Mol_Inf

-complex mediates fidelity, middle substrates are secreted

-Part of T3SS

-Function in the host cell contact-dependent regulation of the T3SS

-allows translocation into host cell

-assist the injection of other virulence effectors into the host cytoplasm

-E3 ubiquitin ligase

-inhibition of TNFa, IL-12p35, IL-15 and IL-18

-Inhibition of RSK by hyperphosphorylation

-Inhibition of Caspase-1 results in inhibition of host cell pyroptosis

-acts as multi-protein-inhibitor

-key for paralysis of macrophages, PMNC, DCs

=> Anti-inflammatory

-Inhibition of phagocytosis due to synergistic mode of action of Yersinia effector proteins

-interfere with regulation of host cell cytoskeleton

-Ser/Thr kinase

-Binds to actin, Rho and Rac

-inhibits Actin polymerisation

=> uses G Actin as bait for actin regulating proteins

Anti-phagocytic activity

=> cell detachment

Tyrosine Phosphatase

FAK Interaction

Dephosphorylisation of Fyb, SKAP-HOM

interference with adhesion-related signal transduction (in macrophages)

Bottom-up Proteomics

Trypsin

Arginin und Lysin + modifiziertes Cystein

ESI = Elektrospray-Ionisation

Weitere Methode: MALDI-TOF

-Tabakpflanzen in den Niederlanden und Russland von Tabakmosaik-Krankheit unbekannter Ursache befallen (1892)

-erste Untersuchungen durch A. Mayer und D. Iwanowski liefern Hinweise auf nicht-bakteriellen Krankheitserreger

-M. Beijernick stellt Ultrafiltrat von Homogenisaten infizierter Pflanzen her (Chamberland-Filter)

=> Pilz- und Baterienfries Filtrat infektiös

=> Agens bleibt unklar, Begriff “Virus” (lat. Gift/Schleim) gewählt (1898)

-Friedrich Löffler und Paul Frosch weisen in Flüssigkeitsproben von an Maul- und Klauenseuche erkrankten Tieren durch Filtrationen nach, dass Krankheitsauslöser kein Giftstoff sein kann, aber partikulär sein muss

=> Schlussfolgerung: derartig “winzigste Lebewesen” könnten möglicherweise auch für andere Infektionskrankheiten wie Pocken & Masern verantwortlich sein

1. Der Infektionserreger muss immer mit Krankheit assoziiert sein und sollte nicht in gesunden Organismen vorhanden sein.

2. Der Erreger muss sich in Reinkultur anzüchten lassen.

3. Der aus Reinkultur gewonnene Erreger muss die Fähigkeit haben, die Krankheit in einem gesunden Organismus auszulösen.

4. Der re-isolierte Erreger muss mit dem ursprünglichen identisch sein

Virulence Plasmids

-pXO1

=> Toxin genes: pagA, lef, cya

=> atxA (Anthrax toxin activator): regulation of toxin genes

-pXO2

=> genes for capsule formation: capB/C/A/D/E

Aktivierung durch CO2 und Temperaturunterschiede (außer AtxA)

pagA: Protective antigen (PA83)

lef: lethal factor

cya (adenyl cyclase): edema factor

=> all are non-toxic precursors

LF+PA(63) = LT (lethal toxin, causes septic shock and death, Proteolysis MAPKK/MEK)

EF + PA(63) = ET (edema toxin, causes oedema, sharp increase cAMP conc.)

B. anthracis might cause systemic infectious disease

-ET and LT provoke vascular shock

-LT induces non-haemorrhagic circulatory collapse

-ET induces systemic vascular dysfunction, e.g. entry of blood into organs (skin: black-coloured lesion, spleen: black coloured)

-Monomeric PA(83) binds to host cells receptors

-proteolytic cleavage of pro-domain enables oligomerisation of PA into heptameric or octameric pre-pore

-3-4 LF or EF molecules bind to pre-pore PA oligomere

=> AB-toxin complex endocytosed

-PA(63) oligomer forms translocon pore in phagosome membrane allowing for translocation of EF and LF into host cell cytoplasm

-subsequent acidification of the endosome/phagosome triggers formation of pore by conformational changes of PA(63)

-PA(63) oligomers form channel in endosome/phagosome membrane

-low pH value of endosome/phagosome (required for pathogen elimination) and pH gradient towards host cell cytoplasm promotes translocation of unfolded EF or LF

-refolding of EF/LF in host cell cytoplasm enables in virulent enzymatic activity

ETEC:

-heat labile toxin (LT)

-heat stabile enterotoxin a & b (STa + STb)

=> STa via GC-C receptor

=> ADP-ribosylation of G-protein

=> disruption of ionic balance

-Actin pedestal proteins

=> Intimin

=> Tir (EPEC)

=> Nck

=> N-WASP

=> ARP2/3

EHEC:

-Shiga toxin (Stx)

=> incorporation via endosomal pathway

=> inhibits innate immune response

=> in Paneth cells: prevents protein synthesis? Induces Apoptosis?

