• Toxins/Infection phases
• Secretion Systems
ADP-ribosyltrans-
ferases(G-protein constantly
active, increased
cAMP/Choleratoxin)
Proteases(inhibition of presyn-
aptic vesicle fusion/
BotulinumtoxinA)
Endotoxin: LPS, LTA
N-Glykosidases(inhibition of protein
synthesis, rRNA-depu-
rination/Shiga-toxin)
Adenylate cyclases(increased cAMP-level/
edema factor
of Anthraxtoxin)
External function
Membrane damaging toxins
Internalized toxins
Exotoxinsspecific secreted proteins
Short Repetition
Genotoxins(arrest of cell cycle
cytolethal distending
toxins)
General mechanism of neuronal Signaling
Short repetition: Botulinum-Toxin: Zn2+ /Metalloproteases
Mechanism of botulinum toxin activity:
Toxin type: zinc metalloprotease
- hydrolysis of integral proteins
of synaptic vesicles
(membrane-docking-complex)
- cleaves synaptobrevin
(=VAMPs), NAP25, syntaxin
- blocks fusion of acetylcholine-
containing synaptic vesicle
with synapses at peripheral
nerve endings
- blocks transmission of
neuronal stimulus at
neuromuscular nerve endings
Result: flaccid paralyses of
muscles (respiratory paralysis)
Short repetition: Botulinum-Toxin: Zn2+ /Metalloproteases
Botulinumtoxin A
• acts at presynaptical
membran activating neurons
• inhibition of acetylcholine
release
Paralysis of muscles
Brock Mikrobiology
Tetanustoxin
• transport to inhibitory
interneurones
• inhibition of Glycin/GABA
release
Non-controled release of
acetylcholin into synaptic
groove leads to spastic
paralysis
Short repetition: Zn2+ /Metalloproteases
Mechanism of toxin activity
• toxin is endocytosed
• released from ER into cytoplasm
• ADP-ribosylation of trimeric
GTP- binding protein Gs
- modified Gs cannot
dephosphorylate GTP
(Inhibition of GTPase activity,
G-protein remains active)
- Gs massifely stimulates host
adenylate cyclase, leading to
constitutive synthesis of cAMP
- unphysiological activation of
cAMP- dependent protein kinase
A induces disease symptoms
protein kinase A
receptor
αs
ß γ
GTP GDP
Gs
Cholera toxin
A-Subunit
Adenylate
cyclase+ATP cAMP
ADP-ribose
NAD+
Nicotinamid
Cholera Toxin
GM1
Activation of chloride channels
block of chloride- and sodium
absorption
Outpouring of intracellular water,
extreme loss of fluids
membrane
Short repetition: ADP-Ribosyltransferases
Expl:
Cholera-Toxin
Corda, Girolamo, 2003, EMBO J.
Consequences of toxin activity
• dysregulation of ion channels
leading to massif loss of chloride and
hydrogene carbonate ions, and water
• In addition: inhibition of Na+/H+-transport
channel leads to loss of sodium ions
• symptoms: dehydration
> loss of up to 25 L water/day
> loss of electrolytes
Normal
Blo
od
stre
am
Lu
me
no
fin
tes
tin
e
Na+
H2O
Na+
+ Cholera Toxin
Na+
Cl-
Na+
Cl-
H2O
Na+
Cl-
K+
K+Cl-Na+ ↑cAMP
Increased activity of
adenylate cyclase
Dia
rrh
ea
Compare to: Pertussistoxin (Bordetella pertussis,
Keuchhusten)
• ribosylation of inhibiting small GTPase (Gi),
inhibition of adenylate cyclase-inhibiting G-protein,
uncontroled muscle contraction, whooping cough
Compare to: Diphteriatoxin fromCorynebacterium
diphteriae, causes severe throat infections,
• ribosylation of EF2, inhibition of protein synthesis
Short repetition: ADP-Ribosyltransferases
Expl:
Cholera-Toxin
Protective Antigen (PA) = B-subunit:
• secreted as single peptide, mediates inter-
nalization of anthrax-toxin, highly immunogenic
• binds to cell surface anthrax-toxin receptor (ATR),
host cell protease cleaves 20 kDa peptide from toxin
• formation of heptameric prepore
• binding of the edema factor and lethal factor to
receptor-PA-complex induces endocytosis
Mourez, 2001, Trends Microbiol.
