Study Guide, Exam 5 2021

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MICR 470/670, Microbial Pathogenesis, Exam 5 Study Questions Lectures 23 - 26 ************************************************************************************* Apoptosis Multicellular organisms make extensive use of the cell suicide mechanism termed apoptosis. What are three normal processes that involve apoptosis in multicellular eukaryotes? 1. Normal Development 2. Maintenance of tissue homeostasis 3. Effective immune system What are two markers that apoptotic cells express on their surface and what are they used for? 1. Vitronectin 2. Phosphatidylserine Recognized by professional phagocytes rapidly engulf apoptotic bodies The FAS ligand can induce apoptosis in target cells. What are the first three proteins in target cells that are activated by FAS ligand? 1. Fas 2. FADD (adaptor protein) 3. Procaspase Caspases play an essential role in apoptosis. There are two major kinds of caspases. What are these two kinds called and what are their basic roles? 1. Activated capase-8 proteolyzes executioner procaspase-3 into an active form. 2. Activated caspase-3 proteolyzes numerous targets, including destroying I-CAD (inhibitor of CAD), thus freeing CAD. One target of caspase-3 is the CAD::I-CAD complex. What does caspase-3 do to this complex and what happens next? Destroys I-CAD (inhibitor of CAD), thus freeing CAD. Free CAD (Caspase-Activated DNase) cut chromosomal DNA What are two intrinsic signals that elicit apoptosis? 1. Apaf-1 (apoptotic protease activating factor) 2. Cyt C (Cytochrome C) Page 1

MICR 470/670, Microbial Pathogenesis, Exam 5 Study Questions Describe the two proteins and all of the small molecules that are in an apoptosome. Cytochrome C binds Apaf-1 along with dATP/ATP creating a heptameric scaffold which binds procaspase-9 How does an apoptosome lead to apoptosis? Apoptosome containing activated caspase-9 then proteolytically processes executioner procaspase-3 and procaspase-7 into active forms that proteolyze substrates There are about 20 Bcl-2 family members that fall into three subfamilies (Bcl-2 subfamily, Bax subfamily, and BH3 subfamily). These three subfamilies are placed into two major groups that control apoptosis. What are those two major groups? 1. Pro-survival members 2. Pro-death members What are two activities that Bcl-2 does to prevent/antagonize apoptosis? 1. When Bcl-2 dimerizes with pro-apoptotic member it prevents pro-apoptotic protein from forming homodimers. 2. Bcl-2 members also bind Apaf-1 and localize it to the mitochondrial membrane preventing apoptosis What is the primary way that Bax leads to apoptosis? Bax promotes membrane perturbation of mitochondria, releasing Cyt C, which activates Apaf-1, leading to caspase activation. What is one way that levels of Bax protein increase in cells destined for apoptosis? Bax is predominantly a cytosolic monomer in healthy cells but during apoptosis it undergoes conformational changes at both termini, translocates to the outer mitochondrial membrane, and oligomerizes. What is the pathway that leads from excessive levels of Ca 2+ in the cytoplasm to free Bax, which can then lead to apoptosis? Calcineurin is activated by increases in concentration of Ca2+ in cytoplasm which occurs during many kinds of stress. Neutrophils have a short lifespan of 1-2 days when fighting infections. Why is this good for the host? Maintenance of tissue integrity  Homeostasis  preserve organ function Page 2

MICR 470/670, Microbial Pathogenesis, Exam 5 Study Questions ************************************************************************************* Apoptosis and Disease Some microbes promote apoptosis in host cells, while others block apoptosis in host cells. What are the benefits to microbes to promote apoptosis? Induction of apoptosis may further invasion or spread through cell barriers, kills immune cells. Some microbes promote apoptosis in host cells, while others block apoptosis in host cells. What are the benefits to microbes to block apoptosis? Inhibition of apoptosis can ensure survival of a host cell that harbors an intracellular microbe, such as viruses and some bacteria. High levels of cAMP lead to apoptosis. Describe three components that are involved and lead to apoptosis in cells with high levels of cAMP (don’t describe how cAMP levels become high). 1. Activate protein kinase A (PKA), which inhibits activity of AKT 2. Low AKT decreases expression of pro-survival Mcl-1, resulting in apoptosis. 3. Low AKT also reduces phosphorylation of Bad, which allows active Bad to bind Bcl-2 members and promote apoptosis. How do Shigella flexneri bacteria induce pyroptosis of macrophage? Shigella is phagocytosed by macrophage and escapes vacuole. IpaB is secreted via T3SS and binds to and activates capase-1 (ICE), which then initiates pyroptotic cell death. Inhibition of eukaryotic translation by bacterial toxins eventually activates apoptosis. Why? Apoptosis is probably caused by decreased synthesis of pro-survival Bcl-2 members Describe four steps in the process whereby small doses of pore forming bacterial toxins cause apoptosis. 1. Small changes in cytoplasmic membrane that can lead to apoptosis due to fluxes of ions (Na+, Ca2+) and hyperpolarization of the membrane 2. 3. 4. Page 3

