Bacterial Pathogenesis Flashcards
Commensal bacteria
Most microbes are never pathogenic
Opportunistic bacteria
- many microbes are potentially pathogenic
- Gain ‘mistaken’ access to deeper tissues
immunocompromised patients - staphylococcus epidermis
- acquire ‘extra’ virulence factors
- escherichia coil
Obligate
- very few microbes are always pathogenic
- Entirely adapted to pathogenic lifestyle
- mycobacterium tuberculosis
Why is it important to understand infection at the molecular level?
Essentially so we can treat them
- e.g. Tuberculosis:
- Vaccine is 100 years old last year
- Most TB drugs predate the moon landing
- Vast increase in MDR-TB (XDR-, TDR-)
- Mtb has evolved resistance to every known TB drug
- Most people die vaccinated
Current antibiotics and their targets
Look at diagram on notes
Key host-pathogen interactions (and why are pathogens harmful?)
- bacterial diseases result from the interactions with the host
- pathogens may use ‘virulence factors’ to subvert or overpower host defences, allowing access to nutrient rich environments
- adherence is the first step in infection - it is required to initiate disease but it is not sufficient to initiate disease because the host has many innate defences that can thwart infection
Steps of infection
- Exposure to pathogens
- Adherence to skin or mucosa
- Invasion through epithelium
- Multiplication - growth and production of virulence factors and toxins
The disease process
Toxicity - toxin effects are local or system
Or
Invasiveness - further growth at original and distant sites
Lead to systemic damage
Extracellular pathogens
Extracellular pathogens do not invade cells, but proliferate in the extracellular space
(Vibrio cholerae, Staphylococcus aureus, Bacilllus anthrasis)
Facultative intracellular pathogens
Invade host cells when it gives them selective advantage
(Legionella pneumophila, Listeria monocytogenes, Neisseria spp)
Obligate intracellular pathogens
Cannot replicate outside host cells when it
(Mycobacterium leprae, all virus)
Persisters, latency, dormancy
- persiters (bacterial), phenotypically drug tolerant, not resistant - associated with state of dormancy
- pathogens may enter a state of dormancy or non-replicating persistence making them difficult to detect and treat with standard drugs that target growth mechanisms
- latent infections where the pathogen may not be demonstatable except when reactivation occurs
- different drugs may be used to target active and latent forms of a disease
- persistent (viral) infections slow but still progressing
Characteristics of bacterial virulence factors
- often specialised:
- Not constitutively expressed but regulated in response to the host environment
- Often co-regulated by same signals and transduction systems = ‘global regulation’
- these characteristics do not apply to all virulence factors nor to all pathogens - genetic context
- May be carried on extra chromosomal plasmids or bacteriophage
- may be grouped in ‘pathogenicity islands’ on chromosome - function
- Secreted onto the bacterial cell surface and/or into the surrounding environment
- facilitate host interaction
- host cell destruction
- interfering with host cell function
Virulence factors in salmonella
Look at diagram in the notes
Colonisation often associate with mucous membranes
- because it is an easy place to gain access to deeper tissues
- once adhesion occurs it can start colonising and in some cases making biofilms
Adherence
Can be mediated by lots of different proteins
Often requires interactions between specific molecules on either ‘side’
E.g. capsule, pili, flagella, fimbriae
Secretion systems mediate interactions with the host
All are considered very promising drug targets as if you can target systems that secretes virulence factors you can cut down pathogenesis of bacteria
The sec secretion pathway
- transports unfolded proteins across cytoplasmic membrane
- Post-translational mechanism cia SecAB SecB-N-terminal signal interaction:
- proteins got to inner membrane or periplasm, where other SS may transport across OM- Co -translational mechanism via SRP-FtsY SRP-N-terminal signal interaction proteins go to inner membrane
The Tat secretion pathway
- Sec and Tat facilitate transport across cytoplasmic membrane
- Transports folded proteins across cytoplasmic membrane
- certain post translational modifications can only be performed in the (reducing the environment of the) cytoplasm and requires already folded proteins. Essential for virulence in many pathogens
Protein secretion in gram negative bacteria
More membranes; check notes for diagram
Adhesion: enteropathogenic E.coli (EPEC) builds specialised structures via T3SS
- bacteria kind of sit on bean bag
- Upregulation of T3SS
- T3SS forms a translocon (TL)
- Effectors (e.g. Tir) are trafficked into host via TL
- Tir inserts into host plasma membrane and interacts with Ec Intimin on bacterial surface
- this interaction promotes Tir clustering»_space;
- recruitment and remodelling of host actin»_space;
- formation of pedestals
Key pathogen interactions and why are the pathogens harmful?
