Mcb C103 Bacterial Pathogenesis Midterm Two Flash Cards

Front 1

Toxins

Back 1

a toxin is a secreted factor with a quantifiable LD 50 that is capable of producing pathology on its own at a distance from the bacterial cell that secretes it (most are proteins and heat-labile)

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Toxoid

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toxin that has been rendered non-toxic but is still immunogenic

Front 3

cholesterol-dependent cytolysins

Back 3

bind cholesterol, oligomerize and form large pores

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superantigens

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bind to MHC Class II molecules and activate subsets of T-cells which release IL-2 and indirectly TNF; can activate as many as 1/5 of T-cells

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effectors

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substrates of auxillary secretion systems such as Type III; secreted directly into host cells, do not act at a distance, and are not toxic

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corynebacterium diphtheriae characteristics

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1. high G + C
2. gram positive
3. obligate aerobe
4. non-motile
5. non-encapsulated
6. arrange like chinese letters

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diphtheria toxin mode of action

Back 7

ADP-ribosylates EF-2 preventing elongation of a nascent polypeptide chain from the ribosome; susceptible amino acid is a post-translationally modified histidine

Diphtheria toxin is an AB toxin

Front 8

what are the three categories of toxins?

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enzymes, pore-formers, or superantigens

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AB Toxins

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A subunit is the catalytic subunit while the B subunit acts as a translocation pore for A subunit

Front 10

What are the classes of DT-resistant mutants

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1. Receptor mutants
2. Vacuolar ATPase mutants
3. Diphthamide mutants

Front 11

How can a non-toxigenic strain of diphtheria be converted into a toxigenic strain?

Back 11

Specialized transduction; the genes encoding DT are located on a lysogenic bacteriophage called beta-phage

Front 12

Diphtheria vaccine

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Diphtheria toxin with specific mutations rendering it non-enzymatically active

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Bacillus anthracis characteristics

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1. spore-forming
2. poly-D-glu-encapsulated
3. gram-positive
4. non-motile
5. facultative anaerobe

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Bacillus anthracis virulence factors

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1. capsule; encoded by plasmid pX02, blocks phagocytosis, composed of poly-D-glutamic acid
2. Anthrax toxin: triparite toxin encoded on plasmid pX01 consisting of three components (LF, EF, and PA)

Front 15

Protective Antigen mode of action

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binds to cellular receptor and is then cleaved by a host furin-like protease; proteolysis in necessary for activation

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Lethal factor (Anthrax toxin)

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enzyme that is a zinc metalloprotease that cleaves MADPKK in host cytosol ultimately resulting in apoptosis

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What are the unique features of the gram-positive cell wall?

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1. lack a periplasm
2. surface proteins covalently attached to the cell wall by a sequence LPXTG near the C-terminus

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streptococcus pneumoniae characteristics

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1. gram-positive
2. diplococcus shape
3. encapsulated; 80 serotypes
4. facultative anaerobe
5. non-motile
6. obligate parasites
7. low G + C

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s. pneumoniae capsule

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capsules are composed of large polysaccharide polymers; essential for virulence and block phagocytosis by neutrophils.

Anti-capsular antibody provides protective immunity by opsonization

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s. pneumoniae vaccine

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consists of purified polysaccharide capsule from 23 different serotypes; new vaccine consists of purified polysaccharide capsule conjugated to DT and is thus a T-cell dependent antigen

Front 21

S. pneumoniae virulence factors

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1. capsule: antiphagocytic (not recognized by TLR)
2. M-like proteins for adherence
3. pneumolysin: member of the CDC family of pore-forming hemolysins

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staphylococcus aureus characteristics

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1. non-motile
2. facultative anaerobe
3. grow in grape like clusters
4. can tolerate high salt
5. extremely hardy
6. low G + C

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staphylococcus aureus determinants of pathogenesis

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1. protein A: classic LPXTG protein; plays a role in mediating pathogenesis because it binds Fc portion of IgG coating itself in host antibody and preventing immune recognition; may also capture antibody specific to S. aureus and prevent it from binding productively to the bug

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streptococcus pyogenes characteristics

Back 24

1. Gram-positive
2. growth in chains
3. low G + C
4. fastidious
5. facultative anaerobe
6. capsule composed of hyaluronic acid
7. 100s of serotypes

Front 25

streptoccal toxic shock toxin

Back 25

superantigen that stabilizes the MHC class II complex between T-cells and APC's even in the absence of productive antigen response. Results in activation of up to 20% of T-cells and a massive inflammatory immune response.