-Actin pedestal proteins

=> Intimin

=> Tir (EHEC)

=> Nucleolin (via Stx)

=> IRTKS

=> TccP

=> N-WASP

=> ARP2/3

EPEC

-intimin-induced clustering of Tir

=> phosphorylation of Tyr474

-phosphotyrosine 474 binds to adaptor proteins Nck1+2

-recruitment of N-WASP

=> activation of the Arp2/3 complex

EHEC

-intimin-induced clustering of Tir

-Tir binds I-BAR proteins IRTKS and IRSp53

-Recruitment of EspF(u)

=> N-WASP multimerization

=> activation of the Arp2/3 complex

a) Actin pedestal

b) Zipper mechanism

c) Trigger mechanism

d) Microvillus-like cell extension

-Vaccinia fusioniert mit Plasmamembran

=> rekrutiert Clathrin und AP-2

-dadurch Polarisation des viralen Proteins A36

=> Infektion weitet sich aus

-Virionen binden von außen und stimulieren Arp 2/3 Komplex abhängige Polymerisation

-Scr und Abl phosphorylieren Tyrosine 112 und 132 des viralen Membranproteins A36

-phosphoryliertes Tyrosine 112 rekrutiert Nck

=> interagiert mit WIP Komplex und dieses wiederum mit N-WASP

-N-WASP stimuliert Arp 2/3 Komplex vermittelte Aktin Polymerisation

-phosphoryliertes Tyrosine 132 führt zur Anreicherung von Grb2

=> stabilisiert Nck, WIP und N-WASP

-Cdc42 aktiviert N-WASP

=> formt Aktinschweif

Upon egress of intracellular enveloped virus through fusion with the plasma membrane, the viral protein A36 is positioned in the plasma membrane underneath the cell-associated extracellular virus (CEV).

Non-receptor tyrosine kinases of the Src/Abl families phosphorylate A36, thereby generating docking sites for the adaptor proteins Nck1 and Grb2.

Nck1 and Grb2 mediate the recruitment of a complex of the WASP-interacting protein WIP and the nucleation-promoting factor N-WASP, that in turn recruits and activates the ARP2/3 complex

ARPC protein isoforms (of Arp2/3 complex)

=> 8 isoforms

Long actin tails: ARPC5L + ARPC1B

Short actin tails: ARPC1A + ARPC5

Legionella

(LCV = Legionella containing vacuole)

-in 50% der Fälle von Immunsystem ausgelöscht

Mycobacteria crucial role for Rab22a in conversion from Rab5  to Rab7 decorated phagosomes => lack of conversion leads to arrest of phagosome maturation

Macrophage pyroptosis

secretion of the proinflammatory cytokines interleukin 1β (IL-1β) and IL-18

invade from basolateral side of the epithelial cells

I. Diarrheic diseases

-EPEC: enteropathogenic E. coli (Actin cytoskeleton adherence, protein translocation via T3SS, pedestal formation; small bowel)

-EHEC: enterohaemorrhagic (Shiga toxin, large bowel)

-ETEC: enterotoxigenic (secretion of LT/ST Enterotoxins, small bowel)

-EAEC: enteroaggregative (clustering/biofilm on epithelial cells, not deadly, but damage due to toxins)

-EIEC: enteroinvasive (lysing phagosome of epithelial cells, travelling within cell, replication in cytoplasm, migration to next cell)

-DAEC: Diffuse adherent (finger-like structure wind around bacteria)

II. Urinary tract infections

-UPEC: uropathogenic

III. Meningitis

-MNEC: Meningiti-associated

+

AIEC: adherent invasive

EIEC + AIEC: reinfection from under epithelial layer => immune cells/macrophages as vesicles (EIEC) or replication ground (AIEC)

-Actin pedestals (e.g. EHEC/EPEC)

-Zipper mechanism (e.g. Listeria, Rickettsia)

-Trigger mechanism (e.g. Shigella, Burkholderia, Salmonella)

-Microvillus-like cell extension (Vaccinia virus)

1. Trigger mechanism: membrane ruffles in M cells

2. Endocytosis by resident macrophage

3. Escape from vacuole

4. Release of pro-inflammatory cytokines

5. Destabilization of epithelia

6. Escape from macrophage

7. Induced micropinocytosis

8. Escape from vacuole

9. Bacterial replication

10. Actin-based motility & cell-cell-spread

11. Escape from secondary vacuole

Listeria Virulence Factors (Selection)

-PlcA & PlcB (phospholipases) + pore-forming toxin listeriolysin O: disruption of primary vacuole

-PlcA & PlcB: impair formation of pre-autophagosome structures which protects bacteria from phagolysosomal degradation

-Phosphatidylcholine-/Phosphatidylinositol-specific phospholipase (PC-PLC/PI-PLC)

