SBM 2044: Lecture 3

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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.