Front 26

s. pyogenes: M-proteins

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contains a membrane anchor region (LPXTG), conserved region, variable region and hypervariable region; anti-phagocytic

Front 27

Streptolysin O

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member of the CDC family of hemolysins, prevents chemotaxis of neutrophils

Front 28

vibrio cholerae characteristics

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1. gram-negative
2. facultative anaerobe
3. comma shaped
4. highly motile with single polar flagella sheathed with LPS

Front 29

v. cholerae animal models

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1. human volunteers
2. rabbit ligated ileal loop
3.infant mouse

Front 30

cholera toxin binding

Back 30

CT binds via the B-subunits to the GM1 ganglioside

Front 31

cholera toxin mode of action

Back 31

enzyme that catalyzes an ADP ribosylation reaction; locks the stimulatory G alpha subunit in the active state resulting in persistant adenylate cyclase activity and therefore, activation of cAMP

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Why does v. cholerae produce cholera toxin?

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Answer: effects on immunity; CT blocks inflammation by acting on dendritic cells and promoting IL-10 production

Front 33

Toxin co-regulated pilus (TCP) role in virulence

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presumed that TCP mediates attachment or it mediates bacterium-bacterium interaction by promoting biofilm formation

Front 34

ToxR

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membrane spanning protein that is also a transcription factor that directly activates the CT promoter

Front 35

ToxT

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transcription factor that controls the transcription of as many as 20 genes

Front 36

cholera toxin bacteriophage

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CTX region encoded by a lysogenic bacteriophage; transfer of phage shown to occur in GI tract

Front 37

bordetellae pertussis characteristics

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1. gram-negative
2. obligate aerobe
3. not invasive
4. obligate pathogen of humans
5. bacteria colonize cilia of upper respiratory tract

Front 38

pertussis toxin mode of action

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ADP-ribosylation of a cysteine in the alpha subunit of an inhibitory G-protein and prevents the Gi complex from interacting with receptors so the complex remains bound to GDP and is unable to inhibit adenylate cyclase.

Front 39

filamentous haemagglutin and pertactin

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FHA: mediates adherence and is a target of antibody
Pertactin: surface protein that is target of antibody

Front 40

trachael cytotoxin mode of action

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peptidoglycan fragment; acts by inducing IL-10 by respiratory epithelial cells which in turn induces nitric oxide that has the toxic effect; blocks chemotaxis and phagocytosis of neutrophils

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adenylate cyclase

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enzyme that converts ATP to cAMP with the help of host calmodulin; may be considered a single chain AB toxin like DT; acts on phagocytic cells and renders them non-phagocytic

Front 42

regulation of B. pertussis virulence genes

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two component regulatory system called Bvg.

BvgS is the periplasmic sensor histidine kinase that phosphorylates BvgA, the transcriptional activator.

Bvg-activated genes are called Vags while Bvg-repressed genes are called Vrgs.

Front 43

B. pertussis vaccine

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mutated pertussis toxin mixed with FHA and pertactin

Front 44

Roles of Bvg+ and Bvg -

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Bvg + necessary for rat colonization
Bvg - necessary for survival in environment perhaps?

Front 45

Role of flagella expression and virulence

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BvgAS promotes virulence both by activating genes required for colonization and by repressing genes that inhibit the development of infection

Front 46

Why are strains ecotopically expressing flagella avirulent?