-Listeriolysin O (LLO): pH sensitive cholesterol-dependent cytolysin

Listeria induces mitochondrial fragmentation

=> induced mitophagy as virulence mechanism

=> LLO induces ER-mediated fragmentation of mitochondrial networks

Virulence Factors of Listeria - Histone dephosphorylation

-SIRT2: lysine deacetylase

-LntA: nucleomodulin, which translocates to the nucleus and inhibits BAHD1 resulting in enhanced ISG (IFN-stimulated genes) expression

BtBimA uses Arp2/3 complex

BpBimA and BmBimA use WH2 motif

-BimA trimerization enables beta barrel formation and subsequent integration into outer membrane of the bacterial cell

-bimA (bim gene cluster) is essential for actin tail formation

-Hypothesis: Cell-to-cell spread more efficient with BimA_Bpm, BimA_Bm

L. pneumophilia induced vacuole as replication niche

-Legionella penetrates host cell membrane using Dot/lcm translocation machinery

-Pathogen inhibits processing of phagosome and induces generation of specialised Legionella-containing replication vacuole (LCV)

-vacuole is decorated with membrane system similar to rough ER

-upon massive cell replication bacteria escape from LCV and induce lysis of host cell to disseminate in host organism

R. rickettsii

=> Rocky mountain spotted fever

R. typhi

=> Epidemic Typhus

R. akari

=> rickettsial pox

LC = Liquid Chromatography

Tandem MS

Analysator 1

Im ersten Analysator werden, abhängig vom gewünschten Ziel, die Ionen nach m/z (Masse-zu-Ladungsverhältnis) selektiert (Produkt-Ionen-Scan) oder es wird nur m/z bestimmt (Vorläufer-Ionen- und Neutral-Verlust-Scan).

Stoßkammer

Die Stoßkammer entspricht im einfachsten Fall einer mit Inertgas (z.B. Helium) gefüllten Röhre, durch die die Ionen fliegen. Aufgrund von Teilchenkollisionen fragmentieren die Ionen und gelangen als Fragment-Ionen in den zweiten Analysator.

Analysator 2

Im zweiten Analysator werden die in der Stoßkammer gebildeten Fragment-Ionen nach m/z analysiert. Damit lassen sich beispielsweise alle Produkte scannen und man erhält genauere Informationen über die Struktur des Moleküls (Produkt-Ionen-Scan). Wahlweise kann auch nur ein einzelnes Fragmention betrachtet werden, was eine sehr sensitive Analyse ermöglicht.

The molecules of a given sample are ionized and the first spectrometer (designated MS1) separates these ions by their mass-to-charge ratio (often given as m/z or m/Q). Ions of a particular m/z-ratio coming from MS1 are selected and then made to split into smaller fragment ions, e.g. by collision-induced dissociation, ion-molecule reaction, or photodissociation. These fragments are then introduced into the second mass spectrometer (MS2), which in turn separates the fragments by their m/z-ratio and detects them. The fragmentation step makes it possible to identify and separate ions that have very similar m/z-ratios in regular mass spectrometers

Quantification of Proteins via

-HPLC-LC-MS (e.g. MRM, PRM)

-Isotopic labelling

-Fluorescence/Colour reaction

-differential 2DE

-Labelfree Quantification (LFQ)

Label-free quantification (LFQ)

-Sample preparation: Tryptic digest, LC-MS/MS

-Quantification by integrating the elution profiles of the peptides

-Yersinia protein kinase A

-serine/threonine protein kinase

-binds to and phosphorylates the α subunit of the heterotrimeric G protein complex, Gαq, resulting in inhibition of Gαq signaling

via YopH

Cell detachment

dephosphorylates FAK and other factors to dissassamble attachment complex

GTPase activating protein (GAP)

antiphagocytosis activity

Targets RhoA => Actin stress fibers

Rac1 => Actin Vytoskeleton, signalling

Rac2

Cysteine protease

-anti-phagocytic

-cleaves Geranyl-Geranyl-Cystein rest of GTPases of the Rho family (essential for membrane adherence)

=> cells cannot keep up cytoskeleton

Serine/threonine-protein acetyltransferase

SUMO (= Small Ubiquitin-like MOdifier) protease?

=> removes SUMO from target

Inhibition of MAPK-kinases

=> prevents activation by phosphorylation of MKKs and IKK(s) by acetylating their activation loop.

Inhibition of lymphocyte proliferation

Poly-D-y-Glutamine (built by CapD)

Surface Layer Proteins (pXO1)

-semi-permeable monolayer of glycoproteins showing para-crystalline structure

-surface array protein (Sap) as main component during exponential growth phase

-extractable antigen 1 (EA1) main component during stationary growth phase -Bacillus S-layer-associated proteins (BSLs), 19 different genes

Capsule: weak immunogenic + inhibits phagocytosis