Bacterial Pathogenesis Flashcards
What is the main entry point for pathogens?
Mucosal Layers
What must pathogens overcome to colonise a host? What is required?
- Overcome physical barriers, innate immunity
- Establish self in host
- Outcompete normal flora
- Requires adherence
What can mediate adhesion?
o Pili/fimbriae
o Afimbrial adhesins
o Capsules
What allows bacteria to adhere?
- Depends on bacterial factors, host receptor availability
- Common host structure and accessible is glycocalyx
- Bacteria need to pass mucin layer (goblet cells produce)
- Target areas with reduced mucous (M cells e.g.)
- Adhesins target common sugar structures (vary enough for some specificity)
- Can target common protein receptors (β1 integrin)
- Mimic natural ligand for receptor
What are fimbrial and afimbrial adhesins?
Fimbrial Adhesins
• Complex surface structures made up of repeating protein subunits
Afimbrial Adhesins
• Outer membrane proteins (G-)
What are the features of gram negative Type I/Pap fimbriae?
Type I/Pap pili
• Assembly requires usher/chaperone system
• Pilus = fibre of six strutrual proteins
• Thick rod, thin tip
• UPEC
What does type I pili bind?
mannose (glycoporteins)
What does Pap pili bind?
bind Gal α (1-4)Gal (glycolipids)
How is the biogenesis of type I and Pap pili regulated?
- Switch mechanism for regulation: all on/off, all elements needed to form pilus
- Pap gene cluster (regulation, major pilus subunit, rod terminator, outer membrane usher, periplasmic chaperone, adaptor/initiator as part of tip/adhesion, Gal α (1-4) binding adhesin)
- Fim gene cluster (same with mannose binding adhesin)
What occurs during pili biogenesis?
- Subunits made in cytoplasm, transported to periplasm
- Pilin subunit must bind chaperone (or degraded)
- Ushered to outer membrane
- Subunits connected on cell surface
What are pilicides?
• Antibiotics, prevent pili construction (interrupt ushers, polygenesis on surface)
What are the features of type IV pili?
- Different biogenesis (no usher, chaperone)
- Polar location
- Form bundles
- Inter bacterial interactions, mucosal adherence, biofilms
- Conserved features
- Twitching motility
What type of pili does EPEC have? What is it essential for?
Type IV
For adherence/colonisation
What are the features of Bordetella pertussis?
o Whooping cough
o Pertussis toxin
o Fimbrial and afimbrial adhesins
o Acellular vaccine less powerful than whole cell (less adjuvants to make response last)
o Vaccine with inactivated toxin, afimbrial adhesins
What type of adhesins does Bordetella pertussis have?
o Fimbrial and afimbrial adhesins
What is FHA?
• Filamentous Haemagglutinin (FHA part of acellular vaccine for Brodetella pertussis)
o 250 kDa
o Adherence
o Binding domains (central RGD motif: mimics β1 integrin), Mimics extracellular matrix protein binding to integrin
What are the features of adhesins in Streptococcus and Staphylococcus?
o Bind extracellur matrix proteins
o Recognise ECM proteins: tissue tropism
o Fibronectin-binding proteins with similar organisation and several repeat regions
What is twitching motility? What organism has it?
o Pseudomonas aeruginosa
o Bacterial crawling
o Pili retract (Type IV)
What is LEE?
- Pathogencity island
- In all A/E pathogens, has all A/E genes
- Genes for T3SS
- Proteins for virulence:
- Intimin
What is intimin?
o Outer membrane protein
o Afimbrial adhesin
What is TIR?
o Translocated intimin receptor
o Secreted by T3SS, put in host membrane
o Phosphorylated by host cell kinases
o Binds intimin
What are the functions of EspA, EspB and EspD?
• EspA, EspB, EspD (secreted proteins)
o EspA = pilus like surface organelle (hollow tube)
o EspB and EspD in host cell membrane, form pore
How do A/E lesions change cytoskeletal structure?
- Intimate attachment causes cytoskeletal change
- A/E pedestal body has lots of structural cytoskeletal proteins (F-actin, talin, ezrin, vilin)
- Host proteins for recruitment: Arp2/3, N-WASP
What is the structure of actin? At which end does polymerisation occur?
o F-actin: polymerised monomers, two twisted linear protofilaments, dynamic
o Polymerisaition at + end (barbed)
o Depolymerisation at – end (pointed)
o Arp2/3 nucleates actin to begin filament
What does N-WASP do?
o Bind Arp 2/3 complex, induce actin polymerisation
o Tip of A/E lesion
What is the process that leads to actin recruitment?