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5 possible answers:
1. flagellin can be seen by TLR 5
2. Nai S and Ipath -> activates inflammasome
3. generate antibodies to flagella -> opsonization and clears infection
4. slows down replication (why bother making flagella if you don't need it)
5. type III secretion and flagellar system are similiar

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Type III secretion (B. pertussis)

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type III secretion suppresses acquired immunity (type III secretion mutants were defective in long-term colonization and elicited higher levels of anti-Bordetella antibodies)

Front 48

neisseria characteristics

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1. obligate pathogen of humans- don't infect animals with similiar disease
2. fastidious growth requirements

Front 49

meningitis vaccine

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conjugate of diptheria toxoid and N. meningitides capsular polysaccharide

Front 50

mechanisms of pathogenesis in neisseria

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1. adherence to epithelial cells
2. Transcytosis from apical to basolateral side
3. gonoccocus- growth in subepithelial space
4. meningococcus- bloodstream, growth in meninges
5. both produce inflammatory immune response
6. meningococcus- acquired immune response (protective)
7. gonoccocus acquired immune response ineffective at preventing recurrent infection

Front 51

PilE

Back 51

pillin protein; major protein for type IV pillus; encoded by two genes (PilE1 and PilE2)

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PilT

Back 52

required for twitching motility

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PilC

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important for adhesion to host cell and pilus stability

Front 54

what are the molecular determinants of pathogenesis in neisseria?

Back 54

1. lipooligosaccharide- derivative of LPS in that it lacks standard O-antigen and can be modified by attachment of host sialic acid. Advantage is camouflage (resists complement and phagocytosis)
2. capsule (only relevant in meningoccous as gonococcus does not have a capsule)- 13 serotypes with different capsular polysaccharide (from A,C,Y, W135). Purpose of capsule is to inhibit complement and phagocytosis
3. IgA protease (both gono and meningo)- protease that cleaves IgA, inhibits function of antibody
4. Type IV pili- pulls bacteria in to make contact with cell and functions in aggregation, adhesion, twitching motility, and natural transformation with DNA
5. opacity proteins (opa)- opa expression affects colony opacity (appearance on a plate) and function in adherence to host cell

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meningitis vaccine

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capsular polysaccharide + DT toxoid

Front 56

Why doesn't the adaptive immune response prevent reinfection in neisseria gonorrheae

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Answer: ability of bacteria to vary in pilus structure and expression of opa proteins

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phase variation

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complete presence/absence of proteins (ex. bacteria switches from a pilus + state to a pilus - state)

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antigenic variation

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switching from one type of pilus to another (ex. PilE1 to PilE2)

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mechanisms of phase and antigenic variation possibilites

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1. genetic recombination with locus called pilS (silent)
2. natural transformation with DNA
Experiment to test which one added DNAase to culture to see if frequency of DNA stayed the same or decreased. Results were that DNA frequency stayed the same and therefore, the major contribution is through intracellular recombination as opposed to natural transformation

Front 60

What does antigenic and phase variation look like with regard to the pilus?

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1. antigenic variation: new pilE assembled into "different" pilus (looks different to antibody)
2. phase variation: new pilE fails to assemble (how you can switch from pil + to pil -

Front 61

what is the mechanism of opa phase variation

Back 61

Answer: slipped strand mispairing

Front 62

Yersiniae characteristics

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1. gram negative
2. in the enterobacteriaceae family
3.

Front 63

What is a more appropriate term for invasion?

Back 63

pathogen-mediated internalization

Front 64

how can one show bacteria are inside cells?

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1. bacteria added to a monolayer of mammalian cells and invasion is quantitated by the number of gentamicin resistant cfus and expressed as % of bacteria internalized
2. flourescent assays
3. electro microscopy of thin section cells

Front 65

why does gentamicin function to kill extracellular bacteria without affecting intracellular?