Bacterial diseases result from interactions with the host
pathogens use ‘virulence factors’ to subvert or overpower host defence, allowing access to nutrient rich environments
Types of virulence factors
Look at diagrams in the notes
What happens after invasion
Spread through epithelial cells layer
Intracellular survival and replication
spread through the epithelial cell layer
- Some (e.g. Shigella, listeria) escape from entry into the cytosol
- intracellular movement, replication and intercellular spread
- movement required the host cytoskeleton
intracellular survival and replication
- Other bacteria (e.g. Salmonella) survive and replicate within intracellular vacuoles
- Some survive in macrophages, allowing them to spread locally and throughout the body by the host defence system
- inhibition of phagolysosome fusion
- survive oxidative burst
Host pathogen tug of war
Hostile environment
Macrophages
Hostile environment
Host sequesters essential iron, but……
- Pathogens bind iron at higher affinity than the host by secreting iron binding molecules and re-importing them
acid in the stomach kills many pathogens, but……
- E.g. Shigella (dysentery) and helicobacter pylori (ulcers) resist low pH by pumping out H+ out of their cells
- Commonly made of polysaccharide, e.g. Streptococcus pneumoniae, this helps avoiding phagocytosis by: being non-immunogenic and lacking affinity for complement factors
Damage caused by bacteria
Direct - from bacterial action (invasive process/ production of toxins)
Cytolysins: interact with host cell membranes
- enzymatic degradation of membrane phospholipid
- phospholipids e.g. clostridia’s alpha toxin
Function - target cell lysis, disrupt host cell signal transduction
Whiny?
- disable immune cells
- assist tissue damage and spread
Enzymatic cytotoxins target fundamental cellular functions
ADP ribosylating enzymes
Mimicry of host adenylate cyclase
N-glycosidation
cleave host SNAREs
ADP ribosylating enzymes
- Cholera and pertussis toxins target adenylate cyclase - disturb cAMP levels, signal transduction and ion balance
- diphtheria toxin targets translation elongation factor 2, blocks protein synthesis
Mimicry of host adenylate cyclase
- Anthrax toxin
- similar consequences to ADP ribosylating enzymes
N-glycosidation
- Anthrax toxin
- similar consequences to above
cleave host SNAREs
- Tetanus and botulinum neurotoxins
- cleave membrane fusion apparatus, disrupt neurotransmission
Direct damage by enzymatic exotoxins
Diphtheria AB toxin blocking host translation
Look at diagram in the notes
Enzymatic cytotoxins
botulinium toxin prevents release of acetylcholine
Check diagram in the notes
Chemistry of exotoxins
- Proteins secreted by certain gram-positive or gram-negative Bacteria; generally heat-labile
Mode of action of exotoxins
Specific; usually bind to specific cell receptors or struc-tures; either cytotoxin, enterotoxin, or neurotoxin with defined, specific action on cells or tissues.
Toxicity of exotoxins
Often highly toxic in pictograms to microgram quantities, sometimes fatal
Immune response of exotoxins
Highly immunogenic; stimulate the production of neutralizing antibody (antitoxin)
Toxoid potential of exotoxins
Heat or chemical treatment may destroy toxicity, but treated toxin (toxoid) remains immunogenic
Fever potential of exotoxins
Nonpyrogenic; do not produce fever in the host
Genetic origin of exotoxins
Often encoded on extrachromosomal elements or lyso-genic bacteriophages
Chemistry of endotoxins
Lipopolysaccharide-lipoprotein complexes, released on cell lysis as part of the outer membrane of gram-negative Bacteria, extremely heat-stable
Mode of action of endotoxins
General: fever diarrhoea, vomiting
Toxicity of endotoxins
Moderately toxic in tens to hundreds of microgram amounts, rarely fatal
Immune response of endotoxins
Relatively poor immunogens; immune response not sufficient to neutralize toxin
Toxoid potential of endotoxins
None
Genetic origin of endotoxins
Encoded by chromosomal genes
Fever potential of endotoxins
Pyrogenic: often induce fever in the host
Endotoxins: LPS
- toxins that are constituents of the cell not secreted
- LPS found on outside of Gram negative bacteria
- is very toxic
- problem when want to purify proteins - make sure no residue of LPS if you want to take them further downstream and do immunological assays with them - they will cause a big reaction
Indirect damage cause by host responses
Indirect - cause by the host response to the bacteria
Acute inflammation
- In response to sensing alarm signals (surface proteins; lipopolysaccharide (endotoxin); cell wall; bacterial DNA
- can be very serious
Chronic inflammation
- Relatively uncommon in bacterial disease
- extended responses to persistent infection
- e.g. Mycobacterium tuberculosis
- e.g chlamydia trachomatis
- ongoing tissue destruction, repair and inflammation
Evade recognition
- mimic or mask with host components
- e.g. Syphilis - treponema pallidum
- shut off or switch expression of surface proteins by phase variation
- e.g. Salmonella flagellum proteins
- more complex DNA rearrangement mechanisms generate antigenic variation
- e.g Neisseria surface proteins
Inactive antibody
- secretory antibody cleaved and inactivated by specific proteases of mucosal pathogens
- e.g. S.pneumoniae, H.influenza
- bacteria bind and inactivate antibodies directly
Bacterial virulence ideas
Many ways that actin can be hijacked and manipulated by pathogens
- host cell entry
- intracellular life: moving, hiding
- escape disseminate
Listeria monocytogenes
- Can be ingested via contaminated food
- not really a problem , but problem in pregnant woman and immunocompromised people
- All listeria spp are motile at low temperatures, facultative anaerobes, nonsporulating.
