Study Guide, Exam 3 2021

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MICR 470/670, Microbial Pathogenesis, Exam 3 Actin Cytoskeleton What are four different cellular structures that actin is involved in? 1. Filopodia 2. Lamellipodia 3. Podosomes 4. Microvilli Describe three features of the F actin structure. 1. F-actin is polarized with a plus end and a minus end 2. G-actin::ATP can form F-actin. 3. F-actin will hydrolyze its bound ATP to ADP + Pi and release Pi. Describe how G-actin becomes activated for addition to an F-actin filament. G-actin::ATP can form F-actin. It is added to an actin filament at the “plus” (growing) end. F- actin will hydrolyze its bound ATP to ADP + Pi and release Pi. ATP hydrolysis is required for depolymerization. How does CapZ bind to actin filaments and what effects does this have? CapZ binds to the plus end, inhibiting polymerization. If actin filaments continue to dissociate from minus end of filament with Cap Z at plus end, actin filaments will shrink. How does phalloidin bind actin filaments and what effect does it have? Phalloidin binds sides of actin filaments, stabilizing them, filaments don’t shorten or lengthen. What effects do -actinin and filamin have on actin filaments? They are cross-linking proteins and they organize actin filaments into bundles or networks What is the activity of gelsolin? Attach to actin filaments and cause depolymerization. A key regulator of actin filaments assembly and disassembly. Binds sides of actin and also binds calcium ions it severs the filaments. What is the structure of the Arp2/3 complex? Arp2/3 complex has 7 proteins that include Arp 2 and Arp3, as well as five smaller proteins. 1

MICR 470/670, Microbial Pathogenesis, Exam 3 Describe how the structure of the actin cytoskeleton in lamellipodia is formed. Filament severing can be a mechanism for increasing the number of plus ends to which actin can polymerize. This produces more filament formation and hence formation of lamellipodia podosomes and forward movement. What is the function of podosomes in leukocytes? Podosomes are the primary sites of integrin-stimulated actin polymerization in leukocytes. Necessary for chemotaxis and movement out of blood vessels at sites of inflammation. Describe how profilin functions in formation of actin filaments. Profilin performs a 1:1 complex with G-actin, binding promotes ADP to ATP exchange. This exchange increases the local concentration of G-actin-ATP, the form able to polymerize. Profilin functions as a carrier donating G-actin-ATP monomer to the plus end of a filament. Describe two ways that PIP2 regulates an increase in actin filament formation. 1. PIP 2 ::Profilin concentartes Profilin to where it is needed. 2. PIP 2 also binds to gelsolin and coflin Describe three molecules that WASP binds to. 1. Profilin::G-actin 2. G-actin 3. Arp2/3 Describe how WASP becomes activated. PIP 2 and Cdc42 bind WASP, localizing it to membrane and activating WASP. What does activated WASP do to promote new actin filament formation? WASP may determine where in a cell actin polymerization will first occur. ****************************************************************************** ******* Intracellular Motility and Intercellular Spread Some bacteria use actin for intracellular motility and to spread to a neighboring cell (actin-based motility). What are two advantages that this provides? Allows them to not be exposed to antibodies, complement, or other immune system components. 2