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MICR 470/670, Microbial Pathogenesis, Exam 5 Study Questions H. pylori VacA causes vacuolation, which is swelling of late endosomes. How can VacA lead to apoptosis? Vesicles eventually rupture and release VacA into cytosol. VacA and p37 translocate to the mitochondrial matrix where they can interact with the mitochondrial permeability transition pore (mPTP). mPTP is involved in Cytochrome C (CytC) release during apoptosis. CrmA, which is made by Cowpox, prevents apoptosis. How? CrmA is a competitive inhibitor of caspase activity. CrmA inhibits caspases 8, 9, and 10 thus inhibiting initiation of apoptosis. How does the Adenovirus E1B protein prevent apoptosis? E1B homology to Bcl-2 prevents apoptosis Rickettsia bacteria inhibit apoptosis in endothelial cells by activating NF-kB. What are two different ways that NF-kB activation prevents apoptosis? 1. Increasing expression of pro-survival Bcl-2 genes 2. Prevents activation of the caspase cascade by increasing expression of cIAP2 ************************************************************************************* Pyroptosis Pyroptosis, apoptosis, and necrosis are all forms of cell death. What are two cell morphologies common to cells undergoing pyroptosis and necrosis but not to apoptosis? 1. Cell Lysis 2. Cell swelling Pyroptosis and apoptosis are both forms of programmed cell death. Nonetheless, they have significant differences. What is a major difference between their effects on the immune system? Pyroptosis results in the release from cells of PAMPs and cytokines that activate pro- inflammatory immune cell mediators, unlike apoptosis. Pyroptosis occurs when cells recognize pathogen-associated molecular pattern (PAMPs). What are two host receptors, discussed in class, that recognize PAMPs and elicit pyroptosis? 1. NOD-like receptors (NLRs) a. NLRP3 b. NLRC4 Page 4

MICR 470/670, Microbial Pathogenesis, Exam 5 Study Questions Pyroptosis occurs when cells recognize pathogen-associated molecular pattern (PAMPs). What is the next step for these receptors after binding to a PAMP? NLRP3 and NLRC4 are incorporated into inflammasomes which then activate caspase- 1 leading to pro-cytokine processing, pore formation, and cell death by pyroptosis. Name three different host proteins that are in an inflammasome. 1. Multimers of NLR 2. PYCARD adaptor protein 3. Pro-caspase-1 (or 11) Caspase-1 has a role inducing an inflammatory signal. What is that role? Caspase-1 starts as pro-caspase-1  oligomerize in an inflammasome activates to caspase-1  cleaves IL-1β and IL-18  induces inflammatory response What are the steps from Caspase-1 to pore formation during pyroptotic cell death? Gasdermin D is proteolyzed and activated by caspase-1  inserts into membrane forming pores  pyroptotic cell death What is the pore-induced intracellular trap (PIT) and how does it help fight intracellular bacteria? PIT = membrane pores small enough to release soluble cytosolic contents but retain organelles and bacteria. Viable bacteria remain trapped within How does pyroptosis act as a defense mechanism against infection? Inducing pathological inflammation ************************************************************************************* Biofilms Bacterial biofilms are permanent bacterial communities that exhibit resistance to different stress factors. What are three factors that bacteria in biofilms are able to resist? 1. antibiotics 2. disinfectants 3. phagocytosis About how prevalent are biofilms in bacterial infectious diseases in the US? ~65 – 80% of all bacterial infections Page 5