- only L.monocytogenes is pathogenic
- temp-dependent expression of virulence factors
Early observations of L.monocytogenes
- comment tails form behind each of the bacteria
- these are always at one end (pole)
- they are formed from actin (by labelling)
What is known about the biochemistry of actin
Look at notes for the diagram
- globular (G-) actin is a soluble monomer that polymerises to form actin filaments (F-actin)
- the filament exhibits polarity due to the way the monomers fit together
- polymerisation is favoured at one end (‘plus’ or ‘barbed’ end)
- under the right conditions (e.g. screening electrostatic repulsion) purified G-actin can assemble into F-actin
- this is slow in a tube! (~60 minutes!)
- actin is assembled and disassembled in cells in seconds
- in cells, proteins promote polymerisation and disassembly to speed things
Comet tails
- The bacteria move away from the tail(push v pull)
- assembly occurs near to the bacteria
- disassembly occurs away from bacteria
- F- actin filaments are linked in the cell
Harnessing the power of bacterial genetics to study host -pathogen interactions
transposon (Tn) mutagenesis: a method for generating a pool of bacterial deletion mutants
Tn-seq for identification of (conditionally) essential genes
- some cells die as soon as transposon is inserted
- condition A give all nutrients that it requires
- b - morphometric to see which are essential
Look at notes for a diagram
Using mutants to identify bacterial factors involved in tail formation (not essential so screening needed)
- infect cells with each mutant
- identify mutants by screening on egg yolk plates, confirm with plaque assay*
- identify mutants that do not form tails
- Infections of fibroblasts, observe areas of dead cells on plates, speculated to be associated with cell-cell spread, which requires tail formation*
- identify genes in question (cloning and sequencing or WGS)
- in this case, only one gene was identified: actA
Look at notes for a diagram
ActA is a surface protein
- contains a signal sequence in its N-terminal region that directs secretion
- if signal sequence is deleted there is no tail formation
ActA binds F-actin
Multiple regions important for interaction between F-actin and ActA in the N-terminal domain
ActA is a surface protein
Anchored to the membrane by this C-terminal region
If this region is deleted there is no tail formation
Now we know that Listeria hijacks host cell actin using ActA
ActA is the only bacterial protein necessary for tail formation, but is ACTA sufficient fro tail formation?
We can completely reconstitute tail formation using purified ActA and Xenopus cell extracts, i.e. Dispense with pathogen and host cel
ActA is necessary and sufficient for tail formation
What factors are required for tail formation?
ActA binds the Arp2/3 complex and stimulates rapid actin assembly
Actin and pathogens
several pathogens exploit actin for their own purpose, to adhere, to enter and to move within and between host cells, e.g Escherichia coli, Listeria monocytogenes, Shigella flexneri, burkholderia spp. And Rickettsia spp.
Discovery of Listeria ActA revealed a family of cellular proteins called ‘nucleation (polymerisation) promoting factors’
They share amino acid similarity to ActA but are found in cells
Involved in stimulation cellular Arp2/3 dependent actin assembly
Like ActA they bind and activate Arp2/3 complex
Important in key cellular signalling mechanisms relevant to cell movement and receptor signalling
Summary
Host actin co-localises Lmonocytogenes, which behave differently in the absence of actin polymerisation
Staining reveals actin tail in rapidly moving bacteria
Transposons mutagenesis identifies one bacterial gene, ActA, an essential for tail formation
ActA together with Xenopus cell extracts is necessary and sufficient for tail formation ActA
Fractionation of cell extracted and reconstitution of complexes identify y essential host factors
