1. of protein virulence factors
SBM 2044: Lecture 3
Weapons delivery & deployment
Secretion & targeting
of protein virulence factors

2. Protein secretion in bacteria
Membranes act as a barrier to the movement of large molecules into or out of the cell
Gram-positive and Gram-negative bacteria have many important structures which are located outside the wall
So how are the large molecules from which some of these structures are made transported out of the cell for the assembly?
How about exoenzymes and other proteins? How are they released through the membrane?
Mechanisms of protein secretion are important and can be exploited for vaccine development.

3. Protein secretion in Gram-Negative Bacteria
Different cell layers for Gram + and Gram – bacteria
For Gram +, the secreted proteins must be transported across a single membrane. Then through a relatively porous peptidoglycan into either:
the external environment
become embedded /attached to the peptidoglycan
For Gram –, the secreted protein must be transported across the IM; escape protein-degrading enzymes in the periplasmic space; and finally across the OM

4. How are the large molecules being transported out across the plasma membrane?
General secretory pathway (GSP) is a protein translocation mechanism
GSP consists of cytosolic chaperones, an integral membrane translocase consisting of several proteins operating cooperatively and signal peptidase
Require energy from hydrolysis of ATP or GTP, and sometimes by proton motive force
Exported proteins are recognised by having a signal sequence at their N-terminus, which is cleaved by signal peptidase.

5. General Secretion Pathway (GSP)
Need to emphasise that this is in E. coli and details may differ in other species, particularly Gram +
SecB = chaperon: maintains protein in secretion-competent
state by preventing premature folding in cytoplasm

6. GSP: Sec-dependant secretion
Gram-positive bacteria
Gram-negative bacteria
Sufficient to get protein
out of the cell
Proteins reach periplasm, but
OM is additional barrier -
need other mechansims to
get protein thro’ OM. OM
sec IM
sec
Signal-peptide

7. How do Gram-neg. bacteria get proteins thro’ OM ??
> 5 quite different mechanisms identified to date
- any particular protein excreted by one of these
‘overall’ mechanisms
Proteins secreted first to
periplasm by GSP (Sec)
and then thro’ OM
Sec-dependent
Type II
Type IV
Type V
+ various others
– e.g. fimbrial systems
Sec-independent
Type I
Type III
Secreted proteins get directly from
cytoplasm to outside without entering
the periplasm

9. Tat-Pathway Twin-arginine translocation pathway
Tat translocase is composed of the membrane proteins TatABC
Translocate folded proteins across membrane
Optional Reading:
Palmer & Berks (2003). Moving folded proteins across the bacterial cell membrane. Microbiology 149, 547–556

10. Type II protein secretion
Present in pathogens such as Klebsiella pneumoniae, Pseudomonas aeruginosa and Vibrio cholerae
Secrete degradative enzymes pullulanases, cellulases, pectinases, proteases and lipases
Secrete cholera toxin and pili proteins
Complex pathway with proteins for translocation through OM
May also use a different plasma membrane transportation system, the Tat pathway (for folded proteins)

11. Type IV protein secretion
Sec-independent
Secrete protein and transfer DNA from donor bacterium to a recipient during bacterial conjugation

12. Type IV: Conjugal transfer in Agrobacterium tumerfaciens
DNA transfer is sec-independent, but sec-dependant Pertussis
toxin is secreted from periplasm using homologous of
many (not all) of the Agrobacterium Type IV components

13. Type V protein secretion
In periplasmic space, many proteins may are able to form channel in the OM, through which they transport themselves

14. Essentially ‘autosecretion’ thro’ OM.
Type V secretion
Essentially ‘autosecretion’ thro’ OM.
relatively rare
Example: IgA proteases secreted by Neisseria gonorrhoeae
Mature protease released
by autocatalytic cleavage OM
Very few proteins can do this
Also IgA protease of Haemophilus influenzae, seriine protease of Serratia marcesens and vacuolating cytotoxin (VacA) of Helicobacter pylori
expressed as ‘preproprotein’ precursors - the ‘pro’ referring to OM-spanning domains that insert into OM and allow rest of molecule thro’ - details still unclear.
autocatalytic cleavage detaches the ‘mature’ protease from the OM-spanning ‘pro’ or ‘Beta’-domain
Some have additional domains (alpha and gamma) which are also removed after secretion – functions of these unclear, but may well act as ‘chaperon sequences’ to maintain protein in appropriate conformation during secretion process
Some cases of the ‘transporter’ domain encoded by separate gene – haemolysin in Serratia marcesens
sec
C-terminal g, a and b domains
b domain = OM-spanning sequence
a + g domains – chaperon sequences??
N-terminal
signal-peptide