Anthrax Toxin – a toxin with three players:
•Anthrax toxin: A2B- toxin
- 2 A subunits: Edema Factor (EF) & Lethal Factor (LF)
- 1 B: subunit: Protective Antigen (PA)
• factor combination induces endocytosis: PA and LF = Lethal Toxin (LTx)
PA and EF = Edema Toxin (ETx)
Short repetition: Adenylatecyclases (Anthraxtoxin)
Anthrax Toxin – a toxin with three players
Edema factor (EF)• bacterial adenylatecyclase activity increases level of cAMP
• increase of cAMP activates protein kinase A, induces water influx
•Anthrax toxin: A2B- toxin
- 2 A subunits: Edema Factor (EF) & Lethal Factor (LF)
- 1 B: subunit: Protective Antigen (PA)
• factor combination induces endocytosis: PA and LF = Lethal Toxin (LTx)
PA and EF = Edema Toxin (ETx)
Short repetition: Adenylatecyclases (Anthraxtoxin)
Lethal factor (LF)
• complex of LF with PA generates lethal toxin
• Zn-dependent metallo protease activity cleaves
MAPKK and reduces kinase activity
• Consequence is necrotic cell damage (black)
Short repetition: Adenylatecyclases (Anthraxtoxin)
Anthrax Toxin – a toxin with three players
•Anthrax toxin: A2B- toxin
- 2 A subunits: Edema Factor (EF) & Lethal Factor (LF)
- 1 B: subunit: Protective Antigen (PA)
• factor combination induces endocytosis: PA and LF = Lethal Toxin (LTx)
PA and EF = Edema Toxin (ETx)
ADP ribosyltransferases(transfer of ribosylgroup)
target expl.: Rho,Rac,CDC42,EF2
Bacterial Adenylatcyclases(generation of second messenger cAMP)
Bac.adenylate-cyclase
Proteases(cleavage of proteins)
target expl.: synaptobrevin, syntaxin, SNAP-25, E-Cadherin, desmoglein
Short Repetition
• subset and activity of virulence factors determines virulence and host adaptation
Phases of Infection
Initial phase Establishment Chronification
Host entry
• Vectors
• Wounds
Adapataion
to host,
proliferation
• Metabolism
• Siderophores
• …
Persistence
• spores, antibiotic-
• resistences
Invasive
Dissemination,
Host damage
• Invasins
• Secretion systems
• Toxins
(i.e. proteases)
S. pyogenes invasion in
epithelial cells (M. Rohde, HZI)
Immune defense
• protection against
phagocytosis via capsules
• IgG-cleaving enzymes
• Inhibition of Complement
Phagocytosis of N. meningitidis by human granulocytes
(sciencedirect.com)
Tissue-
adhesion
• Adhesins
• cellprotrusions
(Pili, Fimbria)
Pneumococcus adhesion to
epithelial cells (M. Rohde, HZI)
Phases of Infection
Initial phase Establishment Chronification
• subset and activity of virulence factors determines virulence and host adaptation
Wound-
infections
hivwebstudy.org
tissue-
infections
microbiologyspring2010.wikispaces.com
Organ-
infections
peir.path.uab.edu
Systemic
infections/
Sepsis
beforeitsnews.com
Chronic
infections
emedmd.com
Symptomless
infected
mevis-research.de
Host entry
• Vectors
• Wounds
Adapataion
to host,
proliferation
• Metabolism
• Siderophores
• …
Persistence
• spores, antibiotic-
• resistences
Invasive
Dissemination,
Host damage
• Invasins
• Secretion systems
• Toxins
(i.e. proteases)
S. pyogenes invasion in
epithelial cells (M. Rohde, HZI)
Immune defense
• protection against
phagocytosis via capsules
• IgG-cleaving enzymes
• Inhibition of Complement
Phagocytosis of N. meningitidis by human granulocytes
(sciencedirect.com)
Tissue-
adhesion
• Adhesins
• cellprotrusions
(Pili, Fimbria)
Pneumococcus adhesion to
epithelial cells (M. Rohde, HZI)
On schedule for 15th of july:
• Zipper- and trigger mode of bacterial uptake
• Overview secretion systems
A) Zippering
1. step: tight receptor binding by bacterial adhesins
2. step: cytoplasmic receptor domain induces signaling cascades
3. step: signaling results in cytoskeleton-mediated zippering of host
plasma membrane via filopodia
4. step: Engulfment and uptake of the bacterium
B) Triggering
1. step: bacterial colonization of the cells
2. step: Injection of effectors via type III or type IV secretion systems
(e.g. Salmonella, Shigella) into host cell
3. step: effectors trigger host signalling, i.e. activation of Rho-
GTPases, which leads to reorganization of cytoskeleton,
induction of membrane ruffling via lamellipodia
4. step: Internalization of this bacteria into a vacuole
Trigger- or Zipper ???: EM of invading C. jejuni (yellow arrows), membrane ruffles (red arrow), and filopodia (blue arrow).
