Study Guide, Exam 4 2021

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MICR 470/670, Microbial Pathogenesis, Exam 4 Study Questions Sec and TAT Systems The general secretory pathway (Sec) in Gram-negative bacteria requires SecA, SecB, SecY, SecE, and SecG. What is the signal sequence used by the Sec system? N-terminal signal domain. Substrates of the Sec-dependent pathway are targeted to the secretion apparatus by an N-terminal signal domain with three parts (N, H, and C regions) What are three secretion pathways for proteins that use the Sec system? 1. Monomeric 2. Two-partner 3. Trimeric The TAT system in Gram-negative bacteria requires TatA, TatB, and TatC. What is the signal sequence used by the TAT system? Same N, H, and C regions except they have an additional special peptide sequence What are two secretion pathways for proteins that use the TAT system? 1. 2. T2SS T2SS is the primary pathway for the secretion of extracellular degradative enzymes. How many proteins are involved in this secretion system and where are they located? There is a total of 14 secretion factors necessary for secretion. Seven of these proteins are located in the cytoplasmic membrane and others are outer membrane proteins. How do proteins secreted by the T2SS get to this system? Translocators such as PulD What are two virulence factors, presented in your handouts, known to be secreted by the T2SS? 1. LT I (E. coli) 2. cholera toxin (V. cholerae) ****************************************************************************** ******* T5SS There are three types of T5SS secreted proteins, Type Va, Type Vb, and Type Vc. All three types have three functional domains. Describe the function of each one. 1. Monomeric system (type Va or 5a) 2. Two partner system (type Vb or 5b) 3. Trimeric system (type Vc or 5c) What is main difference between the secretion of Type Va and Type Vb proteins? Trimerization occurs in the B barrel pore of Vc and does not in Va. 1

MICR 470/670, Microbial Pathogenesis, Exam 4 Study Questions What is main difference between the secretion of Type Va and Type Vc proteins? Describe three steps for the secretion across the outer membrane (not the cytoplasmic membrane) for Neisseria gonorrhoeae IgA1 protease. IgA1 protease acquires active conformation, released in a preform from the membrane-bound helper by autoproteolysis, matures further into functional protease leaving alpha protein fragment behind. What kinds of virulence factors are typically secreted by the type Vc system and name one example given in your handouts. Adherence, invasion, proteolysis, cytotoxicity, serum resistance, and cell to cell spread. Adhesins such as YadA Vc. T1SS All T1SSs consists of three types of proteins. Describe the function of each type. 1. Pore forming outer membrane protein (OMP) – forms the pore 2. Membrane fusion protein (MFP) – fuses the outer membrane to inner 3. Inner membrane ATP-binding cassette (ABC) – inner membrane binding stabilizing What are the four different types of well-studied virulence proteins secreted by T1SSs? 1. Alpha-hemolysin 2. Leukotoxin 3.Adenylate cyclase (CyaA) 4. E. coli has four genes, hlyA, hlyB, hlyC, and hlyD involved in secretion of alpha-hemolysin. What is the function of each one? HlyA is a lipid-modified prior to secretion and inserts into plasma membrane causing a small pore formation and release of cytoplasmic contents HlyC is enzyme that fatty-acid acylates HlyA HlyB is the ABC for HlyA HlyD is the MFP for HlyA TolC is the OMP The T1SS secretes virulence factors independently of the T2SS. Describe two differences in the secretion process between protein secretion for T1SS and T2SS (besides the proteins that constitute the secretion machineries). 1. No periplasmic intermediates, continuous process, no Sec or TAT 2. Proteins secreted by T1SS are not processed during secretion What is the secretion signal for effectors secreted by T1SSs? 60 amino acids long within the C terminal end of the secreted effector molecule interacts with the ABC transporter protein 2