MICR 470/670, Microbial Pathogenesis, Exam 3 What is the propulsive force, which moves bacteria using actin-based motility. Polymerize actin filaments to generate actin tails on their surface to drice the movement Describe the functions of the four main host proteins that Listeria monocytogenes bacteria use to create actin-based motility. 1. Express mimics of WASP family members (Listeria monocytogenes ActA, Burkholderia thailandensis BtBimA, Rickettsia RickA). 2. Express surface proteins that recruithost WASP proteins (Shigella flexneriIcsA). 3. Express mimics of Ena/VASP proteins (Burkholderiapseudomallei BpBimA and Burkholderia mallei BmBimA). 4. Express mimics of formin proteins (e.g., Rickettsia Sca2) Arp2/3 complex, VASP, profilin, and a-actinin. Describe the functions of the six main host proteins that Shigella flexneri bacteria use to create actin-based motility. Arp2/3, VASP, Profilin, a-actinin, WASP, and vinculin are host proteins required for Shigella motility. Listeria bacteria can move within a cell as well as from one host cell to another using host actin. The rate of actin filament formation and therefore the speed of movement are controlled by two host proteins. Name those two proteins (other than G-actin) and describe how they function. ActA Profilin What was done to demonstrate that the rate of Listeria movement could be modulated experimentally? Deleting proline repeats one at a time decreases speed of bacterial movement due to reduced VASP binding and hence reduced profilin binding. Describe how Rickettsia RickA protein creates actin-based motility. Immediatley after infection Rickettsia bacteria use RickA to polymerize actin into comet tails. RickA creates short, curved actin tails that create meandering movement early in infection. 3

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MICR 470/670, Microbial Pathogenesis, Exam 3 Describe how Rickettsia Sca2 protein creates actin-based motility. Later during infection Sca2 is used to polymerized actin into comet tails, replacing RickA. Actin tail polymerization occurs, creating long, straight tails comprised of two or more long filaments arranged in nearly parallel arrays that wrap around each other in a helical fashion. Describe how RickA and Sca2 function to control actin-based motility and their roles in Rickettsia infection. RickA is involved in the immediate polymerization and composition of motility during initial infection, later suring infection that bacteri aswitches to Sca2 as a source of motility which increases virulence of the pathogen. Describe three features of actin-based motility that occur with Burkholderia thailandensis . 1.Actin polymerization by Burkholderia thailandensis depends on BtBimA, which is a polarized protein that promotes actin nucleation similar to ActA protein. 2. BtBimA binds Arp2/3, which polymerizes branched actin filaments. 3.Arp2/3 complex is localized throughout actin tails formed by these bacteria, apparently causing actin-based motility. Describe three features of actin-based motility that occur with Burkholderia mallei . 1. Actin polymerization byB. pseudomallei and B. malleidepends on BpBimA and BmBimAthat are related to each other but are distinct from BtBimA and do not use Arp2/3. 2. BpBimA and BmBimA mimic host Ena/VASP and bind directly to actin. 3. Actin tail is bundled and long and results in mostly straight paths. Describe three events that occur when Listeria moves from one cell to a neighboring cell. 1. Listeria removes ActA when inside the new vacuole, thereby delaying motility for a short period of time before ActA expression resumes. 2. Delayed motility is presumably beneficial because it increases time bacterium is in a new cell and hence increases replication time and number of bacterial offspring in each cell. 3. Listeria escapes double-membrane vacuole by expressing the same LLOit uses during entry by the phagocytic processes plus two phospholipases (PlcA and PlcB) to disrupt the double- membrane vacuole. Describe the mechanism used by Burkholderia to facilitate cell-to-cell spread. Burkholderia spread occurs through bacterial-mediated fusion of host cells caused by additional bacterial virulence proteins, resulting in generation of multi-nucleated host cells. 4

MICR 470/670, Microbial Pathogenesis, Exam 3 ****************************************************************************** ******* Pathogenicity Islands Genomic islands (GEIs) are often classified into five types, what are they? 1. Pathogenicity islands 2. Symbiosis islands 3. Metabolic pathway islands 4. Antibiotic resistance islands 5. Fitness islands The functions encoded by genes on genomic islands (GEIs) include four categories of functions. What are those four categories? 1. Mutations 2. Rearrangements 3. Deletion events 4. Horizontal gene transfer Most GEIs share seven features, what are they? 1. Relatively large DNA 2. Nucleotide Statistics 3. Inserted at 3’ end of tRNA genes 4. Flanked by 16-20bp direct repeats 5. Functional or cryptic genes encoding transposases and integrases, or factors related to conjugation systems. 6. Insertion sequences or transposons responsible for mobilizing genetic material. 7. Carry genes offering a selective advantage for the bacteria 5

