Secretion systems in bacteria II

Question 1

Bacterial injection machines - delivery of effector proteins

Answer 1

• The type III, IV and VI protein secretion systems inject ‘effector proteins’ into the
cytoplasm of host cells

• Not to be confused with toxins, which are also secreted (T2SS)

• Effector proteins are highly diverse between different bacteria. The injection machine
itself, however, is better conserved

Question 2

What do effector proteins do?

Answer 2

• Promote invasion of non-phagocytic cells
• Inhibit phagocytosis by phagocytes
• Induce apoptosis
• Modulate intracellular trafficking
• Suppress host cell defences (plants)

Question 3

How do effector proteins do it?

Answer 3

• Structural mimicry- some effector proteins ‘impersonate’ host proteins, modifying their
function
• Covalent Modifications- eg tyrosine phosphatase; ser/thr kinase; acetylation

Question 4

Give an example of a structural Mimicry

Answer 4

• S. typhimurium SptP is a GTPase activating
protein
• It binds to the Rac1 GTPase in such a way that
a critical Arg residue can recognize bound
GTP
• Host GTPase activating proteins have a
different scaffold, but place the Arg residue in
the same position in space
• The GTPase activating activity of SptP is
therefore an example of convergent evolution
of this function by a bacterial effector protein
• This results in recovery of the actin cytoskeleton after bacterial internalization

Question 5

The type III secretion system - the injectisome

Answer 5

• An injection nanomachine
• Function- injection of bacterial effectors into host cells

• Multiple components: 25 proteins needed to construct, 9 of which are highly conserved,
and 8 of those show homologies with flagellum proteins

=> There is extensive evidence that the T3SS and the flagellum share a common evolutionary
origin

Unlike other secretion systems, part of the T3SS can be isolated directly: the needle complex

Question 6

Chaperones and effector proteins

Answer 6

Effector proteins have dedicated
chaperones which maintain them
in a partially unfolded state in the periplasm

They then dissociate from the chaperone before passing down the needle channel
SptP-SicP chaperone complex

Question 7

The Needle

Answer 7

Atomic model for the Shigella needle: note that the interior diameter is small (
effector proteins are unfolded as they pass down it

Needle length control
A protein called YscP acts as a ‘molecular ruler’, ensuring consistency in the needle length. The needle is built from the tip (cf flagellum) and substrate specificity switching must occur to
build the T3SS complex, and then deliver effector proteins.

Question 8

Explain the Tip

Answer 8

Three specialized tip proteins form a translocon pore in the host cell membrane

Question 9

Summaries key points of T3SS

Answer 9

• Complex, regulated nanomachine
• No signal peptide required
• Many details not well understood:
– Control of assembly
– Mechanism of translocation
– Regulation of passage of effector

Question 10

The type IV secretion system

Answer 10

Three functional types:

• conjugation; transfer of DNA into the host eg Agrobacterium tumefaciens
• transformation; DNA uptake eg Helicobacter pylori ComB system
• protein transfer; eg Bordetella pertussis, Legionella pneumophila, Brucella spp

Question 11

Cryo-EM structure of the TraN/VirB7, TraO/VirB9, and TraF/ VirB10C-ST core complex

( Type 4)

Answer 11

Tripartite complex forms a channel with 14-fold symmetry whichconnects the cytoplasm to the external surface/pilus interior

The crystal structure of the O layer components has also been determined.

Transmembrane portion of VirB10 forms the OM channel and consists of alpha helices (cf Wza in last lecture)

There was evidence of flexibility in the structure,
which could be linked to opening of the OM aperture

T4SS- Summary
• Although the structures of many of the T4SS components are known, the precise mechanism of secretion/uptake is

Question 12

Summaries T4SS

Answer 12

T4SS- Summary
• Although the structures of many of the T4SS components are known, the precise mechanism of secretion/uptake is

• No signal peptide required

• T4 pili measure 8-10nm across, with an internal diameter of 2nm

• This is enough for translocation of a DNA duplex, but the mechanism of transport of
protein substrates is less well understood

Question 13

Type 5 Secretion systems pathways - the auto transporters

Answer 13

• Much simpler secretion system; prototypical autotransporter has a C-terminal outer membrane-spanning region, which promotes translocation of the N-terminus
• N-terminal section can then be tethered to OM or cleaved, for secretion
• Many virulence factors are secreted by this pathway

Question 14

Type V how do auto transporters work?

Answer 14

• 12-stranded C-terminal beta barrel contains an alpha helix which links it to the passenger domain

• Initially it was thought that the barrel is inserted into the membrane, and the passenger
domain then threads through it

• Direct evidence for this is still lacking, and current thinking is that other proteins may be
involved- there is evidence for involvement of Omp85 for example (cf Bam complex in
lecture 1)

Question 15

Type VI secretion systems

Answer 15

• Found in about 25% of Gram negative bacteria, including E. coli, P. aeruginosa and V.
  cholera
• T6SS gene clusters encode 12-25 different proteins
• Two proteins homologous to T4SS components (DotU and IcmF)
• ClpV ATPase- related to ClpB (ATP-dependent proteolysis). Similar function for ClpV?

Question 16

T6SS secreted proteins

Answer 16

Two main groups: Hcp and VgrG families
• Hcp is a hexamer, of unknown function (see
left)
• VgrG is similar to the tailspike proteins from
bacteriophages- they may be involved in
puncturing the bacterial cell envelope

Question 17

T6SS Duelling bacteria

Answer 17

• The presence of a T6SS in one bacterial species (eg Vibrio cholerae) stimulates another (Pseudomonas aeruginosa) to use its T6SS to ‘attack’ the first bacterium

• Attack is mediated by effector proteins
translocated into other cells by the T6SS