Bacterial Protein secretion

Question 1

Describe types of secreted virulence factors:

(including exotoxin subtypes)

Answer 1

Extracellular enzymes:

Spreading factors: Hyaluronidase, Collagenase.

Coagulase (fibrin from fibrinogen)

(strepto/staphylo)Kinases (plasminogen to plasmin –> digests fibrin)

Exotoxins (3 types)

1) Cell surface active [e.g. superantigens]
2) Membrane damaging [e.g. haemolysins]

3) Intracellular: AB type toxins [B binding, A active]
Effector proteins directly injected by injectosome [T3SS]

Question 2

Components and 3 stages of SecY secretion pathway? (across cytoplasmic membrane, inner in gram -ve)

Answer 2

SecYEG trimeric pore/translocon.

Stage 1: Sorting and targeting pre-proteins: N-terminal Signal sequence on pre-protein recognised by SRP co-translational [signal recognition peptide] (for membrane proteins) or Sec B chaperone post-translational. [extra-cellular proteins]

Stage 2 translocation: SecA ATPase drives SecB extracellular post-translational pre-proteins.

Or co-translational binding of SRP to hydrophobic regions of signal sequence, migration of ribosome to FtsY receptor. Translation only continues once bound (as membrane protein won’t fold properly in cytoplasm)

Stage 3: Release and maturation: mediated by Signal Peptidase-1 (cleaves off signal sequence)

Question 3

Features of a bacterial signal sequence?

Answer 3

At N-terminus, about 25 amino acids long.

N-terminal N-region positive residues

H region - hydrophobic residues (bound by SRP or SecB)

C-region (cleavage signal??) AxA recognition site immediately precedes site of cleavage by SignalPeptidase-1

Question 4

Details of SecA dependent secretion (SecY channel):

Answer 4

SecA and SecYEG both occur as dimers:

SecY has a translocating and non-translocating copy: translocating copy has a plug which is released by signal sequence interaction with SecY.

Rounds of ATP hydrolysis by SecA pump peptide through translocating copy of SecY.

Question 5

Describe similarities and differences between T3SS and SecYEG secretion systems:

Answer 5

T3SS only on gram -ve.

Both have signal sequences but T3SS sequence is unclear/variable and remains uncleaved

Effector specific chaperones used for T3SS instead of SecB.

Both secrete unfolded proteins. (although T3SS can fit basic 2ndry structures)

T3SS often found on mobile genetic elements (pathogenicity islands)

Question 6

Control of T3SS gene expression in P.aeruginosa:

Answer 6

Partner switching of Exs proteins.

expression of T3SS maintained normally at low level until cell contact.

this causes ExsE (bound to ExsC) to be secreted.

This allows free ExsC (anti-anti-activator) to bind ExsD (an anti-activator bound to ExsA) releasing ExsA

ExsA activates transcription of T3SS genes.