MICR 470/670, Microbial Pathogenesis, Exam 3 What are three sources for the origin of GEIs? 1. Plasmids 2. Prophage 3. Conjugative trasnposons What are the four mobile genetic elements that carry virulence factors? 1. Transposons 2. Plasmids 3. Bacteriophages 4. PAIs Give an example of one virulence factor (as presented in your handout) carried on each of the four mobile genetic elements from E. coli . 1. Transposons – heat stable enterotoxin 2. Plasmids – heat-labile enterotoxin 3. Bacteriophage – Shiga Toxin 4. Pathogenicity Island – LEE, Pai I and Pai II E. coli is normally a non-pathogenic member of the human intestinal tract. However, there are several different pathogenic variants of E. coli that have larger genomes than the non-pathogenic strain. Give three different mechanisms or sources of DNA for how such pathogenic variants acquired the larger genomes. 1. Virulence-associated plasmid 2. Phage carrying Shiga-toxin gene 3. PAI When the E. coli pathogenicity island called LEE is transferred in the lab to a benign E. coli strain, what phenotype do those recipient bacteria get? In one step, acquisition of attaching and effacing phenotype. 6

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MICR 470/670, Microbial Pathogenesis, Exam 3 Salmonella has at least 10 PAIs that carry virulence factors, including pathogenicity island 1 (SPI-1) and SPI-2. Describe the effects on virulence for mutant Salmonella lacking SPI-1 compared to strains lacking SPI-2. Virulence factors on SPI-1 are required to break through the intestinal barrier but not essential for subsequent pathogenicity. When inoculated orally and intraperitoneally in mice, mutants with defective SPI-2 are avirulent. Virulence of V. cholerae is associated with the synthesis and secretion of cholera toxin. What genes are required for encoding cholera toxin and where are those genes located? TCP is an important adhesin for V. cholerae to colonize intestines. TCP gene cluster is present for VPI. Some Vibrio cholerae cause cholera (severe diarrhea) however, most V. cholerae bacteria living in natural aquatic environments are not virulent and do not cause cholera and cannot infect human intestines. Non-virulent V. cholerae can become pathogenic in a three-step process. What are those three steps? 1. VPI acquisition- allows adhesin via TCP 2. CTX lysogenization via binding to TCP Փ 3. Expression of phage ctxA and ctxB genes to make toxin-expression from prophage DNA. The Gram-positive bacteria Staphylococcus aureus have a pathogenicity island called SaPI. Analysis of the genes present in the SaPI reveals the origin of this pathogenicity island. What is the origin? Prophage SaPIs are related to certain temperate phages with which they share several essential functions Describe those six functions. 1. Integrase 2. Replicon 3. Morphogentic genes 4. Packaging module 5. Regulatory genes 6. SaPIs lack genes encoding structural capsid proteins 7

MICR 470/670, Microbial Pathogenesis, Exam 3 Describe the model for SaPI transfer. SaPIs must parasitize an infecting wild-type phage, which induces the SaPI to excise and replicate. SaPI DNA is encapsidated efficiently into small-headed, phage-like particles preferentially over wild-type phage DNA. Phage particles with SaPI DNA infect another bacterial cell, resulting in very high frequencies of inter-cell transfer. ****************************************************************************** ******* Endotoxin What is the normal function of endotoxin for bacteria? Endotoxin is LPS and is attached to the outer membrane of Gram-negative bacteria. LPS is essential for structural integrity of the outer membrane and is essential for bacterial growth and survival What are the three major parts of LPS? 1. Lipid A 2. Core polysaccharide 3. O polysaccharide (O antigen) What are the 6 detailed pathophysiological distress reactions given in your handout? 1. Fever, prostration (sometimes diarrhea) 2. Changes in white blood cell counts 3. Disseminated intravascular coagulation 4. Hypotension 5. Shock 6. Lethality due to ARDS (acute respiratory distress syndrome) What host responses lead to the pathophysiological distress reactions caused by LPS? Inflammatory response What part of endotoxin is responsible for its toxic effects and how is this part recognized by the host? Lipid A component of LPS is the main cause of toxic activities 8

