoskeleton makes a pedestal to cradle the E. coli bacteria. What roles do type IV pili and intimin play in adherence of enteropathogenic E. coli (EPEC) to host epithelial cells? Initial adherence is via a type IV pilus Tight, intimate adherence then occurs when two bacterial-encoded proteins, intimin and Tir interact. Describe four steps for Tir to be used for EPEC adhesion. 1. Tir translocates from E. coli cell to host cell sytoplasm using a type 3 secretion system 2. Tir inserts into host plasma membrane 3.Tir becomes phosphorylated on tyrosine by a host kinase 4. Tir now serves as an attachment site for EPEC cells Tight junctions help maintain polarization of intestinal epithelial cells Describe the structure of such cells. Tight junctions maintain polarization between apical and basolateral surfaces of cells. Bacteria often adhere to the apical surface of the epithelium by bridging mechanism involving lectin-carbohydrate interactions. Describe the structure and function of M cells. M cells are specialized intestinal cells in Peyer’s patch and have a role in presenting luminal antigens to the immune system. They have no villi, do not secrete mucus or digestive enzymes and lack glycocalyx surface. What deleterious effect happens when neutrophils (PMNs) migrate to an infection site at the intestinal epithelial layer in search of bacteria to phagocytosis? 6

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MICR 470/670, Microbial Pathogenesis, Exam 2 Study Guide Transmigration of PMNs destabilizes the epithelium and favors further bacterial invasion from lumen of the intestines. Integrin and cadherin redistribute to apical surfaces and bacteria bind allowing them to be internalized. Local inflammation paradoxically favors infection. Mammalian adhesion molecules are most commonly classified into four main groups. What are those four groups? 1. Integrins 2. Cadherins 3. Selectins 4. Ig-superfamily members What are generalizations for the functions of  1- and  2-integrins?  1-integrins dimers usually bind ECM components and leads to actin polymerization.  2-integrins are often involved in cell-cell contact of leukocyte during inflammation by binding to ICAM-1 and is critical for antigen-dependent T cell activation and PMN transmigration. What is the typical function of cadherins? Function in cell-cell interactions at adherens junctions What are Ig members normally involved in? Cell-cell interactions. Some form homotypic binding Selectins are lectins that interact with carbohydrate residues on the surface of certain host cells. Describe the most common function of selectins. Attachment and rolling of leukocytes What four steps, as presented in your handout, are involved in blood stream neutrophil migration to a site of infection? 1. Tethering and rolling 2. Activation 3. Firm adhesion 4. Transmigration 7

MICR 470/670, Microbial Pathogenesis, Exam 2 Study Guide Describe the process that involves adhesion of LFA-1 integrins on PMNs to ICAM-1 on endothelial cells. Chemokine activation of neutrophils causes LFA-1 to bind tightly to ICAM-1 on endothelial cells Adherence of pathogens to phagocytic cells can have a variety of effects. What are the three effects discussed in class (other than the normal phagocytic process)? 1. Turning off normal phagocytic process that leds to pathogen destruction 2. Internalization by different process that avoids the destructive phagocytic process 3. Killing of the phagocytic cell by apoptosis Describe two different but typical effects when pathogenic bacteria bind specifically to non-phagocytic cells. 1. Adhesion can promote efficient bacterial internalization (invasion). 2.Adhesion of bacteria to the surface of a normally non-phagocytic host cell. 9. Vesicle Trafficking in Eukaryotes During the transport of newly synthesized proteins destined for secretion and for delivery to the lysosome, where are they sorted into separate vesicles? Trans-Golgi network (TGN) Describe the main signal on proteins destined for the lysosome and three other protein components involved in the process, as described in class, for getting to the lysosome. Main Signal is: mannose-6-phosphate (M-6-P) residues. Overall Structure is Lysosomal protein-M-6-P::M-6-P Receptor::AP1::Clathrin Where in the host vesicle trafficking do AP1, AP2, and AP3 function? AP1: TGN AP2: Plasma membrane AP3: Endosomes 8