15. How do Gram-neg. bacteria get proteins thro’ OM ??
> 5 quite different mechanisms identified to date
- any particular protein excreted by one of these
‘overall’ mechanisms
Proteins secreted first to
periplasm by GSP (Sec)
and then thro’ OM
Sec-dependent
Type II
Type IV
Type V
+ various others (e.g. fimbriae)
Sec-independent
Type I
Type III
Secreted proteins get directly from
cytoplasm to outside without entering
the periplasm

16. Type I secretion pathways
Discovered in studying E. coli a-haemolysin (HlyA)
HlyA lacks an N-terminal secretion signal-peptide, but
is nonetheless secreted efficiently
secretion involves a sec-independent pathway
Employed by various Gram-neg. species
Each pathway specific for a single protein - although can be > 1 Type I pathway in cell to secrete different proteins.
Each involves 3 ‘accessory’ proteins, one being an ‘ABC’
(ATP-binding cassette) transporter (e.g. E. coli HlyB)

17. Type III protein secretion
Sec-independent
Inject virulence factors directly into host cells
Secrete (inject) toxins, phagocytosis inhibitors, stimulators for cytoskeleton reorganisation in the host cell.

18. Type III Secretion Involves sets of ~ 20 genes - many share homology
between different species, suggesting common ancestors
& functions
In all cases, genes involved are clustered together:
- on virulence plasmids in Yersiniae, Shigella, & EIEC
- in ‘Pathogenicity islands’: LEE in EPEC & EHEC
SPI-I & SPI-II in Salmonella
First discovered 10 years ago in studies on Yersiniae sp. & subsequently found to be employed by various Gram-neg. pathogens to secrete certain virulence proteins
Probably ‘acquired’ by horizontal transfer & ‘adapted’ by different
species to secrete different sets of ‘effector’ (virulence) proteins

19. Type III Secretion - some examples
Differences mainly in the nature & function of the
‘effector’ proteins - at least some of the proteins involved
in secretion ‘apparatus’ very similar in diff species
secreted
Pathogen effector proteins Function
Yersiniae sp. YOPs killing phagocytes
Shigella sp. IpaA-D Bacterial invasion
Salmonella SIPs + SOPs Bacterial invasion
EPEC & EHEC Tir A/E Lesions

20. Type III secretion system and other virulence genes of Yersinia are encoded on the pYV plasmid
Note the similar basal body structures in both the TTSS injectisome and the flagella

21. Yersiniae Type III secretion apparatus
Euk cell membrane
Pore
Yersiniae Type III secretion apparatus
Needle OM
Peptidoglycan
Periplasm
Scanning tunneling electron microscopy shows injectisome tip - lock
Basal body IM

22. EM of purified Type III secretion complexes

23. S. typhimurium Type III ‘needle complex’
Note: ‘Needles’ very much thinner & shorter than EPEC ‘filaments’,
but apparatus spanning IM & OM probably very similar

24. Infers need for a signal
Type III Secretion Systems
Unlike other systems, proteins not secreted as soon as they
are translated, but can accumulate in cytoplasmic ‘pools’.
Infers need for a signal
to trigger secretion
Shigella sp. secrete invasion proteins called IpaA - D. Found
> 90% remained cell-associated in broth cultures (small
quantities released - possible ‘leakage’ rather than secretion).
However, rapidly secreted in presence of mammalian cells

