sbm 2044: lecture 3 weapons delivery & deployment secretion & targeting of protein virulence...
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SBM 2044: Lecture 3
Weapons delivery & deploymentWeapons delivery & deployment
Secretion & targeting of protein virulence factors
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.
• 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
Protein secretion in Gram-Negative Bacteria
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.
General Secretion Pathway (GSP)
SecB = chaperon: maintains protein in secretion-competent state by preventing premature folding in cytoplasm
IMsec
Gram-positive bacteria Gram-negative bacteria
Sufficient to get proteinout of the cell
GSP: Sec-dependant secretion
Proteins reach periplasm, butOM is additional barrier -need other mechansims to get protein thro’ OM.
OM
sec
Signal-peptide
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
Sec-independentType IType III
Sec-dependentType IIType IV Type V+ various others – e.g. fimbrial systems
Secreted proteins get directly fromcytoplasm to outside without enteringthe periplasm
Proteins secreted first to periplasm by GSP (Sec)and then thro’ OM
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
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 12-14 proteins for translocation
through OM • May also use a different plasma membrane transportation
system, the Tat pathway (for folded proteins)
Type IV protein secretion
• Sec-independent
• Secrete protein and transfer DNA from donor bacterium to a recipient during bacterial conjugation
Type IV: Conjugal transfer in Agrobacterium tumerfaciens
DNA transfer is sec-independent, but sec-dependant Pertussistoxin is secreted from periplasm using homologous of many (not all) of the Agrobacterium Type IV components
Type V protein secretion
• In periplasmic space, many proteins may are able to form channel in the OM, through which they transport themselves
Type V secretion
Essentially ‘autosecretion’ thro’ OM.• relatively rare
• Example: IgA proteases secreted by Neisseria gonorrhoeae
OM
sec
C-terminal and domains • domain = OM-spanning sequence • + domains – chaperon sequences??N-terminal
signal-peptide
Mature protease releasedby autocatalytic cleavage
Very few proteins can do this
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
Sec-independentType IType III
Sec-dependentType IIType IV Type V+ various others (e.g. fimbriae)
Secreted proteins get directly fromcytoplasm to outside without enteringthe periplasm
Proteins secreted first to periplasm by GSP (Sec)and then thro’ OM
Each pathway specific for a single protein - although
can be > 1 Type I pathway in cell to secrete different
proteins.
Type I secretion pathways
Employed by various Gram-neg. species
Each involves 3 ‘accessory’ proteins, one being an ‘ABC’
(ATP-binding cassette) transporter (e.g. E. coli HlyB)
Discovered in studying E. coli -haemolysin (HlyA)
• HlyA lacks an N-terminal secretion signal-peptide, but is nonetheless secreted efficiently
secretion involves a sec-independent pathway
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.
Type III Secretion
• 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
• Involves sets of ~ 20 genes - many share homology between different species, suggesting common ancestors & functions
Probably ‘acquired’ by horizontal transfer & ‘adapted’ by different species to secrete different sets of ‘effector’ (virulence) proteins
Type III Secretion - some examples
Shigella sp. IpaA-D Bacterial invasion
Salmonella SIPs + SOPs Bacterial invasion
EPEC & EHEC Tir A/E Lesions
• 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
Note the similar basal body structures in both the TTSS injectisome and the flagella
Type III secretion system and other virulence genes of Yersinia are encoded on the pYV plasmid
Yersiniae Type III secretion apparatus
Scanning tunneling electron microscopy shows injectisome tip - lock
Pore
Basal body
Needle
OM
IM
PeriplasmPeptidoglycan
Euk cell membrane
EM of purified Type III secretion complexes
S. typhimurium Type III ‘needle complex’
Note: ‘Needles’ very much thinner & shorter than EPEC ‘filaments’, but apparatus spanning IM & OM probably very similar
Type III Secretion SystemsUnlike other systems, proteins not secreted as soon as theyare translated, but can accumulate in cytoplasmic ‘pools’.