MICR 470/670, Microbial Pathogenesis, Exam 2 Study Guide Describe three aspects of micropinocytosis. Micropinocytosis is initiated by growth factors, resulting in formation of unguided ruffles that first extend outward then fold back against cell surface, accidentally engulfing extracellular fluids and particles. Results in loose phagosome around particle. Mechanism is actin dependent. Parts of the cytoplasmic membrane are internalized and recycled back to the cytoplasmic membrane. How much of the membrane is internalized every hour and how much of it is recycled? Equivalent of entire plasma membrane is internalized every hour and over 90% of internalized lipids and proteins are recycled back to the cell surface with rapid kinetics Some receptors internalized from the cytoplasmic membrane are recycled and some are not. Describe the process for one receptor, presented in your handout, which is recycled. LDL and transferrin receptor are both internalized constitutively whether or not they bind their ligands. If they have bound their ligands, they are released from receptor and trafficked to lysosome for degradation. After releasing LDL or ferric ions in early endosome, the receptors are recycled back to plasma membrane to undergo additional rounds of endocytosis. Typically, such receptors can cycle between plasma membrane and early endosome about 100 times before being degraded. Describe one receptor, presented in your handout, in the cytoplasmic membrane that is not recycled and when it is internalized and why it is not recycled. EGF receptor is internalized upon ligand binding. Both receptor and ligand pass through early endosome then to late endosomes and then to lysozyme to be degraded. It is a mechanism of down regulation to reduce the number of growth factor receptors on cell surface. Describe the pathway for a vesicle endocytosed in a clathrin-coated pit to the lysosome. Endocytosis  Early endosome  TGN (or skip this)  late endosome  lysosome Describe what happens to the clathrin. Clathrin coat rapidly depolymerizes and is recycled to make new vesicles Describe the role that microtubules, dynein, and kinesin play in vesicle trafficking. Dynein moves vesicles on microtubule tracks indegradtive endosomal pathway Kinesin moves vesicles on microtubules for exocytosis to plasma membrane 9

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MICR 470/670, Microbial Pathogenesis, Exam 2 Study Guide What is transcytosis? Transcytosis: Molecules internalized from one membrane can be returned back to same membrane or sorted to opposite membrane. What does secretory IgA (sIgA) bind to in epithelial cells and what happens to the sIgA? poly-Ig receptor; sIgA::Poly-Ig Receptor complex is internalized from basolateral membrane and transcytosed across cells in an endocytic vesicle to the apical membrane where it is secreted 10. Pathogen Internalization What are replicative phagosomes? The fusion of phagocytic vesicles carrying bacteria with host vacuoles and multiply intracellularly. Some pathogens survive and divide after normal phagocytic process. What is one protein, presented in your handout, which Legionella uses to do this and what happens to bacteria that lack this protein? SdhA protein is translocated by T4SS and is essential to maintain the phagosome. Without it the phagosome is disrupted, and bacteria are degraded in cytoplasm and infected phagocytic cell dies. Describe the structure of cord factor and how it functions after Mycobacterium are phagocytized. Cord factor is trehalose dimycolate, a glycolipid, which can form a crystalline monolayer on its cell surface. Cord factor prevents fusion of phagosome containing Mycobacterium with host vacuoles. Describe how Neisseria gonorrhoeae survive internalization by phagocytes. Bacterial internalization is not by the normal phagocytic process. Bacteria are internalized by Opa-mediated binding to CD66. CD66 receptors are CEACAMs (members of Ig superfamily). When Opa binds to CEACAM3 on granulocytes, internalization is very efficient and requires the actin cytoskeleton. Internalization occurs at membrane ruffles and avoids the normal phagocytic uptake. Neisseria can become internalized into epithelial cells by a process that is much different than internalization into macrophages. Describe this process. Neisseria Opa protein binds to CEACAM1 (or CEACAM6) on apical surface of epithelial cells and is then internalized in pseudopods. This does not involve the cytoskeleton. This occurs at specialized membrane domains called lipids rafts. 10

MICR 470/670, Microbial Pathogenesis, Exam 2 Study Guide Yersinia and Listeria both promote internalization into non-phagocytic cells. Describe two features that are unique to each pathogen and one feature which is common to both. Yersinia are internalized after binding to integrins. Yersinia adhesin “invasion” binds to β 1-interins.Listeria are internalized after binding to cadherins. Listeria adhesin “internalin A” binds to E-cadherins Binding to both host receptors activates actin cytoskeleton promoting uptake of bacteria. In both cases binding results in actin polymerization at site of bacterial adherence. Describe three common features of the zippering mechanism that results in the internalization of Yersinia and Listeria into non-phagocytic cells. 1. Incremental interactions between bacterial ligands and cell adhesion molecules. 2. Receptor recruitment and clustering at bacterial cell interfaces is the first step. 3. Increasing attachments occur between the host and bacterial cells. Describe four features of the macropinocytic process Shigella uses to be internalized into cells. 1. Bacteria induce extended membranes (membrane ruffles) that enclose a bacterial cell and internalize it. 2. Bacterium is internalized into a large loose vacuole. 3. Shigella inject proteins into host cells using T3SS 4. Requires activation of GTPases. Shigella activates Rac, Cdc42, and Rho. Describe three features of Listeria internalization via a growth-factor receptor. 1. Listeria internalin B (InlB) adhesin binds to host Met growth-factor receptor. 2. Activation of the Met recruits PI3K and Rac that promote actin polymerization. 3. Describe components of lipid rafts and how pathogens use them. Lipid microdomains in the cytoplasmic membrane are called lipid rafts and are enriched in specific lipids: cholesterol, glycosphingolipids, and sphingomyelin. Lipid rafts are clustering devices that compartmentalize signaling proteins into small domains within cytoplasmic membrane. Pathogenic microbes take advantage of them by binding receptors within rafts, such as growth factor receptors. 11