25. Activation of Type III secretion
Studies on several pathogens (Yersiniae, Shigella, EPEC)
have shown that Type III secretion activated in proximity
to host cells
What is the trigger ?
Various studies suggested that adhesion to host cells is
the activation trigger ‘contact-dependant secretion’
However, may not be that simple - evidence that some
Type III secretion systems can be activated by ‘soluble’
signalling molecules
e.g. EPEC in tissue culture medium, but not L-broth
Quorum sensing recently implicated

26. Quorum sensing
Remarkable ability of bacteria to sense their own cell
population density & respond by activating and/or
repressing appropriate sets of genes
Prototype system:
Bioluminescence in Vibrio fischeri - emits light
at very high cell densities of light in organ of host but not when free in sea -

27. AHL = N-acetylated-homoserine lactone
Small molecules that diffuse freely through cell membrane
Concentrations inside and outside cell equilibrate
Shading reflects
[AHL] in media
Low cell density
High cell density
High cytoplasmic [AHL]
Low cytoplasmic [AHL]
No induction
‘Auto-induction’ of lux operon
AHL often called an ‘AI’ (auto-inducer)

28. Similarities + Differences Type I Type III
Sec-independent - secretion
apparatus spans IM + OM
Sec-independent - secretion
apparatus spans IM + OM
3 ‘accessory’ secretion proteins
~ 20 ‘accessory’ secretion proteins,
(identified by isolating mutants)
Single protein secreted
Multiple proteins secreted, tho’ all
for similar ‘end’ (e.g. invasion)
Target protein secreted rapidly
upon translation
Secreted proteins can ‘accumulate’
in bacterial cell before secretion in
response to ‘external’ signal
Secreted protein released into the
bacterial cell environment – before
any interactions with host cells
Secreted proteins injected directly
into host cell - appears to be main
function of Type III systems

29. Any QUESTIONS so far?

30. Sec-dependant General secretion pathway (GSP)
Gram-negative bacteria
Gram-positive bacteria
Proteins reach periplasm, but
OM is additional barrier -
need other mechanisms to
get protein out thro’ OM.
(Types I - V secretion)
Sufficient to get protein
out. In this case, other
mechanisms needed to
retain wall - associated
proteins OM IM
sec
sec
Type II
secretion
Signal-peptide

31. Targeting secreted proteins to Gram-positive cell walls
Four distinct mechanisms identified to date:
Rare:
Binding to wall teichoic acid
Binding to membrane anchored LTA
Lipoprotein ‘anchors’
C-terminal wall-associating signals
More widespread:

32. 1. Binding to cell-wall teichoic acid
Streptococcus pneumoniae and Streptococcus suis
Pneumococcal surface protein A (PspA)
Pneumococcal autolysin (LytA)
S. suis autolysin- [homologous to pneumococcal LytA]
C-terminal ends share homologous choline-binding
domains – enable binding to TA of these species
PspA and LytA – Very distinctive proteins – only common feature is conserved C-terminal choline-binding domain
S. suis – a pig pathogen

33. n Reminder of the structure of teichoic acid: poly-ribitol phosphate
Polymer of either Glycerol phosphate or Ribitol phosphate, with
various substituents (R)
poly-ribitol phosphate
O P O C C C C C O P O C O
H H H H H O H
H O OH O H H
R R’ n
In most species studied to date
R = D-alanine R’ = N-acetylglucosamine
In S. pneumoniae and S. suis
R = phosphodiester linked choline
- chemically more stable than
ester-linked D-Ala

34. 2. Binding to membrane anchored LTA
Single example recognised only recently
InlB of Listeria monocytogenes – has C-terminal
domain that ‘targets’ LTA – mechanism??
Exogenous addition of InlB allows it to associate with wall and mediate invasion – hence wall ‘targeting’ sequence, analagous to the C-terminal wall targeting sequence of lysostaphin (secreted by Staph. simulans and targets cell walls of other staph species)

35. attached at outer surface of cytoplasmic membrane by a lipid anchor
3. Lipoproteins
attached at outer surface of cytoplasmic membrane by a
lipid anchor
Examples include penicillinase in S. aureus
Similar mechanisms used in both Gram-pos. & Gram-neg.
Distinctive N-terminal signal peptides
distinct Sec apparatus with specialized signal
peptidase (called signal peptidase II)
recognized by