MICR 470/670, Microbial Pathogenesis, Exam 4 Study Questions Describe three steps involved in leukotoxin secretion. LtxA leukotoxin binds to LtxB (ABC), LtxD (MFP) then fuses to TdeA (OMP), which functions like TolC T3SS It appears that type III secretion systems (T3SSs) are partially derived from components of bacterial flagella apparatus. Describe the function which is common to both the T3SS and bacterial flagella. 1. Protein sequence similarities 2. Protein secretion in common 3. Flagella and T3SS are made up of a single protein 4. Homology of inner membrane of T3SS and flagellar apparatus proteins Describe three physical characteristics of T3SS needle structures. 1. Straight 2. Apparently rigid 3. Hollow Seven characteristics of T3SSs were discussed in your handouts and in class. Describe them. 1. Host contact 2. Dedicated cytoplasmic chaperones 3. Secretion signal (mRNA signal) 4. No periplasmic intermediates 5. No proteolytic processing 6. Secretion regulated transcriptional expression 7. Regulated posttranslational and cotranslational protein translocation Describe the secretion signal for effectors to be targeted to the T3SS machinery and be secreted and one experimental evidence for identifying the secretion signal. The first 15 or so codons in mRNA Single base change in codon 3 of Yersinia YopQ prevents secretion of an NPT In general, what kinds of effectors are translocated early and what kinds are translocated later? Early effectors are needed in a timely manner to prevent or promote phagocytosis Late effectors are needed once the bacteria is where it needs to be to promote survival What proteins do Shigella bacteria use to make pores in host cells as part of their T3SS and what is the name of the structure created by these proteins? IpaB, IpaC, IpaD Bacterial pore, membrane attack complex (Translocon) How fast does translocation of preformed effectors into the host cytoplasm occur after contact between bacteria and host cells. Within 80 – 200 seconds 3

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MICR 470/670, Microbial Pathogenesis, Exam 4 Study Questions What proteins are the first to be secreted by a T3SS and when does this happen? Translocators are secreted when the tip of the needle docks on the host membrane and they create a pore called a translocon in the host membrane Some chaperones for T3SS effectors are said to be dedicated, what does that mean? An individual chaperone usually promoting translocation of a single cognate effector or two effectors. What happens to the virulence of Yersinia that have a deletion of the SycE chaperone and why? Deletion of syce gene causes impairment in YopE translocation which gives rise to an avirulent strain of Yersinia Describe three changes that happen to the YopE protein in cells lacking SycE. 1. Translocated YopE are ~40 fold lower 2. Loss of virulence without translocated YopE 3. 450-fold-higher LD 50 The Salmonella SicA chaperone is bound to its effectors (SipB, SipC) before activation of its T3SS. What does the SicA chaperone do when the T3SS is activated? SicA chaperone binds effectors SipB and SipC as well as InvF to turn on transcription of some anti-host virulence genes Chaperones have one or more of four possible functions. What are those four possible functions? 1. Passive protection 2. Secretion competency 3. Chaperone-Effector complex as targeting motif 4. Temporal function After T3SS-dependent translocation of effectors into host cells, there is an increase in transcription of certain bacterial virulence genes, such as those that encode T3SS effectors. What is the mechanism for this transcriptional activation? Transcription is activated when chaperones release their effector proteins for translocation, the chaperones are free to bind to the AraC – like TA and turn up gene expression Describe the model for temporal translocation of effectors. 1 st Translocation: chaperone::effector (already made) translocated effector and free chaperone binds to AraC-like TA; Chaperone::AraC-like TA new transcription of effector genes 2 nd Translocation: Newly synthesized effectors cotranslationally translocate without chaperone List four different functions or enzymatic activities of effector proteins translocated into the host cytoplasm by T3SSs. 1. Effectors that block pro inflammatory responses 2. Effectors that promote bacterial internalization 3. Effectors that prevent phagocytosis 4. Effectors that control transcription 4