MICR 470/670, Microbial Pathogenesis, Exam 2 Study Guide 11. Intracellular Parasitism What are the four significant advantages for bacteria to live intracellularly? 1. Microbes become inaccessible to antibody and complement attack. 2. Microbes become inaccessible to direct phagocytic attack. 3. Microbes no longer require specific adherence mechanism. 4. Microbes have access to a large variety of nutrients. What are the three basic processes that microbes use to get internalized into host cells? 1. Normal phagocytosis 2. Induced phagocytosis 3. Active invasion Describe three features of the invasion of host cells by Toxoplasma . 1.Toxoplasma creates its own entry vacuole, the parasitophorous vacuole (PV), which does not mature into an acidic, lysosomal compartment. 2.Toxoplasma adhesins often mimic host ECM proteins. MIC2 adhesin uses its exterior domain to bind to glycosaminoglycans (GAG). Binds parasites actin cytoskeleton allowing it host cell entry. 3. Describe three features of Toxoplasma rhoptries. 1. Rhoptries are club-shaped organelles that participate in invasion and establishment of the intracellular parasitic lifestyle. 2. PV formation is initiated by secretion from the rhoptries. 3. Toxoplasma invasion results in a parasitophorous vacuole (PV) and PV membrane. Where do the proteins in the PV membrane come from? Secreted parasite proteins that originate in the secretory organelles of the parasite What secretory organelles secrete their protein contents concomitant with parasite invasion? 12

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MICR 470/670, Microbial Pathogenesis, Exam 2 Study Guide Rhoptries secrete their protein contents concomitant with parasite invasion. Toxoplasma secretes many proteins into host cells that it invades. Where are three locations that those proteins go? 1. Host cell 2. Forming vacuoles 3. Surface of the PV What are the three basic intracellular niches for microbes that live inside host cells? 1. Cytoplasmic – pathogens reside within the cytosol 2. Intravacuolar – pathogens persist in modified endosomes or in sequestered, non- acidic non-endosomal vacuoles. 3. Intralysosomal – pathogens persist in lysosome How does Shigella escape, or try to escape from its phagosome in macrophages and neutrophils and why is there difference? Shigella flexneri can escape macrophage vacuoles due to hemolysin-like enzymes translocated into the host by a T3SS. Shigella cannot efficiently escape from the phagocytic vacuoles of neutrophil because they produce elastase, which is a protease that is extremely active against the virulent proteins that Shigella secretes. How does Listeria escape from its phagosome? Listeria escapes vacuoles after entry using at least three enzymes that can disrupt membranes. Listeria has two phospholipases that help degrade vacuolar double membranes. Listeria monocytogenes has a third enzyme, listeriolysin O (LLO) which forms pores in vacuolar membranes. Describe the process for Trypanosoma cruzi to escape from its parasitophorous vacuole. Trypanosoma escapes its vacuole during acidification, which activates TcTox a hemolysin enzyme. TcTox forms pores in membranes. To aid in acidification it also recruits lysozymes to the site of internalization that then fuse with PV. Mycobacterium produces ammonia to block the normal maturation process of their phagosomes and prevent fusion with lysosomes. How does this work? 13

MICR 470/670, Microbial Pathogenesis, Exam 2 Study Guide Production of ammonia prevents acidification, which is necessary for efficient fusion of compartments. Describe the internalization process into macrophages and subsequent survival and growth induced by Legionella . Legionella induce internalization into macrophages by a coiling phagocytic process that results in sequestered vacuole. Coiling phagocytosis involves unilateral pseudopods repeatedly rotating around microbes. After entry it recruits the ER and ribosomes to help with survival. Describe the adhesion and internalization processes into host cells by Chlamydia . Enter non-phagocytic cells and produce vacuoles. First they adhere to host cell via OmcB binding host glycosaminoglycan. Then use pmp21 adhesin to bind to epidermal growth factor receptor (EGFR). Internalization happens via clathrin-coated pits. Toxoplasma protozoa secrete proteins into the parasitophorous vacuole to aid in nutrient accumulation by two mechanisms, what are they? 1. Sequestration of cellular organelles, ER, and mitochondria 2. Formation of pore in vacuolar membrane for transport of nucleotides, amino acids, and peptides from host cytoplasm. As Plasmodium invades blood cells it inserts a protein into lipid rafts of the host membrane. What is the protein and what effect does it have on the blood cells? It inserts its PfEMP1 protein into lipid rafts. This protein binds CD36 on endothelial cells, which causes aggregation of blood cells to blood vessels and platelets. Where does Coxiella survive and grow inside host cells and how does it do this? Coxiella reside in acidic vacuoles that are part of the endosomal network. Their phagosomal vacuoles fuse with other vesicles and contain fully active lysosomal hydrolases. They replicate inside the phagolysosome. What are two advantages for Coxiella to reside in a lysosomal compartment? 1. Access to degradative products, in particular purines. 2.Resistant to lysosomal hydrolases. 14

Study Guide, Exam 2 2021

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