Presentation is loading. Please wait.

Presentation is loading. Please wait.

Bacteria and the cytoskeleton The human body is a dangerous place for a bacteria to be! Antibodies Neutrophils Complement Innate response - lysozymes.

Similar presentations


Presentation on theme: "Bacteria and the cytoskeleton The human body is a dangerous place for a bacteria to be! Antibodies Neutrophils Complement Innate response - lysozymes."— Presentation transcript:

1

2 Bacteria and the cytoskeleton

3 The human body is a dangerous place for a bacteria to be! Antibodies Neutrophils Complement Innate response - lysozymes

4 Many bacteria find it much more comfortable inside the cells of its host.

5 Some bacteria gain entry to cells by forcing them to phagocytose them. Bacteria secrete Factors that stimulate Macrophagocytosis Once engulfed Bacteria digest the phagolysosome Now cells can grow Within the cell

6 Fibroblast protrusion, Louise Cramer University College London

7 GFP-actin. Stimulated Macropinocytosis

8 Killing phagocytosis tight compartment Stimulated phagocytosis loose compartment

9 Other bacteria (EPEC) stimulate the production of an elaborate adhesion (Pedestal), that prevents phagocytosis and removal by flushing

10 Salmonella Escherichia Yersinia Shigella Staphylococcus Legionella Listeria Many bacteria subvert normal cytoskeletal function in order to parasitize their eukaryotic host through either adhesive complexes or inducing macro-pinocytosis (Most nasty bacteria are named After people!!)

11 Yersinia pestis was responsible for the Great Plagues. During the 6th century AD, the plague ravaged the known world over a 50 year period causing 100 million deaths. The "black death" again devastated Europe during the 14th century over a 5 year period causing 25 million deaths (25% of the European population). The bacterium was named after Yersin who identified it as being the causative agent of an outbreak of plague in Hong Kong

12 Xenopsylla cheopis Dirty Rat Rattus norvegicus Dirty Rat Homo sapiens

13 WHO reports 1,000 to 3,000 cases of plague every year!

14 Other Yersinia cause disease. Yersinia enterocolitica Typically, only a small number of human cases of Yersiniosis are recognized. Symptoms are like that of appendicitis and out breaks are often detected by a sudden increase in appendectomies in a particular region. The Center for Disease Control & Prevention estimates that about 17,000 cases occur each year in the United States.

15 Bacteria inject toxins into cells to subvert their activities The hypodermic syringes that they use are modified flagella

16 The three main types of bacterial secretion Type III is most often associated with pathogenic bacteria A A Type II Type III Type I A Outer membrane Inner membrane Periplasm

17 Abbrev. Full nameCommon name and features inocolum Source ETECEnterotoxin E.coliMontizuma’s revenge, traveller’s tummy 10 8 Faecal usually comparatively mild, ( Diacalm grade) contamination EIECEnteroinvasive Invades, Shigella pathogenicity islandhigh Food & waterborne EPEC Enteropathogenic Pedestal formation, infant diarrhoea10 8 - 10 Nosocomial community EHECEnterohaemorrhagic (O157)“Hamburger disease” Shiga toxin3Cattle faeces, meat The most common pathogenic E.coli

18 Interactions of the common pathogenic E.coli with epithelial cells

19 Interaction of EPEC with epithelium first through EspA filaments (a), then through intimin (b). Knutton et al, 1998. Nucleolin is a third binding site.

20 Scanning E.M. of EPEC and epithelium. EspA filaments appear to insert into cell (arrows in A), possibly to deliver EspB. EspA may be part of the Type III secretion pathway, it is needed for EspB delivery. Note the pedestals are all of equal length.

21 Some pathogenic E.coli (EPEC, EHEC) put down their own “Welcome mat” Tir (translocated intimin receptor) is injected into host by Type III secretion Tir binds to host  -actinin, talin and vinculin all components of the focal contact. Nucleolin is a bacterial binding site for EPEC.

22 Cell death & loss Cell division Movement of fresh cells up from crypt Lumen of gut By targeting nucleolin E.coli are able to attach to the cells that will exist for the longest time. E.coli bind dividing cells in the crypt and stay attached as the are conveyed to the tips.