36. Short hydrophobic sequence
Lipoprotein signal peptides N-
Short hydrophobic sequence
Signal peptidase II
cleavage site
1-3 positively
charged a.a.
-Leu-x-y- Cys-
x and y usually
small, uncharged
residues
A diglyceride is attached
to the N-terminal Cys of
the mature protein
Diglyceride
Contrast with ‘typical’ GSP secretion signal-peptide ( Lecture 3 )

37. 4. ‘Sorting’ via C-terminal wall-associating signals
Vast majority of Gram-pos. wall-associated proteins share
structurally similar C-terminal wall-associating signals
Hydrophobic /Charged ‘tail’
membrane ‘anchor’ -C
First recognized by comparing C-terminal sequences of S. aureus Protein A & streptococcal M proteins
Pro-rich region
hydrophobic
residues
5 - 10
mostly
charged
LPxTG
motif

38. C-terminal wall-associating signals
Studies of S. aureus Protein A,
showed that membrane ‘anchor’
plays a transient role in a more
complex wall-associating pathway
Pro-rich
‘flexible’
wall-spanning
Hydrophobic
Originally suggested that wall-association due simply to ‘anchoring’ of C-terminal ends of proteins in membrane
BUT this was vast oversimplification
Membrane ‘anchor’
Charged ‘tail’
Care: do not be misled by some textbooks/reviews which say proteins
anchored in membrane.

39. wall-associated ‘Sortase’ N C Wall-associating signal Signal peptidase
N-terminal signal peptide
-L-P-x-T
Cleavage at
LPxTG
Cross-linked
to cell-wall G
In S. aureus, the Thr X-linked to the terminal glycine of the pentaglycine x-bridge, probably prior to incorporation of the peptidoglycan precursor into the wall N
Some, but not
necessarily all,
covalently
linked to wall
(e.g. InaA, Prot. A) C
Minority
simply
‘anchored’?
(e.g. ActA in Listeria)
Majority
‘cleaved’
at LPxTG
mRNA

40. Retaining secreted proteins in Gram-positive cell walls
1. Binding to wall teichoic acid
Limited to a very few species (e.g. S. pneumoniae, S. suis)
2. Binding to membrane anchored LTA
Single example recognised only recently (InlB of Listeria monocytogenes)
3. Lipoprotein ‘anchors’
A minority of wall-associated proteins in many species anchored
to outer surface of cell membrane via an N-terminal lipid anchor
4. C-terminal wall-associating signals
Vast majority of wall-associated proteins studied to date
share structurally similar C-terminal wall-associating signals

41. Retaining proteins at Gram-negative cell-surfaces
First step: Sec-dependent secretion to periplasm (GSP)
Then:
Targeting of integral OM proteins - OM-interacting
‘surfaces’ result from folding in periplasm
(may involve periplasmic Dsb and Ppi enzymes) OR
Individual biogenesis pathways – e.g. fimbriae

42. E. coli fimbrial adhesins: > 40 distinct adhesins identified
Most are variations on common theme - common ‘ancestor’
Each encoded by a cluster of genes encoding regulators of
expression, structural components and additional proteins
for fimbrial biogenesis
Type I (common) fim genes
B E A I C D F G H
Regulators
(in cytoplasm)
Chaperone
Major subunit
‘Usher’ (OM)
Minor subunits

43. Type I fimbrial biogenesis
Minor subunits:
FimH = adhesin
‘Tip’ structure
Fim A
major
subunit
FimF + G
Fim G - also regulates fimbriae length? OM
Fim D ‘Usher’
assembly &
attachment
Fim C periplasmic chaperone
With exception of regulatory gene products (which remain in cytoplasm) – all Fim gene products expressed as precursors with typical N-terminal signal peptides
FimC Chaperone: maintains other components in appropriate unfolded conformation & delivers them to OM ‘usher’ IM
Sec
Secreted thro’ IM by Sec-apparatus
All components

44. References
Prescott’s Microbiology Chapter 3, Paragraph 3.8 ONLY: Prokaryotic Cell Structure and Function
Optional
Sherris Medical Microbiology Chapter 3 p37-40 ONLY
and some relevant paragraphs in Chapter 10.