MICR 470/670, Microbial Pathogenesis, Exam 5 Study Questions About how many biofilm-related bacterial infections occur each year in the U.S. and about how many deaths occur each year? ~17 million new biofilms infections occur each year in the United States Up to 550,000 people die each year with or from biofilm infections What is the definition of a biofilm? Bacterial biofilms are structured consortia of bacteria encased in a self-produced matrix. Name four different kinds of macromolecules that are commonly found in bacterial biofilms (as described in your handout). 1. Exopolysaccharides 2. Proteins 3. Teichoic acids 4. Extracellular DNA (eDNA) What are four mechanisms that can account for why bacteria in biofilms are tolerant to antibiotics? 1. Failure of antibiotic penetration 2. Accumulation of high levels of antibiotic degrading enzymes 3. Slow or non-growing cells are more resistant to antimicrobial agents 4. Genetic changes creating or transferring antibiotic resistance Why are clinical biofilm infections difficult to detect? Complex polymicrobial biofilm communities that can only be detected by culture techniques when bacterial cells happen to detach a sufficient bolus of planktonic cells that can be isolated and grown on conventional culture media. Prosthetic medical devices are risk factors for biofilm infections. What are the four typical clinical characteristics of such infections? Slow onset, middle-intensity symptoms, chronic evolution, and resistance to antibiotic treatment Orthopedic biofilm infections may lead to host damage caused by the host immune system. What are two such host damages? 1. Osteomyelitis – devastating effects on bone and soft surrounding tissues 2. Implant infections – Replacement of implant is the only real chance to eradicate the infection Page 6

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MICR 470/670, Microbial Pathogenesis, Exam 5 Study Questions Biofilm formation is classically viewed as a four-step process. Briefly describe those four steps. 1. Initial attachment of bacterial cells 2. Cell aggregation 3. Biofilm maturation 4. Detachment of cells from the biofilm What are two different functions of autolysin in biofilm formation? 1. Adhesive 2. Enzymatic Polysaccharide intercellular adhesin (PIA) is prominent in biofilms. What is the biochemical structure of PIA and what genes encode its formation? PIA is linear b-1,6-linked glucosaminylglycan (poly-N-acetyl-glucosamine or PNAG), synthesized by enzymes encoded by icaADBC locus. It is possible to grow PIA-producing bacteria in the lab and isolate PIA-non producing variants. The non-producing variants can be grown and make PIA-producing variants again. What accounts for this? Transposition of IS256 out of icaADBC locus Bap (Biofilm Associated Protein) can participate in making biofilms. What are three major steps that occur during this process? Bap is processed and N-terminal fragments self-assemble into functional amyloid aggregates (at acidic pHs and low [Ca2+]) to build a biofilm matrix. GelE is a gelatinase, which is part of a process that results in release of genomic DNA from bacteria in biofilms. Describe the steps from GelE to release of DNA. GelE is a protease which cleaves many proteins, including cleaving and activating autolysin. There is a process independent of the GelE pathway, discussed in class, that contributes to release of genomic DNA from bacteria in biofilms. What is that other process? AltE autolysin is a peptidoglycan hydrolase Page 7

MICR 470/670, Microbial Pathogenesis, Exam 5 Study Questions What bacterial proteins are involved in dispersion of biofilm cells as planktonic bacterial cells? Proteases and β-type PSMs (phenol-soluble modulins). PMNs are known to attack biofilms. What are the major characteristics of these PMNs? PMNs at site of infection exhibit upregulation of surface receptors required for bacteria recognition and killing PMNs are known to attack biofilms, what problem does this create for the host? PMNs are not able to control the infection. Living bacteria can still be isolated from infected sites. PMNs do cause damage to host tissues at site of infection Describe what NETs are and how they may be able to fight bacterial infections. Neutrophil extracellular traps (NETs) trap microbes and are loaded with proteases, thereby killing microbes. ************************************************************************************* Quorum Sensing What is quorum sensing? Accumulation of signaling molecules in environment enables a single bacterial cell to sense the bacterial cell density. What is the basic quorum sensing process? Basic process is signaling from one bacterial cell to another, which controls gene expression according to population density QS signaling molecules have four important characteristics. What are they? 1. Production of QS signal 2. QS signal accumulates 3. Critical threshold concentration 4. Cellular response controls population behaviors What is an autoinducer? Autoinducers are QS signaling molecules that are released by bacteria into extracellular medium Page 8

