Unveiling Protein Secretion Mechanisms in Bacterial Cells

1. 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) 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 IM sec 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.Microbiology149, 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 12-14 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. 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 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. colia-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 Probably ‘acquired’ by horizontal transfer & ‘adapted’ by different species to secrete different sets of ‘effector’ (virulence) proteins

19. secreted Pathogen effector proteins Function Yersiniae sp. YOPs killing phagocytes 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 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. Euk cell membrane Yersiniae Type III secretion apparatus Pore 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. 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 ~ 20 ‘accessory’ secretion proteins, (identified by isolating mutants) 3 ‘accessory’ secretion proteins Multiple proteins secreted, tho’ all for similar ‘end’ (e.g. invasion) Single protein secreted Secreted proteins can ‘accumulate’ in bacterial cell before secretion in response to ‘external’ signal Target protein secreted rapidly upon translation 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-positive bacteria Gram-negative bacteria Sufficient to get protein out. In this case, other mechanisms needed to retain wall - associated proteins Proteins reach periplasm, but OM is additional barrier - need other mechanisms to get protein out thro’ OM. (Types I - V secretion) 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

33. O H H H H H O H O P O C C C C C O P O C O O H O OH O H H R R’ n Reminder of the structure of teichoic acid: Polymer of either Glycerol phosphate or Ribitol phosphate, with various substituents (R) poly-ribitol phosphate 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??

35. 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. 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 15 - 20 hydrophobic residues Pro-rich region 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 Membrane ‘anchor’ Charged ‘tail’ + + Care: do not be misled by some textbooks/reviews which say proteins anchored in membrane.

39. C N Wall-associating signal Signal peptidase wall-associated ‘Sortase’ N-terminal signal peptide -L-P-x-T Cleavage at LPxTG Cross-linked to cell-wall G Some, but not necessarily all, covalently linked to wall (e.g. InaA, Prot. A) N C Majority ‘cleaved’ at LPxTG Minority simply ‘anchored’? (e.g. ActA in Listeria) 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 minorityof wall-associated proteins in many speciesanchored 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 Chaperone Regulators (in cytoplasm) Major subunit ‘Usher’ (OM) Minor subunits

43. Type I fimbrial biogenesis Minor subunits: FimH = adhesin Fim A major subunit ‘Tip’ structure FimF + G Fim G - also regulates fimbriae length? OM Fim D ‘Usher’ assembly & attachment Fim C periplasmic chaperone 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.