MICR 470/670, Microbial Pathogenesis, Exam 4 Study Questions Yersinia effectors cause paralysis of phagocytic function preventing engulfment of bacteria. Describe the functions of three effectors that help accomplish this. YopH is a very active protein tyrosine phosphatase. Disrupt normal immune responses YopE binds RhoA, Rac, and Cdc42 and displays GAP activity that turns off these small GTP- binding proteins. Massive depolymerization of actin stress fibers YopT also inactivates RhoA, Rac, and Cdc42. It is a protease that cleaves the small GTP-binding proteins at their C’ end. Function is redundant with YopE. Describe the function of the Yersinia effector that prevents transcriptional activation of the inflammatory response. YopP is a protein acetyltransferase. YopP blocking MAPK kinases prevents a pro-inflmmatory response and indirectly blocks the activity of NF-kB which blocks turn on of the immune response. Salmonella induces internalization into epithelial cells. Describe the functions of two effectors with different activities that help accomplish this. SopE and SopE2 have guanine nucleotide exchange factors (GEF) activity and bind and activate Rho-family proteins causing membrane ruffling and bacterial internalization Salmonella SopD effector has the opposite effect on host cells as SipA, SipC, SopB, SopE, and SopE2. How does this benefit the bacteria? SopD inactivates Cdc42 and Rac1, reversing SipA, SipC, SopE, SopE2, and SopB effects.This seems counter intuitive but works for Salmonella because of timing. Salmonella has two T3SSs. What stage of Salmonella infection requires the T3SS-1 and what stage requires the T3SS-2? After Salmonella are internalized by epithelial cells, the T3SS-1 gene expression is down regulated. T3SS-2 gene expression is induced 1-4 hours after bacterial internalization and is maintained continuously to insure bacterial survival and intracellular replication over long periods of time. How does Salmonella coordinate the use of T3SS-1 and T3SS-2? T3SS-1 is for initial rapid translocation of several effectors but over the longer term (30-60 minutes) ruffling is shut off by SopD and the cell resumes its normal morphology with the bacteria safely inside. T3SS-2 gene expression is induced 1-4 hours after bacterial internalization and is maintained continuously to insure bacterial survival and intracellular replication over long periods of time. T4SS What are the three kinds of substrates translocated by the type IV secretion system (T4SS) and what are the three destinations of substrates transferred by T4SSs? 1. DNA/protein complexes transferred from one bacterium into another bacterium. 2. Proteins or DNA/protein complexes transferred from one bacterium into a host cell. 3. DNA fragments transferred into or out of the extracellular space 5

MICR 470/670, Microbial Pathogenesis, Exam 4 Study Questions Describe the three basic types of T4SSs. 1. Conjugation systems: machines that translocate DNA/protein complexes from bacterial donor to bacterial recipient cells by a contact-dependent process. 2. Effector translocator systems: deliver proteins or DNA/protein complexes into eukaryotic cells by contact-dependent process. 3. DNA release or uptake systems: translocate DNA to or from extracellular milieu. Not contact- dependent process. Not discussed here. Describe three functions of the F pilus in conjugation. 1. Forms a specific attachment between donor and recipient 2. Contraction to bring cells close together to facilitate transfer 3. What role do conjugation plasmids play in antibiotic resistance? Conjugation plasmids are typically the transferred DNA and they carry the genes encoding the T4SS machinery. The Agrobacterium tumefaciens T4SS machinery has been well studied. What molecules are transferred by this system and where do they go? transfer part of a plasmid DNA (T-DNA) bound to bacterial proteins into plant cells. Results in a tumor like growth. There are 12 Vir proteins (VirB1–VirB11 and VirD4) that constitute the Agrobacterium T4SS machinery. The VirB components can be divided into three major groups of proteins. Describe the three groups. i. Energizing components (VirB4, VirB11, VirD4; shown in yellow in figure on next page). ii. Core and accessory components (VirB1, VirB6, VirB7, VirB8, VirB9, VirB10; shown in blue). iii.Surface/pilus-associated proteins (VirB2, VirB3, VirB5; shown in red). H. pylori bacteria secrete CagA by a T4SS. What are four effects, described in your handout, that CagA has on host cells? CagA causes NF-kB activation, IL-8 induction, neutrophil infiltration, and apoptosis. What effect does deletion of the Legionella pneumophila Dot/Icm T4SS have on its pathogenesis? Mutants of Legionella lacking the Dot/Icm T4SS are readily destroyed by phagocytes and are non-virulent, revealing essential role in pathogenesis. What are two effects that the T4SS translocated effectors of Legionella pneumophila have on host cells? Legionella first prevent fusion of phagocytic vacuole, in which they reside, to lysosomes. Then they remodel their phagocytic vacuole using endoplasmic reticulum and mitochondrial vesicles, providing nutrients, and creating a vacuole that allows intracellular multiplication. 6

