Practice questions

Question 1

T3SS of yersinia

Answer 1

• Present in all three pathogenic Yersinia species
• Encoded on the pCD1 plasmid
• Expression activated by temperature change to 37ºC and presence of calcium
• Inject multiple toxic yersinia effector proteins (Yops) directly into host cells

Question 2

Three pathogenic Yersinia species

Answer 2

• Y. enterocolitica
• Y. pseudotuberculosis
• Y. pestis

Question 3

Structure of T3SS

Answer 3

• Injectisome
• Needle fixed into the bacterial inner and outer membrane and protrudes from the surface to penetrate host membrane
• Translocon forms a channel through host membrane
• Yop effectors transferred from the cytoplasm into the host cell

Question 4

What does Yop stand for

Answer 4

Yersinia outer proteins

Question 5

Examples of Yops

Answer 5

• YopE
• YopH
• YopM
• YopT
• YpkA/YopO
• YopP/YopJ

Question 6

YopE

Answer 6

Disrupts cytoskeleton

Question 7

YopH

Answer 7

Disrupts focal adhesions

Question 8

YopM

Answer 8

Regulation of host cell necrosis

Question 9

YpkA/YopO

Answer 9

Rounding up of cells

Question 10

YopP/YopJ

Answer 10

Apoptosis of macrophages

Question 11

TopT

Answer 11

Disrupts actin filaments

Question 12

Functions of Yops

Answer 12

• To subvert host cell signalling pathway
• Trigger apoptosis or cell death
• Inhibit phagocytosis
• Suppress immune cells

Question 13

DRAW T3SS

Answer 13

DRAW T3SS

Question 14

Virulence factors Yersinia pestis

Answer 14

• Plasminogen activator
• Murine toxin
• F1 Capsular antigen

Question 15

Virulence plasmids in Yersinia

Answer 15

• pPCP1
• pMT1
• pCD1

Question 16

Virulence factor that pPCP1 encodes

Answer 16

• Plasminogen activator
• Pla protease

Question 17

Virulence factor that pMT1 encodes

Answer 17

• Murine toxin
• F1 capsular antigen

Question 18

Virulence factor that pCD1 encodes

Answer 18

• Low Calcium Response (Lcr) T3SS
• Yops

Question 19

Plasminogen activator virulence factor

Answer 19

• Transmembrane protease
• Encoded on plasmid pPCP1
• Has protease activity that interacts and cleaves host proteins targets
• Targets important in response to infection (e.g. coagulation and fibrin clot)
• Allows bacteria to disseminate from bite site and subversion of immune response

Question 20

Yersinia murine toxin (Ymt) virulence factor

Answer 20

• Phospholipase D (PLD) activity
• Encoded by plasmid pMT1
• Required for survival in midgut of rat flea
• Intracellular PLD activity protects Y.pestis from a cytotoxic digestion product of blood plasma in the flea gut.
• Enables colonisation of the flea midgut
• Acquisition of PLD precipitated the
  transition of Y. pestis to obligate arthropod-borne transmission

Question 21

Low calcium response V (LcrV)

Answer 21

• Mutants deficient in LcrV not cytotoxic
• Lack of LcrV leads to secretion of effectors into the extracellular environment
• Several virulence roles proposed for V antigen
• LcrV or V antigen confers resistance to
  phagocytosis

Question 22

Several virulence roles proposed for V antigen

Answer 22

• Regulation of T3SS
• Translocator (part of the secretion apparatus)
• Immune modulator

Question 23

F1 Capsular Antigen

Answer 23

• Encoded by plasmid pMT1 (pFra)
• Forms a surface located polypeptide capsule at 37°C growth
• Characteristic capsule visible by light microscopy
• Antiphagocytic activity
• Is antigenic and a good vaccine candidate
• Important but not essential for virulence

Question 24

5 Virulence factors of Pseudomonas aeruginosa

Answer 24

1. Type IV pili
2. Exopolysaccharides
3. Exotoxin A
4. T3SS
5. Antioxidant enzymes

Question 25

Type IV in P. aeruginosa

Answer 25

- Retractile appendages - Crucial for the initiation of infection by controlling twitching motility and attachment to host cells