MICR 470/670, Microbial Pathogenesis, Exam 5 Study Questions What role does cell density play in QS? At critical cell densities: concentration of signaling molecules exceeds a threshold value and the molecules activate transcription of a particular set of genes creating a coordinated population response. Some autoinducers bind to cytoplasmic receptors. What do the autoinducer/receptor complexes do? Binding of autoinducers to receptors does not effectively occur until a threshold concentration of autoindeucers is achieved which indicated high density of cells producing that autoinducer. Autoinducer receptors can be transcription factors, while others relay signals to downstream transcription factors. There are 3 well-described QS systems discussed in your handout. What are they and what kinds of bacteria use them? 1. Autoinducer-1 (AI-1) system Gram-negative (Ex. A. fischeri) 2. Autoinducer Peptide (AIP) system Gram-positive (Ex. B. subtilis) 3. Autoinducer-2 (AI-2) Gram-negative and Gram-positive (E. coli) What is the type of signal molecule used in the AI-1 system? Acyl-homoserine lactones (AHLs) What does the A. fischeri QS cycle consist of? LuxI synthesizes the AI, which binds to LuxR, which forms dimers that turn up transcription of certain genes What QS system is specific to Gram-positive bacteria and what are the two different kinds of receptors for the signaling molecules in this system? Autoinducer peptides (AIP) 1. Histidine-protein kinase receptor 2. Cytoplasmic receptor What is the basic type of signal molecule used in the AI-2 system? AI-2s are forms of furanosyl borate diesters. AI-2s are synthesized by LuxS Page 9

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MICR 470/670, Microbial Pathogenesis, Exam 5 Study Questions What are the four virulence processes, presented in your handout, that involve the AI-2 system in E. coli ? 1. Increases biofilms 2. Increases chemotaxis 3. Increases virulence 4. Increases cell aggregation What gene encodes the synthesizer of the autoinducer and what gene encodes the autoinducer receptor in the A. fischeri bioluminescence system? Autoinducer = LuxI Autoinducer receptor = LuxR What is the process controlled by the genes in the lux operon? LuxC, LuxD, and LuxE genes code for components of an acid reductase that converts the long-chain fatty acid tetradecanoic acid into the fatty-aldehyde substrate used by light-producing enzyme luciferase. LuxA and LuxB genes encode subunits of luciferase, which generates light Why is QS off at the edges of a biofilm in Staphylococcus aureus ? Edges of biofilm; GFP was not produced by cells therefore QS was off Maintaining a biofilm on edges maintains protection from antibiotics, immune cells, etc.. How does QS affect the cells at the bottom of a S. aureus biofilm? QS in S. aureus represses biofilm formation and induces degradation, so cells at the bottom (QS on) degrade matrix, freeing cells to disperse. Biofilm off at the bottom allows release of cells to disperse or penetrate tissues. QS has been well studied in Pseudomonas aeruginosa . What is the effect of mutants lacking both AI-1 QS systems in rat infections, as presented in your handout? ~50 fold reduced levels of PAOR bacteria after 4 weeks, compared to rats infected with PAO1 wild type bacteria that had functional QS Page 10

MICR 470/670, Microbial Pathogenesis, Exam 5 Study Questions What are the three basic strategies for inhibiting QS? 1. Target signal generation 2. Target AHL signal dissemination 3. Target the signal receptor What are the three different ways, presented in your handout, to target AHL signal dissemination? 1. Increasing Ph (>7) – AHL molecules undergo lactonolysis 2. Raising temperatures – causes lactonolysis of AHL 3. Lactonases – produced by some bacteria to compete with AHL-producing bacteria What was done to prevent A. hydrophila infections of fish as an alternative to antibiotic treatment? Oral administration of AiiA to fish in their feed significantly attenuated Aeromonas hydrophilia infection in zebrafish, proving AiiA to be an effective alternative to treatment with antibiotics Describe how combination therapy with a QS inhibitor and an antibiotic work in P. aeruginosa . QS inhibitors and antibiotics can increase the efficacy of the antibiotic. Garlic extract components inhibited AHL receptors and also reduced P. aeruginosa tolerance to tobramycin treatment in biofilms. QS inhibitors increased the susceptibility of P. aeruginosa biofilms to tobramycin. ************************************************************************************ Page 11

Study Guide, Exam 5 2021

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