Infers need for a signalto 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
Activation of Type III secretionStudies on several pathogens (Yersiniae, Shigella, EPEC)have shown that Type III secretion activated in proximityto 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
Quorum sensing
Remarkable ability of bacteria to sense their own cellpopulation density & respond by activating and/or repressing appropriate sets of genes
Prototype system: Bioluminescence in Vibrio fischeri - emits lightat very high cell densities of light in organ of host but not when free in sea -
AHL = N-acetylated-homoserine lactone
• Small molecules that diffuse freely through cell membrane
• Concentrations inside and outside cell equilibrate
Low cell densityLow cytoplasmic [AHL]
High cell densityHigh cytoplasmic [AHL]
No induction ‘Auto-induction’ of lux operon
AHL often called an ‘AI’ (auto-inducer)
Shading reflects[AHL] in media
Multiple proteins secreted, tho’ allfor similar ‘end’ (e.g. invasion)
~ 20 ‘accessory’ secretion proteins,(identified by isolating mutants)
Sec-independent - secretionapparatus spans IM + OM
Sec-independent - secretionapparatus spans IM + OM
Type I Type III
Secreted proteins can ‘accumulate’in bacterial cell before secretion inresponse to ‘external’ signal
Similarities + Differences
Secreted proteins injected directlyinto host cell - appears to be mainfunction of Type III systems
3 ‘accessory’ secretion proteins
Single protein secreted
Target protein secreted rapidlyupon translation
Secreted protein released into thebacterial cell environment – beforeany interactions with host cells
• Any QUESTIONS so far?
Type IIsecretion
Sec-dependant General secretion pathway (GSP)
IM
Gram-negative bacteria
Proteins reach periplasm, butOM is additional barrier -need other mechanisms to get protein out thro’ OM. (Types I - V secretion)
OM
sec
Signal-peptide
sec
Gram-positive bacteria
Sufficient to get proteinout. In this case, othermechanisms needed toretain wall - associatedproteins
Targeting secreted proteins to Gram-positive cell walls
• Binding to wall teichoic acid
• Binding to membrane anchored LTA
• Lipoprotein ‘anchors’
• C-terminal wall-associating signals
Four distinct mechanisms identified to date:
Rare:
More widespread:
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-bindingdomains – enable binding to TA of these species
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
O O
Reminder of the structure of teichoic acid:Polymer of either Glycerol phosphate or Ribitol phosphate, with various substituents (R)
poly-ribitol phosphate
R = D-alanine R’ = N-acetylglucosamineIn most species studied to date
In S. pneumoniae and S. suis R = phosphodiester linked choline - chemically more stable than
ester-linked D-Ala
2. Binding to membrane anchored LTA
Single example recognised only recently
- InlB of Listeria monocytogenes – has C-terminal domain that ‘targets’ LTA – mechanism??
3. Lipoproteins
• attached at outer surface of cytoplasmic membrane by a lipid anchor
• Similar mechanisms used in both Gram-pos. & Gram-neg.
Examples include penicillinase in S. aureus
Distinctive N-terminal signal peptides
distinct Sec apparatus with specialized signalpeptidase (called signal peptidase II)
recognized by
Lipoprotein signal peptides
-Leu-x-y- Cys-x and y usuallysmall, uncharged residues
Signal peptidase IIcleavage site
Diglyceride
Short hydrophobic
sequence1-3 positivelycharged a.a.
A diglyceride is attached to the N-terminal Cys ofthe mature protein
Contrast with ‘typical’ GSP secretion signal-peptide ( Lecture 3 )
N-
4. ‘Sorting’ via C-terminal wall-associating signals
Hydrophobic /Charged ‘tail’membrane ‘anchor’
Vast majority of Gram-pos. wall-associated proteins sharestructurally similar C-terminal wall-associating signals
LPxTGmotif
15 - 20 hydrophobicresidues
5 - 10mostlycharged
Pro-rich region
-C
Charged ‘tail’
Hydrophobic
Pro-rich‘flexible’
wall-spanning
Membrane ‘anchor’
Care: do not be misled by some textbooks/reviews which say proteins anchored in membrane.
C-terminal wall-associating signals
+ +
Studies of S. aureus Protein A,showed that membrane ‘anchor’plays a transient role in a morecomplex wall-associating pathway
Cross-linked to cell-wall
Cleavage at
LPxTGG
-L-P
-x-T
mRNA
Signalpeptidase
CN
N-t
erm
inal
sig
nal p
epti
de
Wall-associatingsignal
N C
Minority simply
‘anchored’?(e.g. ActA in Listeria)
Majority‘cleaved’at LPxTG
Some, but notnecessarily all, covalently linked to wall
(e.g. InaA, Prot. A)
wall-associated‘Sortase’
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
Retaining proteins at Gram-negative cell-surfaces
• 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
First step: Sec-dependent secretion to periplasm (GSP)
Then:
Type I (common) fim genes
B E A I C D F G H
E. coli fimbrial adhesins: > 40 distinct adhesins identified
Regulators(in cytoplasm)
Major subunit
Minor subunits ‘Usher’ (OM)
Chaperone
• 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
All components
Secreted thro’ IM by Sec-apparatus
Sec
Fim C periplasmic chaperoneFim D ‘Usher’
assembly &attachment
Fim Amajor subunit
FimH = adhesin
Fim G - also regulates fimbriae length?
Minor subunits:
FimF + G
Type I fimbrial biogenesis
IM
OM
‘Tip’ structure
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.