MICR 470/670, Microbial Pathogenesis, Exam 3 What are the 5 functions of O polysaccharide during infections? 1. Carrier activity 2. Anti-complement activity 3. Antiphagocytic activity 4. Adherence activity 5. Antigenic variation ****************************************************************************** ******* Bacterial Exotoxins Bacterial toxins can be divided into 3 major categories based on their sites of action on host cells and how they get there. What are those 3 categories? 1. Toxins acting at the Cell Surface 2. AB Toxins 3. Translocated Toxins Some bacteria produce toxins called superantigens. Describe how superantigens work. Superantigens are bivalent molecules that bind major histocompatibility complex class II (MHC-II) on APCs and also variable part of the T-cell receptor (Vb or Vg). Superantigens cross- link non-matching MHC-II and T-cell receptors, resulting in activation of T-cells, even in the absence of a specific peptide. Superantigens cause toxic-shock syndrome (induced by toxic shock syndrome toxin 1 (TSST-1), vomiting and diarrhea. What are two physiological effects on hosts caused by superantigens? 1. T-cell activation and inflammatory 2. Excessive, uncoordinated release of cytokines 9

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MICR 470/670, Microbial Pathogenesis, Exam 3 Bacteroides fragilis enterotoxin BFT belongs to a large family of bacterial metalloproteases that act on host cell surfaces. Describe what protein BFT targets and three effects that occur when intestinal epithelial cells are exposed to BFT. Pore-forming toxins work by forming holes in the plasma membrane of host cells, thus breaking the permeability barrier. Such toxins are often called lytic factors or cytolysins (or just lysins). Cell permeablization first causes release of cytokines, activation of intracellular proteases and sometimes apoptosis, ultimately resulting in cell death (necrotic or apoptotic). Describe two aspects for the process of Streptolysin O binding to host cells and its toxic effect. Bind cholesterol-containing membranes they then polymerize to form very large pores that can be up to 35 nm in diameter. 40 or more monomers combine to make one pore. Describe two characteristics of small pore-forming toxins. Form pores 1-1.5 nm in diameter allowing solutes with molecular masses of less that 2000 Da to diffuse through. What modification of E. coli hemolysin must occur for it to be active? RTX toxins are synthesized as protoxins and are activated by fatty-acid acylation on Lys594 and Lys690. The typical function of the B portion of AB toxins is to translocate the A subunit into the host cytoplasm. What are three pathways by which this happens? 1. Directly 2. Endocytosis a) endosomal pathways b) retrograde pathways What are the five different architectures for AB toxins? 1. Single chain AB toxins 2. Binary AB toxins 3. Tripartite AB toxins 4.AB 5 toxins 5. AB 7/8 toxins 10

MICR 470/670, Microbial Pathogenesis, Exam 3 Write the reaction which bacterial toxins with ADP-ribosylase activity catalyze. Target Protein + NAD  Target Protein::ADP-Ribose + Nicotinamide Describe three characteristics of diphtheria toxin (DT) activity and effects on host cells. DT B subunit has two subdomains R subdomain binds to a receptor on host cell surfaces T subdomain translocates the A subunit across the endosomal membrane A subunit has ADP-riboslyase activity and modifies the ribosomal elongation factor 2 (EF2) on a modified histidine residue, which interferes with tRNA binding What is the enzymatic activity of Shiga toxin? Composed of an A and B Subunit B subunit binds glycolipid Gb 3 and is internalized by receptor mediated endocytosis, undergoes retrograde transport to Golgi, ot the ER. A subunit is released into the cytosol and has N-glycosidase activity which cleaves adenine residue on the 28S ribosomal RNA, making it not interact with EF1 or EF2, thus protein synthesis is blocked, and cell dies. Describe three characteristics of cholera toxin activity and effects on host cells. 1. Cholera toxin (CT) modifies Gs causing permanent activation of the G protein 2. G protein activation causes production of a large amount of cAMP 3. Increases Cl-secretion and inhibition of Na + uptake (causing watery diarrhea) Bordetella pertussis produces two AB toxins, pertussis toxin (PT) and CyaA. What is the common effect on target cells by these two different toxins? Increases in cAMP formation, which induce increased Cl-secretion and inhibition of Na + uptake. What host proteins are targets of PT? PT modifies the G i , G 0 , G t , and G gust G proteins on the α subunit. PT modifies a cysteine residue near the C’ terminus of these G proteins. 11