Tir Has Tyr residue which is phosphorylated → recognised by Nick → binds N-WASP → N-WASP binds ARP 2/3 → Actin recruitment
- N + C terminals of Tir interact with cytoskeletal components directly
- Middle region of Tir binds intimin
How do the EPEC and EHEC actin pathways differ?
- EPEC uses NICK (host protein) to recruit pro/N-WASP
- EHEC: Tir not phosphorylates, use bacterial protein (TccP) and recruit GBD/N-WASP
- Both lead to actin recruitment
How does EPEC avoid the innate immune response?
- Inhibits production of IL-8, prevent neutrophils
- T3SS dependent (need secreted proteins)
- NF-KB controls IL-8 production
What is involved in the NF-KB signalling pathway?
- TF’s p65 and p50 in cytoplasm sequestered by IkB proteins (mask nuclear localisation sequence)
- External signal (TNF, IL-1, PAMPs) bind receptors on cell surface
- Adapter proteins recruited
- Downstream mediators TAB2/3 and TAK activated
- IKK (inhibitor of NF-KB kinase) complex phosphorylated and activated
- Ikk phosphorylates IKB
- IKB ubiquitinated and degraded by proteasome
- NF-KB proteins translocate to nucleus, bind DNA NF-KB consensus sites, transcription of IL-8
How does luciferase indicate NF-KB activation?
- More luciferase activity = more NF-KB activation
* Lack of activity suggests protein inhibits the pathway
What is the action of NleE?
- Cysteine methyltransferase (enzyme)
* Modifies TAB2/3
What is the action of NleC?
- Zinc metalloprotease (enzyme)
* Cleaves NF-KB p65 and p50 heterodimer
What are the two invasion mechanisms?
Trigger and Zipper
What is involved in the zipper mechanism?
• Exploit host cell adhesion pathway
• Cause receptor immobilisation and clustering
• High affinity binding bacteria ligand to host cell receptor
• RME
o Uptake mediated by host cell
o In endosome or escape endosome
• Tricks the cell
What is involved in the trigger mechanism?
• Cell ruffling, growth factors and hormones
• Short contact, large scale actin polymerisation, ruffles form on host cell surface
• Ruffles fold over bacteria, engulfed
• Macropinocytosis
o Membrane ruffles trap membrane bound pockets of extracellular medium
o Mediate dby host cell
o In endosome or escape endosome
What are the features of Listeria monocytogenes?
- G+
- Food borne
- CNS, maternofetal infections, abortion
- Invasive (Zipper)
How does listeria invade? Which mechanism does it use?
• Internalised due to actin cytoskeleton
• Internalin protein (InIA) locks into host cell receptor (e-cadherin)
• α and β catenins have direct link to cytoskeleton
• Receptors cluster, invasion
Zipper mechanism
What is the receptor of Internalin/InIA ?
E-cadherin
What are the features of shigella flexneri?
- G-
- Water, person-person
- Bacillary dysentery
- Invasive (trigger)
What did shigella evolve from? How does it differ to it’s relative?
- From e.coli
- Gene loss (flagella, anti-virulence genes) and acquisition (mobile virulence plasmid)
- LNF and non-motile (unlike e.coli)
What does shigella virulence plasmid encode? Which proteins are involved?
- Encodes T3SS
* Secreted proteins (IpaB, IpaC, IpaD) form invasion complex
What allows salmonella to invade?
- Salmonella pathogenicity island 1 (SPI-1)
- Encodes T3SS, Sip proteins, Ipa
- Membrane ruffling
- T3SS homologues of Shigella
- Same function as Shigella proteins, target cytoskeleton and host GTPase
Where does salmonella replicate? How?
Salmonella’s Second T3SS
• Allows replication in endosome
• SPI-2
• Ssa effector proteins
How do SPI-1 and SPI-2 differ?
SPI-1 • Cytoskeletal rearrangements • Diarrhoea • Inflammation • Invasion SPI-2 • Avoidance of anti0microbial activities • Modification of vacuole trafficking • Intracellular survival, replication
What are the main differences between the trigger and zipper mechanisms?
Trigger Passive Bacteria in cell vacuole One protein Listeria (Internalin)
Ruffle Active Bacteria in cell vacuole Require several proteins Shigella (Ipas), Salmonella (Sips)
What are the benefits for pathogens to live intracellular?