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MICR 470/670, Microbial Pathogenesis, Exam 4 Study Questions What does the signal for Dot/Icm-dependent protein translocation consist of? Signal for Dot/Icm-dependent protein translocation consists of amino acids with similar physical and chemical properties in the last 35 residues: i. Large stretches of glutamic acid at positions -10 to -17 from the C’ end; ii. Several hydrophobic residues located at the C-terminal end are the most important. There are three known chaperone proteins for the Dot/Icm T4SS. Describe three characteristics of how they function. IcmS/IcmW and IcmS/LvgA complexes are chaperones that interact with a large subset of T4SS substrates to facilitate their translocation by mechanisms akin to T3SS chaperones. IcmS/IcmW and IcmS/LvgA chaperones bring the substrates into the proximity of transport machinery by directly interacting with a T4SS component, DotL, the coupling protein. The expression of Dot/Icm-dependent substrate genes can occur during at least four different time frames. What are those four time frames? Expression of effector genes can occur during at least four different time frames: i. Prior to internalization; ii. Beginning of intracellular infection; iii.End of intracellular infection; iv.Entire life cycle. Give four differences in the process and regulation of secretion of pertussis toxin (PT) by Bordetella pertussis compared to secretion of most other protein virulence factors by type IV systems (T4SS). 1. Independent of the presence of host cells under laboratory conditions 2. PT secretion is constitutive. 3. secretes pertussis toxin into extracellular space. 4. PT subunits assemble in periplasmic space forming complete toxin. T6SS T3SS and T4SS systems transport substrates through both the inner and outer membranes and directly into the cytoplasm of host cells. The T6SS system is significantly different from the T3SS and T4SS ( independent of the molecular machinery ). Why? T6SS is significantly different from the T3SS and T4SS because it is involved in both:A. Interbacterial competition and B. Pathogenesis The activity of the T6SS helps to reshape microbial communities in two ways. What are those two ways? T6SS activity helps reshape microbial communities by: i) Competing with neighboring bacteria. ii) Promoting biofilm formation. 7

MICR 470/670, Microbial Pathogenesis, Exam 4 Study Questions The T6SS machinery comprises 13 core proteins, named type six secretion (Tss) proteins. What is the function of the TssB and TssC subunits of a T6SS? TssB and TssC subunits (red circle) assemble dynamic cytoplasmic tubules that undergo cycles of assembly, contraction, and disassembly. What other microbial structures does some of the T6SS machinery resemble both structurally and mechanistically? They are physically and mechanistically similar ti phage contractile sheaths All T6SS-carrying bacteria have Hcp and VgrG proteins. What role do VgrG proteins play in the translocation machinery and process ? VgrG pierces target cell membranes and is itself delivered inside target cells What role do Hcp1 proteins play in the translocation machinery and process ? Hcp1 forms donut-shaped hexamers that stack on top of each other to form a tube. Hcp protein is both part of the secretory apparatus but also secreted Two groups of T6SS effectors that target eukaryotic cells have been identified. What are those two groups? 1. C-terminal domain (CTD) toxins borne by specialized VgrG proteins 2. Independent toxins not borne by VgrGs What is the V. cholerae VgrG1 protein effector activity and what effect does this effector have on host cell functions and how does this benefit the bacteria? VgrG1 has an actin-cross linking domain (ACD), which catalyzes the covalent cross-linking of G-actin. Cross-linked actin cannot function like normal filamentous actin. The result alters host cell morphology, prevemts host cell cytoskeleton rearrangements and disables phagocytosis by macrophages. What is the enzymatic activity of Aeromonas hydrophila VIP-2 and what role does it play in pathogenesis of these bacteria? VIP-2 domain catalyzes ADP-ribosylation of actin, which also interferes with cytoskeleton dynamics There are four categories of effectors with antibacterial activities. What are they? 1. Cell wall-degrading enzymes 2. Membrane-targeting proteins 3. Nucleases 4. Mono-ADP-ribosyltransferase Tae effectors have antibacterial activity. What is its enzymatic activity and how does the bacterial cell protect itself from the toxin? Tae are enzymes with amidase activity, which cleaves the peptidoglycan peptide units and their cross-bridges 8