Question 26

Exopolysaccharides in P. aeruginosa

Answer 26

- Alginate, Psl and Pse - Contribute to biofilm matrix - Impair bacterial clearance - Promote establishment of chronic infections

Question 27

T3SS in P. aeruginosa

Answer 27

Critical for the destruction of host defences through the injection of four cytotoxic effectors

Question 28

4 Cytotoxic effectors secreted by T3SS in P. aeruginosa

Answer 28

ExoU, ExoT, ExoS and ExoY

Question 29

Exotoxin A (ETA) in P. aeruginosa

Answer 29

- Most toxic product released by P. aeruginosa - Inhibits host protein synthesis due to ADP-ribosylating activity

Question 30

Antioxidant enzymes in P. aeruginosa

Answer 30

- Help overcome oxidative stress in host - Includes catalases KatA, KatB and KatE

Question 31

Additive

Answer 31

- Result of two drugs is equal to the combined action of each of the drugs used separately - Isobologram is straight line

Question 32

Synergistic

Answer 32

- Result of the two drugs is significantly better than additive - Isobologram curves down

Question 33

Antagonistic

Answer 33

- Result of the two drugs is significantly less than additive - Isobologram curves up

Question 34

Pathophysiology of TB (what are the possible outcomes after exposure to respiratory droplets containing Mycobacterium tuberculosis)

Answer 34

1. Initial infection 2. Innate immune phase 3. Adaptive immune phase

Question 35

Initial infection of TB

Answer 35

- Droplet nuclei inhaled (can contain 1-3 bacilli, and remain airborne from minutes to hours) - After inhalation, droplet nuclei can reach the alveolar membrane

Question 36

Innate immune phase of TB

Answer 36

- Bacilli phagocytosed by alveolar macrophages - Most are contained or destroyed - Some survive phagocytosis by blocking lysosome fusion with phagosome (replicate in macrophages, released when macrophages die) - Macrophages stimulated and produce proinflammatory cytokines and chemokines (drives recruitment of more leukocytes to the site of infection) - Bacilli grow until threshold number of organisms is reached 2-8 weeks after infection - No immediate host response as no toxin produced

Question 37

Major cells types involved in innate immune phase

Answer 37

Macrophages, neutrophils, dendritic & natural killer cells

Question 38

Adaptive immune phase in TB

Answer 38

- Bacteria released from macrophages travel to lymph nodes and present to lymphocytes - Stimulates cell mediated immunity

Question 39

Cell mediated immunity in TB

Answer 39

- Recruitment and activation of T lymphocytes - Release of lymphokines, monokines and cytokines by T cells

Question 40

Effects of the release of lymphokines, monokines and cytokines by T cells

Answer 40

- Promotes recruitment of cells to infection site - Activates macrophages to kill bacilli - Triggers formation of early granuloma - Macrophages fuse to form multi nucleated giant cells or differentiate into foamy cells and surround the granuloma

Question 41

TB granuloma

Answer 41

- Caseating (necrotic) core - B and T cells form outer layer - Epithelial macrophage, macrophage, DCs, NK cells, neutrophils and giant cells form middle layer - Mycobacterium tuberculosis, apoptotic infected macrophage and apoptotic infection epithelial macrophage form inner layer

Question 42

It is estimated one quarter of the world has latent TB, has this changed from previous estimates and what is latent TB

Answer 42

- Yes, previous estimate was one third, has become lower - Latent TB describes people that are infected but NOT sick. They cannot transmit the disease

Question 43

Spread plate method viable organisms per mL in an original culture calculation

Answer 43

Cfu = number of colonies/volume plated in mL x dilution factor

Question 44

Helber chamber total number of cells per ml in the original culture calculation

Answer 44

Number of cells per square / volume of square

Question 45

Frequency of transformation calculation

Answer 45

number of colonies x (broth/plated amount) x fraction of ligation mix x 1000/1000