From: T. Ó Cróinín & S. Backert, Frontier in Infection and Cellular microbiology
(Filopodien)
• Induced by bacterial adhesion to cell receptors
• Used by Gram-Positives and Gram-Negatives!
α5β1-integrin receptors
ECM
Actin
Bacterium
ECM
Actin
Integrin receptors
Example for Yersinia, adapted from Prof. Dr. Dersch, HZI
Colorized EM of Yersinia invasion
(M. Rohde, HZI)
Expl. for direct mode of integrin-mediated uptake via invasin (Yersinia)
= invasin
Induction of own bacterial uptake
by a receptor-independent mechanism
Membrane ruffling
• Requires an secretion system with injectisome/needle!
• So far exclusively described for Gram-Negatives!
• Gram-Positives use sec, tat, holins, type
IV pili, type VII SS, and outer membrane
vesicles (OMV = type 0)
• but no injection-needle-formation, no
trigger-like uptake mechanisms reported!
• Gram-Negatives might use all currently described
secretion systems (type 0 - type IX)
• either membranes-spanning one-step secretion across
both membranes to outer environment or into host cells
• or two step translocation, first from cytoplasma to
periplasma and second from periplasm to
outer environment
• generate injection needels and
induce trigger-like uptake processes,
membrane pedestal formation and
other cytoskeletal rearrangements
Function in Pathogenesis of Bacterial Infection:
• Export of virulence factors (proteases, adhesins, toxins,…)
• Export of bactericidins for species competition in host environment
• Export of transporter proteins for iron and nutrient aquisition
• Translocation of effector proteins into host cytosplasm for host cytoskeleton rearrangements
• Assembyl of pilus and curli for bacterial adherence
• Assembly of flagella and type IV pili for bacterial motility
• Generation of conjugation pili for interbacterial DNA-transfer
• Export of proteins, carbohydrates for biofilm formation
• Export of antibiotics to enhance
antibiotic tolerance / resistance (OMV)
From: Galan and Collmer, „TypeIII secretion“, Science, 1999
Plus T9SSPlus OMV
Transport of factors only into periplasma
Plus T9SSPlus OMV
Transport of factors into exterieur or host cells, crossing IM and OM
Plus T9SSPlus OMV
Formation of pilus filamtens or curli
Plus T9SSPlus OMV
Transport from periplasam to exterieur, +/- pilus
Plus T9SSPlus OMV
Secretion systems & pili types also present in Gram-Positives!
Plus T9SSPlus OMV
Transport of factors only into periplasma
Plus T IX SSPlus OMV
Transport from periplasam to exterieur, +/- pilus
• Importance in virulence and in cell physiology
•Transport of proteases, lipases, toxins (e.g. ETEC heat labile toxin, Cholera toxin)
• Role in pilus biogenesis
• 2 step process:
- Sec or TAT across IM
- TypeII SS to cross OM
- associated with typIV pilus formationArchitecture of TIISSs
• OM complex
– secretin
– pilotin
• IM complex
– platform proteins
– cytoplasmic ATPase
• SDA protein (secretin dynamic
associated)
– interacts with secretin and IM
components
– may act to transduce energy
• Three subtypes: Va,Vb,Vc
• common features: N-terminal leader sequences for IM transport,
formation of periplasmic intermediates, formation of beta-barrel pore in OM
Type Va: Autotransporters (AT) similar to Vc
(Oca-system)• secretion starts wit Sec-translocation through IM
• secreted proteins induces its own translocation:
- N-terminal signal sequence (passenger domain)
for IM-transport
- C-terminal ß-barrel for pore-formation in OM
• Type 5c requires formation of a coiled Oca-
protein heterotrimer in periplasma for transport
Sectreted proteins: adhesins, degradative enzymes, cytotoxins
- Neisseria gonorrhoeae IgA protease
- Yersinia pseudotuberculosis InvA
Type Vb: Two partner secretion
Type Va: Autotransporters (AT) similar to Vc
(Oca-system)• secretion starts wit Sec-translocation through IM
• secreted proteins induces its own translocation:
- N-terminal signal sequence (passenger domain)
for IM-transport