23 Microfilaments are present in ‘stress fibres’ that are attached to ‘Focal adhesions’. They are also present as a gel under the plasma-membrane esp. at the leading edge

24 The pedestal has features in common with both the focal contact and microvilli Pedestal base Myosin II tropomyosin  -actinin Vinculin Villin Ezrin Arp2/3 WASP

25 Light emitted at 384nm measured Pyrene Excited at 366nm Pyrene-actin in quartz cuvette NH-C-CH 2 -S-H 2 C- Actin Pyrene-actin method to measure polymerisation kinetics NH-C-CH 2 I O H-S-H 2 C- Actin + II

26 Actin-Binding Proteins modify actin polymerization

27 CRIB Proline-richWH2 Acidic Basic C R I B Proline-richWH2 Acidic B a s i c N C C N G Arp complex ActinActin P P Inactive Active Wiskott-Aldrich Syndrome Protein (WASP)

28 Arp2 Arp3 p40p35 p19 p18 p14 Zero length crosslink Non-zero length crosslink Yeast two hybrid screen Microfilament binding The Arp2/3 complex An actin-binding group of proteins pivotally involved in the regulation of actin polymerisation.

29 WASP-WT WASP-  C WASP-  GBD Kalman et al, 1999 Nature Cell Biol. 1; 389-391. Analysis of the WASP domains required for Pedestal formation

30 Kalman et al, 1999 Nature Cell Biol. 1; 389-391. Pedestal formation and localization of Arp2/3 complex components.

31 Pedestal formation by EPEC

32 Salmonella Edwina Currie Eggs Salmonella

33 Commensal Salmonella calm the Immune system

34 SipA SipC Actin polymerizing and bundling SptP Cdc42 GTP Inactive WASP Salmonella Type III secretion Arp2/3 complex Plastin SipB SopE SopB Cdc42 GDP Rac GTP Rac GDP Active WASP Pathogenic Salmonella disrupt normal cell function

35 Hints of Plastin’s involvement in signalling & Bacterial invasion. 1). BPB inhibition of plastin inhibits IP 3 dependent Ca 2+ increase in PMNs. 2). Plastin is itself regulated by Ca 2+. 3). Phosphorylation of plastin at Ser5 by PKA results in integrin activation in PMNs stimulated by Fc receptor ligation

36 SipA SipC Actin polymerizing and bundling SptP Cdc42 GTP Inactive WASP Salmonella Type III secretion Arp2/3 complex Plastin SipB SopE SopB Cdc42 GDP Rac GTP Rac GDP Active WASP Pathogenic Salmonella disrupt normal cell function

37 Injection By type III secretion Initial contact Actin polymerization and phagocytosis Phagosome stimulates new protein secretion Through a second type III machine A fresh actin wave of actin polymerization results in the vacuole being covered in actin. Lysosomes can’t fuse Nucleus Some time later an actin ADP-ribosylating enzyme disassembles the structure for unknown reasons (cell lysis & spread?). The Salmonella cycle of infection SPI1 SPI2

38 Gel filration of SipC SipC and actin SipC Actin SipC and actin (Higher power) Hayward, R.D. & Koronakis, V. 1999 EMBO J. 18, 4926-4934.

39 Gel filtration Actin + SipC-C Actin + SipC-N

40 Co-sedimentation of SipC N-terminus with actin bundles. Sedimentation of actin bundles from a mixture of SipC-N and F-actin (both 5 µM), demonstrating formation of an actin-SipC-N complex. Supernatants (S) and pellets (P) after centrifugation

41 SipC C-terminal domainSipC-C inhibition by Cytochalasin D Actin+SipC-C Actin Actin+SipC-C+ Cyto Actin + Cyto E.M. of actin with SipC-C and SipC-N Bundle

42 Induction of cytoskeletal rearrangements in vivo by SipC and SipC-C microinjection. Cultured HeLa cells fixed 30 min after microinjection with purified SipC (upper panels) or SipC-C (lower panels) (3 µM). Cells (DIC; A and D) were stained with polyclonal antibody to SipC and FITC-conjugated anti-rabbit IgG [SipC (B), SipC-C (E)] and with Texas Red-conjugated phalloidin to visualize F- actin [SipC (C), SipC-C (F)]. Injected cells are indicated by arrows.

43 Co-injection of SipC-N with GST-GFP. Cultured HeLa cells (DIC; A and D) fixed 20 min after microinjection with GST-GFP alone (upper panels) or mixed with SipC-N (lower panels) (3 µM). GST-GFP was visualized directly [GST-GFP alone (B); + SipC-N (E)] and F-actin stained with Texas Red-conjugated phalloidin [GST-GFP alone (C); + SipC-N (F)]. Injected cells are indicated by arrows (N = nuclear injection).

44


Download ppt "Bacteria and the cytoskeleton The human body is a dangerous place for a bacteria to be! Antibodies Neutrophils Complement Innate response - lysozymes."

Similar presentations


Ads by Google