MICR 470/670, Microbial Pathogenesis, Exam 3 Bacillus anthracis produces two different AB toxins, edema factor (EF) and lethal factor (LF). Describe the enzymatic activity of LF and two effects LF has on host cells. Cleaves the N’ end of MAPKK1 & MAPKK2, inactivating the kinases and disrupting the MAP kinase cascade which controls cell proliferation and gene expression. Induces macrophages to overexpress proinflmmatory cytokines, apoptosis. Bacillus anthracis produces two different AB toxins, edema factor (EF) and lethal factor (LF). Describe the enzymatic activity of EF and two effects EF has on host cells. Has calmodulin-dependent adenylate cyclase activity, producing a lot of cAMP. Disrupts water hemostasis (edema) and disrupted macrophage functions. What is the “B subunit” for EF and LF and its structure? The protective antigen (PA7/8) that undergoes a change to allow translocation of EF/LF across endosomal membranes into cytosol Describe the enzymatic activity of cytolethal distending toxin and its effects on host cells. CdtB is a DNA nuclease that makes dsDNA breaks, resulting in cell-cycle arrest at G2 checkpoint, leading to cell swelling. Describe the enzymatic activities of Clostridium difficile TcdA toxin and uropathogenic E. coli cytotoxic necrotizing factor 1 (CNF1) and their effects on host epithelial cells. TcdA has UDP-glycosyltransferases that monoglucosylate threonine residues in GTP-binding proteins, inactivating them. CNF1 has glutamine deaminases activity, which deamidates glutamine residues of Rho, Rac and Cdc42 activating them. Inactivated Rho, Rac and Cdc42 result in breakdown of the cellular actin fibers, disrupting epithelial structure. Activated Rho, Rac and Cdc42 promote bacterial internalization though induced ruffling, stress- fiber formation and cell spreading Clostridium bacteria produce many neurotoxins. What host cellular process (not the protein targets) is inhibited by botulinum toxin? Neurotransmitter vesicle docking and membrane fusion. What are the protein targets of tetanus toxin and botulinum toxin A? Tetanus Toxin = VAMP/synaptobrevin Botulism Toxin A = SNAP-25 12

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MICR 470/670, Microbial Pathogenesis, Exam 3 Why does tetanus toxin cause spastic paralysis while botulinum toxin causes flaccid paralysis? Tetanus toxin blocks the release of inhibitory neurotransmitters in central nervous system, which is required to stop the nervous impulse. w/o it, it produces generalized muscular spasms. Botulinum toxin blocks release of stimulators in peripheral nervous system, causing weakness or flaccid paralysis. Describe two different negative effects H. pylori VacA toxin has on epithelial cells. 1. VacA pores increase cellular permeability to organic molecules, iron, and nickel within gastric epithelial cells thereby liberating vial nutrients. 2. VacA can damage mitochondria and induce apoptosis in epithelial cells AB toxins are diverse and target a variety of cellular functions. Name four different enzymatic activities of AB toxins. 1. Phospholipase C (PLC) activity (degrades phospholipids), 2. Acting on G proteins, 3. cAMP production, 4. Inactivates MAP Kinase Cascade components AB toxins are diverse and target a variety of cellular functions. Name four different host cell functions targeted by AB toxins. 1. Cell cycle progression, 2. Protein synthesis, 3. Actin cytoskeleton, 4. Neurotransmitter vesicle trafficking, 5. Late endosomal pathway ****************************************************************************** ******* 13

Study Guide, Exam 3 2021

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