- C-terminal ß-barrel for pore-formation in OM
• Type 5c requires formation of a coiled Oca-
protein heterotrimer in periplasma for transport
Sectreted proteins: adhesins, degradative enzymes, cytotoxins
- Neisseria gonorrhoeae IgA protease
- Yersinia pseudotuberculosis InvA
Type Vb: Two partner secretion
• Similar to autotransporter but 2 proteins
• secreted protein (TpsA) with TPS-domain at N- terminus
• channel forming β-barrel (TpsB protein) in OM,
• TPS proteins in pathogens:
- Haemophilus influenzae adhesins (HMW1)
- Bordetella pertussis hemagglutinin (FHA)
- Serratia marcescens hemolysin ShlA, ShlB
Plus T9SSPlus OMV
Transport of factors into exterieur or host cells, crossing IM and OM
• Inner membrane complex
– ABC-transporter (with ATP-binding cassette)
and trimeric MFP
• Substrate recognition
– C-terminal signal (not cleaved)
– Proteins remain unfolded during transport
• MFP contacts trimeric outer membrane
proteins (OMP)
– conformational change in MFP
– transient complex
• Energy provided by ATP and PMF
• Proteins secreted by T1SS:
- Pore-forming leukotoxins (E. coli hemolysin)
- proteases, lipases
- Pseudomonas aeruginosa HasAp Haemophore
The most complex secretion system so far
- encoded on pathogenicity islands
(SPI-1 and SPI-2 in S. typhimurium)
- Two different major functions:
A) transport and injections of effectors into host cells
B) exportsystem for assembly of flagellae
• Needle-like channel
- needle length controlled by molecular ruler:
length of protein determines length of needle
(EscP in EPEC/EHEC, YscP in Yersinia)
• Effector translocation
- blocked by gate keeper substrate (SepL in EPEC/EHEC)
- relieved upon low calcium signal (host cell contact)
• Architecture:
- Basal complex with ATPase, Needle &
translocation pore
Plus T9SSPlus OMV
Transport from periplasam to exterieur, +/- pilus
?
Found in Gram-Positives and -Negative
but with different functions:
a) Conjugative TIVSS delivers plasmids &
mobile elements (transposons)
b) DNA uptake and release
c) Effector translocation (only Gram-Negatives);
delivers effector proteins into eukaryotic cell:
Proteins, DNA, Protein-DNA complexes
• ATP hydrolysis drives secretion
• Homologous to Conjugation Machinery of
bacteria and to flagellar system of archaea
• Virulence proteins secreted into eukaryotic cell:
- Bordetella pertussis, pertussis toxin;
- Agrobacterium tumefaciens, T-DNA portion of
T1 plasmid
- Legionella pneumophila, Coxiella burnetii, Dot/Icm system
• Phage-tail-spike-like injectisome
•Evolutionary similarity to components of bacteriophage tails
•Highly analogous to TypeIII and TypeIV SS
•Encoded on pathogenicity islands
(Exampl.: Pseudomonas aeruginosa, Francisella
tularensis, Burkholderia mallei, Vibrio
cholera, Campylobacter,
also Agrobacterium tumefaciens)
•Used by bacteria to attack competing bacteria
within the same niche
•Architecture
- protein complex connects IM & OM
- enery sorce is ATP hydrolysis (ClpV),which also acts
as disassembly protease
- Tail is formed by Hcp tube polymerization, covered by VipA/B dimers
- VgrG formes tip of Hcp tube,
Contact with host cell
triggers contraction of
VipA/B sheath
and Hcp-tube is fired
Ates et al., 2016
• present in Actinobacteria & Firmicutes including
pathogenic M. tuberculosis, M. leprae, C. diphteriae,
Staphylococcus aureus
- specialized system to transport substrates through
cell wall with high lipid content (i.e. mycolic acids layer)
- In Gram- negatives: conserved IM protein complex identified
- energy derives from ATP hydrolysis by EccC
- Transport of substrates with conserved C-terminal signal
sequence
- putative membrane channel consists of EccB-E
- OM pore complex unknown, secretion process barely
understood
- It is unknown whether it is a one- or two-step process
Model for Gram-Negatives
Model for Gram-Positives (Mycobacteria)
Functional aspects of Type 7SS in Mycobacteria
• ESX-1 is used by M. tuberculosis, M. marinum, and M.