MICR 470/670, Microbial Pathogenesis, Exam 4 Study Questions The T6SS of Vibrio cholerae is used to attack sister cells. What happens when this occurs? A cognate immunity protein binds to and neutralizes the effector, both cells survive the attack Vibrio cholerae bacteria use a T6SS to allow them to invade and infect host intestines. Describe four features of the process from ingestion of V. cholerae bacteria to onset of disease (cholera), as described in your lecture on T6SSs. 1. V. cholerae T6SS translocates toxins into commensal bacteria in order to kill them 2. Dead commensal bacteria leave room for V. cholerae to colonize intestinal tract. 3. Phagocytic cells attack V. cholerae but are killed by toxins translocated by the T6SS 4. Cholera toxin secretion produces massive watery diarrhea characteristic of this infection, resulting in spread of bacteria. T7SS T7SS is a specialized secretion apparatus that secretes proteins across the mycomembrane of Mycobacterium tuberculosis . Mycolic acids in the mycomembrane protect cells in several ways, described in class. What are four of those ways? 1. Aid virulence due to resistance to chemical damage, dehydration, and hydrophilic antibiotics. 2. Allow bacteria to grow inside macrophages 3. Make extracellular protein transport difficult 4. Specialized for protein secretion across inner and mycomembranes T7SS is a specialized secretion apparatus that is required for the virulence of mycobacteria, such as Mycobacterium tuberculosis . What role does the ESX-1 T7SS play in a murine model of M. tuberculosis infection? ESX-1 system enables bacterial release from the phagosome into cytosol What is the role of EspG in the T7SS? EspG is a cytoplasmic chaperone guiding substrates to the secretion apparatus. What is the secretion sequence for PE substrates? Secreted specifically by their cognate ESX secretion systems. What is one beneficial role of EsxB in Mycobacterium tuberculosis pathogenesis? EsxA::EsxB dimers are cytotoxic to alveolar and endothelial cells, causing respiratory damage and contributing to pathogenesis. Tuberculous meningitis is one of the most severe extra‐pulmonary manifestations of Mycobacterium tuberculosis infections. What are two different routes that M. tuberculosis use to cross the blood-brain barrier? 1. Trojan horse mechanism – Using ESX-1 to escape pagolysosome 2. ESX – 1 – dependent invasion and infection of endothelial cells with subsequent damage of the basal lamina 9

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MICR 470/670, Microbial Pathogenesis, Exam 4 Study Questions Cytolysin-Mediated Translocation Streptococcus pyogenes Cytolysin-Mediated Translocation (CMT) has 2 components, SPN and SLO. SPN is associated with SLO in four ways described in your lecture. What are those four ways? 1. nga gene encoding SPN effector is in an operon with slo gene and are expressed together 2. SPN and SLO proteins are secreted outside of the bacterial cell at the same time 3. SPN and SLO proteins are secreted at the same time 4. SLO expression is required fro translocation of SPN into epithelial cells Diffusion cannot account for SPN translocation. What are four results that demonstrate this? 1. Non-adherent mutants 2. Mixed infections 3. 80% of total secreted SPN localizes to host cytoplasm 4.SLO pore formation and translocation activities can be uncoupled. What is an ExPortal and how is it involved in translocation of SPN into host cells by SLO in Streptococcus ? The ExPortal is a concentration of Sec pathway components in one location which leads to polarized secretion of proteins. There is a cooperation between the ExPortal with CMT pathway for a polarized translocation of effector proteins. S. pyogenes bacteria translocate SPN via CMT. CMT is a multistep process involving five discrete steps that must be coordinated in order to achieve efficient translocation of SPN. What are those five steps? 1. Recognition and attachment 2. Coordinated secretion 3. SLO monomer specifically recognizes SPN 4. SLO/SPN complex then binds host membrane receptor 5. SLO directs translocation of SPN across the host cell membrane What is the enzymatic activity of the effector translocated by the Streptococcus pyogenes CMT? SPN catalyzes hydrolysis of NAD+ to yield nicotinamide plus ADP-ribose or cyclic-ADP-ribose The enzymatic activity of the translocated effector apparently alters host cell signaling pathways and cell functions due to three possible effects. What are those three effects? 1. Cytoplasmic CA 2+ level increases 2. Attachment of ADP-ribose 3. Low levels of NAD + SPN effects also alter host cell biology in three ways. What are those three ways? 1. Inhibiting bacterial internalization 2. Contributing to the damage of cell membranes 3. Inducing apoptosis 10

MICR 470/670, Microbial Pathogenesis, Exam 4 Study Questions SLO has two distinct functions. What are they? 1. CMT – delivery of SPN into cells 2. Creation of pores that cause loss of cytoplasmic contents What is the role of cholesterol in the two distinct functions of SLO? 1. Translocate effectors into host cell cytoplasms 2. Cytolytic activity 11

Study Guide, Exam 4 2021

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