of protein virulence factors SBM 2044: Lecture 3 Weapons 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.

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

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) 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

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

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

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 Pertussis toxin 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

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

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

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)

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 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

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

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

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

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

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

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

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 -

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)

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

Any QUESTIONS so far?

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

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:

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

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

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)

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

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 )

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 15 - 20 hydrophobic residues 5 - 10 mostly charged LPxTG motif

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.

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

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 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

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

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

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.