- No access by complement or antibodies
- No need for adherence
- Nutrient access
- Intracellular parasite
What types of intracellular niches are there?
- Intralysosome (low pH, hydrolytic, rare)
- Intravacuolar (neutral pH)
- Cytosolic (actin based motility)
What kinds of intracellular niche do Shigella and Listeria use? How?
Cytosolic • Invade cells • Escape vacuole and proliferate • Recruit host cytoskeleton • Actin tail formation, propel to cells
What is the process of cell to cell spread by Shigella?
- Invade through ruffles via T3SS, Ipa
- Phagocytosis → phagolysosome lysis → proliferation → actin based locomotion → cell to cell spread (double membrane dissolved)
- Invade M cells, engulfed by macrophages, apoptosis, release pro inflammatory cytokines, recruit neutrophils
- Can only invade enterocytes from basolateral surface
What does IcsA do?
autotransporter, expressed at one end of bacterium ,polar, interacts directly with cytoskeletal machinery
What are the interaction domains of N-WASP?
CDC42 (activates N-WASP), Nick, Actin, Arp2/3
What is the role of ActA? Which bacteria express it?
- ActA at pole
- ActA binds VASP (not N-WASP) for actin polymerisation and Arp2/3
Listeria
How does listeria spread from cell to cell?
- Bacteria adhere tightly, RME
- Internalised in vacuole
- Lyse vacuole
- Recruit actin all around bacteria
- Then actin mobilised to pole, actin tail allows movement through cytoplasm
Why can it be beneficial to live in macrophages?
- Professional phagocytes, easy to get in
- Long lived cells
- Migrate through body
- Although they are APCs and can kill
What occurs during phagocytosis?
- Facilitated by opsonisation
* Killing after phagolysosome fusion, activation of oxygen independent and dependent pathway s
What is oxygen dependent killing?
o Respiratory burst (NADPH oxidase)
o Reactive oxygen speices (ROS) O2, H202, OCl
What is oxygen independent killing?
o Low pH (lysosome) o Proteolytic enzymes o Lysozyme o Lactoferrin o Membrane damaging proteins
What is the process of phagosome maturation?
- Phagosome
- Fuse with early endosome
- Fuse with late endosome
- Fuse with lysosome
What is coxiella burnetti? Why is it weird?
- Vacuole has markers for late endosomal lysosomal compartments (LAMP1, LAMP2, cathepsin D)- looks like lysosome
- Optimal growth at pH < 5
- Obligate intracellular parasite
- Related to legionella
- Type IV secretion system (Dot/Icm)
What are the features of Salmonella in terms of its intracellular niche?
• Tubular phagosome existence
• SCV: Salmonella containing vacuole
o Produce Sifs (salmonella induced filaments)
• Late endosomal, acidic, LAMP 1 marker present
• Prevents fusion of phagolysosome
• Phagosome has no CI-MPR (receptor) and lysosomal enzymes so is not a phagolysosome
SPI
• SPI-1 turned on for invasion, turned off after invasion and SPI-2 turned on
How does SCV mature?
- SPI-2 effectors responsible
- Invasion (SPI-1)
- Early SCV: EEA1, Rab
- Intermediate SCV: Rab
- Late SCV: LAMPs, vATPase, Rab7
- Deviate from normal pathway to form SCV with protruding Sifs
What is SifA?
- Causes Sif formation and keeps integrity of SCV
- Bacteria escape vacuole if it’s deleted
- Host cell targets are Rab7 and SKIP- kinesin binding protein to recruit microtubule motors
How does SPI-2 help salmonella form an intravacuolar niche?
• SPI-2 for inhibition of respiratory burst (prevent NADPH oxidase assembly on phagolysosomal membrane)
How does legionella form an intravacuolar niche?
What are the features of LCV?
LCVs- legionella containing vacuoles
• Phagosomes don’t acidify and fuse lysosomes
• ER like
• Arf1, Rab1, Sec22b (t-SNARE), ER proteins (calnexin, sec61), ribosomes
• No association with markers of endocytic pathway
Dot/icm genes
Why are dot/icm genes important?
- Type IV SS- deliver virulence proteins
- Mutants can’t prevent lysosome fusion, pathogen dies
- Proteins have a lot of functional redundancy
- Proteins cooperative or antagonistic (e.g. target Rab1 GTPase)
- Some target Arf1 GTPase
