Practice questions Flashcards
T3SS of yersinia
- 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
Three pathogenic Yersinia species
- Y. enterocolitica
- Y. pseudotuberculosis
- Y. pestis
Structure of T3SS
- 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
What does Yop stand for
Yersinia outer proteins
Examples of Yops
- YopE
- YopH
- YopM
- YopT
- YpkA/YopO
- YopP/YopJ
YopE
Disrupts cytoskeleton
YopH
Disrupts focal adhesions
YopM
Regulation of host cell necrosis
YpkA/YopO
Rounding up of cells
YopP/YopJ
Apoptosis of macrophages
TopT
Disrupts actin filaments
Functions of Yops
- To subvert host cell signalling pathway
- Trigger apoptosis or cell death
- Inhibit phagocytosis
- Suppress immune cells
DRAW T3SS
DRAW T3SS
Virulence factors Yersinia pestis
- Plasminogen activator
- Murine toxin
- F1 Capsular antigen
Virulence plasmids in Yersinia
- pPCP1
- pMT1
- pCD1
Virulence factor that pPCP1 encodes
- Plasminogen activator
- Pla protease
Virulence factor that pMT1 encodes
- Murine toxin
- F1 capsular antigen
Virulence factor that pCD1 encodes
- Low Calcium Response (Lcr) T3SS
- Yops
Plasminogen activator virulence factor
- 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
Yersinia murine toxin (Ymt) virulence factor
- 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
Low calcium response V (LcrV)
- 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
Several virulence roles proposed for V antigen
- Regulation of T3SS
- Translocator (part of the secretion apparatus)
- Immune modulator
F1 Capsular Antigen
- 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
5 Virulence factors of Pseudomonas aeruginosa
- Type IV pili
- Exopolysaccharides
- Exotoxin A
- T3SS
- Antioxidant enzymes
Type IV in P. aeruginosa
- Retractile appendages
- Crucial for the initiation of infection by
controlling twitching motility and attachment to host cells
Exopolysaccharides in P. aeruginosa
- Alginate, Psl and Pse
- Contribute to biofilm matrix
- Impair bacterial clearance
- Promote establishment of chronic infections
T3SS in P. aeruginosa
Critical for the destruction of host defences through the injection of four
cytotoxic effectors
4 Cytotoxic effectors secreted by T3SS in P. aeruginosa
ExoU, ExoT, ExoS and ExoY
Exotoxin A (ETA) in P. aeruginosa
- Most toxic product released by P. aeruginosa
- Inhibits host protein synthesis due to ADP-ribosylating activity
Antioxidant enzymes in P. aeruginosa
- Help overcome oxidative stress in host
- Includes catalases KatA, KatB and KatE
Additive
- Result of two drugs is equal to the combined action of each of the drugs used separately
- Isobologram is straight line
Synergistic
- Result of the two drugs is significantly better than additive
- Isobologram curves down
Antagonistic
- Result of the two drugs is significantly less than additive
- Isobologram curves up
Pathophysiology of TB (what are the possible outcomes after exposure to respiratory droplets containing Mycobacterium tuberculosis)
- Initial infection
- Innate immune phase
- Adaptive immune phase
Initial infection of TB
- Droplet nuclei inhaled (can contain 1-3 bacilli, and remain airborne from minutes to hours)
- After inhalation, droplet nuclei can reach the alveolar membrane
Innate immune phase of TB
- 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
Major cells types involved in innate immune phase
Macrophages, neutrophils, dendritic & natural killer cells
Adaptive immune phase in TB
- Bacteria released from macrophages travel to lymph nodes and present to lymphocytes
- Stimulates cell mediated immunity
Cell mediated immunity in TB
- Recruitment and activation of T lymphocytes
- Release of lymphokines, monokines and cytokines by T cells
Effects of the release of lymphokines, monokines and cytokines by T cells
- 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
TB granuloma
- 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
It is estimated one quarter of the world has latent TB, has this changed from previous estimates and what is latent TB
- Yes, previous estimate was one third, has become lower
- Latent TB describes people that are infected but NOT sick. They cannot transmit the disease
Spread plate method viable organisms per mL in an original culture calculation
Cfu = number of colonies/volume plated in mL x dilution factor
Helber chamber total number of cells per ml in the original culture calculation
Number of cells per square / volume of square
Frequency of transformation calculation
number of colonies x (broth/plated amount) x fraction of ligation mix x 1000/1000