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1. gentamicin does not enter cells
2. gentamicin goes in a different compartment
3. gentamicin kills at neutral pH but not at acidic pH
4. active efflux pumps

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Discovery of invasion (alternative approaches to how invasion could have been discovered)

Back 66

1. isolate transposon mutants blocked in invasion, then identify mutated genes (transposon mutagenesis); forward genetics
2. Analyze genes up and downstream from transposon insertion by reverse genetics
3. transform heterologous bacterium and assay for invasion. This is how invasin was discovered

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Why should the invasin experiment have never worked?

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1. could have taken too much DNA
2. might need another unlinked gene for invasion
3. if bug was gram-positive would not have worked

Front 68

Yersiniae virulence plasmid

Back 68

Plasmid is calcium-dependent; required for virulence in rodent models; plasmid is associated with extracellular killing of macrophages by y. pestis

Front 69

virulence plasmid encoded proteins (yops)

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Yop expression is dependent on a type III secretion

Front 70

Do yops act from the inside or outside?

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yops are mad on the surface of cells and then are secreted into the cell. Evidence: yops have no s.s. yet are secreted, yops are not classic toxins, yops injfected into cells caused phenotype.

Front 71

what are the effects of yops on host cells?

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1. inhibition of phagocytosis in macrophages
2. inhibition of internalization in Hela cells
3. affects on actin cytoskeleton
4. inhibition for the respiratory burst in neutrophils and macrophages
induction of apoptosis
5. inhibition of TNF release

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summary of plasmid mediated virulence in yersiniae

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1. plasmid is essential for the pathogenesis of all yersiniae
2. plasmid encodes a type III secretion apparatus
3. plasmid encodes effector proteins that are called yops
4. these effects are the magic molecules of pathogenesis

Front 73

what are integrins?

Back 73

proteins involved in cell adhesion

Front 74

how do y. entercolitica and y. pseudotuberculosis enter cells?

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involves invasin, binding is dependent on divalent cations

Front 75

how does one identify host receptors for invasin?

Back 75

experiment steps
1. chemically label all surface proteins with biotin
2. solubilize membrane proteins with detergent
3. pass detergent solubilized proteins over column (proteins don't bind and receptor binds column)
4. release receptor from column using EDTA
5. identify receptor by SDS-PAGE; blot to membrane, bind with biotin-binding protein

Front 76

What is the hypothesized pathway of actin cytoskeleton?

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integrins -> activate small GTPases in the Rho family -> activates WASP proteins -> activates Arp 2/3 complex -> promotes actin assembly

Front 77

What evidence supports the hypothesized pathway for actin cytoskeleton?

Back 77

1. Integrin, Rac, and Arp 2/3 shown to be localized around invading bacteria. Experiment put invasin on plastic bead and showed it taken up by host cell, therefore invasin is sufficient to induce phagocytosis
2. manipulate Rac activity and see if it influences internalization. Experiment: Rac Valine 12 mutant (locked in GTP form) and Rac N17 mutant (inhibits GEF and locked in GDP form). Introduce plasmid with Rac V12 or Rac N17 mutation into cell. Rac V12 promotes invasion and Rac N17 inhibits invasion. Shows Rac's importance for invasion

Front 78

Role of invasin in virulence?

Back 78

1. invasin mutant phenotype (N mutant has 80 fold reduced ability to invade cultured cells and is slower to infect peyer's patches
2. however, LD 50 is similiar to wt therefore, invasin not playing a role in death of mouse

Front 79

what is the role of effector yops? (Yops E, H, O)

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1. inhibit cell adhesion and actin assembly
2. prevent phagocytosis
3. promote apoptosis and inhibit cytokine secretion (antagonize immune system)

Front 80

yop H

Back 80

effector molecule that is a tyrosine phosphatase; promotes cell rounding and dissolution of focal adhesions and stress fibers

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Yop E

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mimics Rho GAP and promotes keeping Rho in the inactive GDP form

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Yop O

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mimics GDI and prevents release of GDP (prevents activation of GTPase)

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Yop T

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protease, cleaves Rho and releases it from membrane