leprae in the macrophage infection cycle
• Only ESX-1 expressing mycobacteria translocate from the
phagosome to the cytosol
• bacteria start to replicate and ultimately induce a necrosis-
like cell death
• escape from phagosomes into the cytosol is essential for
full virulence
• phagosomal escape is not observed for M. bovis BCG or
esx-1 mutants of M. tuberculosis and M. marinum
• ESX-1 system is used to lyse erythrocytes and are
supposed to transport proteins with membrane-disrupting
potential
• Gram-negative specific pathway for
secretion and assembly of prepilins for
fimbriae biogenesis, the prototypical curli
• Also called: extracellular nucleation-
precipitation (ENP) pathway
• TVIIISS differs in that fibre-growth occurs
extracellularly
• thin aggregative fimbriae (Tafi) are the
only fimbriae dependent on the TVIIISS
• Tafi were first identified in Salmonella spp
• Associated with adherence and biofilm
formation
Chapman et al., 2002
PseudomonasSalmonella/E.coli
Lasica et al., 2017
• Also known as Por-secretion system (PorSS) or perioGate
• Describes first in 2017 in Porphyromonas gingivalis (cause of aggressive Parodontitis),
also present in Flavobacterium spec. commensals
• Several lipoproteins identified, but ß-barrel-proteins forming a translocation pore in OM still unknown
• Cargo protein with two sorting signals: N-terminal signal peptide (SP) for sec-translocation and
conserved C-terminal somin recognized by type IX SS
• After translocation of periplasmic intermediate by type IX translocon, C-terminal signal sequence is
cleaved off by PorU-sortase
• OMV/MV pinched off from cell surface as membranous nanoparticles
• OMV/MV now considered as true secretion system of Gram-negative & -positive bacteria
• OMV/MV filled with membrane-associated and soluble proteins, nucleotides, etc.
• involved in a series of biological functions, including nutrient acquisition, iron
scavenging, antibiotic resistance and biofilm formation
• Contribute to pathogenesis by delivering virulence factors
- P. aeruginosa MVs enable long-distance delivery of multiple virulence factors including
alkaline phosphatase, hemolytic phospholipase C and Cif, a toxin that inhibits CFTR-mediated
chloride secretion in the airways
• Importance in antibiotic resistance, response to ß-lactam by secretion of OMV filled with ß-lactamases,
increase ß-lactam tolerance of P. aeruginosa, H. influenzae
• bacterial infection is a multi-step event
• non-phagocytic uptake occurs via receptor-binding mediated
„zipper“mechanism or effector-mediated „trigger“-mechanism
integrin receptors often involved
in receptor-mediated „zipper“-
uptake
type III, IV, and VI-secretion
systems involved in „trigger“- type
uptake (only Gram-Negatives)
• bacteria use up to 9 (10) different secretion systems for export of virulence factors
• Sec, tat, holins and type VII-secretion are ubiquitously used for proteins export
• Gram-negative bacteria express injectisomes spanning both membranes
• exocytosis of outer membrane proteins by both Gram positives and Gram –
negatives is defined as type 0 secretion system
• bacteria secrete exotoxins with specific enzymatic activity and host cell tropism
Questions for Repetition
- Describe the principal toxin mechanism of botulinum and tetanus toxin.
- Name the bacteria specis expressing Cholera toxin and describe the toxin mechanism
- What kind of toxic activity is mediated via the two A domains of the anthrax toxin?
- What properties are required if you would like to synthesize a powerful bacterial toxin? Name 5 properties.
- Explain the trigger and the zipper-like bacterial invasion mechanisms.
- Describe 6 biological functions of bacterial secretion systems.
- Which bacterial secretion systems are involved in trigger-like uptake processes?
- Which secretion system has been described for Gram-positive bacteria?
- Describe the principal architecture of a type VI secretion system, where do we find structural analogies?
- Describe the biosynthesis and the nature of type zero secretion system
- Which secretion system is also used for bacterial conjugation and DNA-transfer?
- Which secretion system is used for twichting motility? What is twichting motility?
- Describe the two energy sources which can be used for energizing secretion processes.
- What are key features/differences between sec, tat, and holin-pathway?
Video
https://www.youtube.com/watch?v=nP4
Ou2eoq4c
Tit-for-Tat (Type 6 Secretion fighting):
Video https://youtu.be/OBf64TEo7gA
Salmonella type III secretion system
3:32