Antimicrobials 1 Flashcards
How does CRISPR work?
- Invastion
- bacteriophage injects DNA into bacteria - adaptation
- phage DNA gets chopped and spacer gets inserted into CRISPR array - production
- the CRISPR array forms CRISPR RNA (guide RNA) - targeting
- RNA guides Cas9 protein (molecular machinery) to the correct sequence and cleaves it
CRISPR/Cas 9 molecular components
3 components
- Cas 9 protein (endonuclease)
- crRNA with 20bp complentary seqeunce for target
- transactivating crRNA (tracrRNA) that provides a bridge between Cas9 protein and guide RNA
CRISPR/Cas 13 system
- RNA dependent RNA nuclease (searches RNA not DNA)
- collateral activity: nuclease domains remain active even after cleaving target sequence so it cleaves anything else that’s close
CRISPR/Cas 9 molecular mechanism
mechanism
- scans DNA for PAM sequence (bacteria don’t have PAM –> not self)
- when it finds the PAM Cas9 binds to it
- Cas9 unwinds the DNA and attempts pairing
- the 20pb sequence of the guide crRNA pairs to the DNA
- Cas9 cleaves it if full match
CRISPR Cas12 system
- RNA dependent DNA nuclease (like cas 9)
- staggered cuts
- different PAM
What are the 2 main classes of CRISPR systems?
Class 1: multiprotein system
Class 2: require only one protein to cleave DNA (ex. cas9)
how many bacteria have CRISPR/cas systems and how is it spread?
- 48% eubacteria
- 95% archea
- spread through conjugation
importance of PAMs?
- self vs non self recognition
- PAMS only present on phage DNA and not CRISPR array (CRISPR array is an immunological record)
How do phages counter CRISPR?
- resistance due to mutations in target sequences following repair
- evolved anti CRIPSR (Acr) proteins that inactive bacterial adaptive immunity
–> evolutionary war between bacteria getting new defences and phages developing resistance
Mechanisms of Acr and protein examples
- crRNA loading interference (AcrIIC2)
- blocking DNA binding
- blocking DNA cleavage (ACrIIA2)
- enzymatically deactivate system (AcrVA5)
–> acetylates PAM-sensing lysine residue
use of Acrs in phage therapy
Acrs can be used to turn off CRISPR systems to enhance phage therapy
How can we use CRISPR/Cas9 as an antibacterial therapy?
- re-program the system by changing the spacer sequence to a bacterial sequence
- can deliver suicide crRNA alone or with Cas enzyme
- can be targeted against antiobiotic resisatnce genes or knock out essential genes –> now sensitve and can use normal antibiotics
Which CRISPR/Cas system should we use as a therapy?
Cas13
- collateral damage –> results in cell death by shredding RNA and killing bacteria
- bactericidal
Challenges and contraindications of CRISPR/Cas antimicrobial therapy
- immune reaction - if cas9 or cas13 entered circulation it could cause an immune response
- off target DNA and RNA cleavage - if it got into human cells it could target similar sites and edit the genome
- stimulate evolution - DNA repair could lead to increased antimicrobial resistance
- delivery - not small molecules anymore so hard to get through biofilm and cell wall
what are antibacterial nanomaterials?
synthetic nanomaterials (<500nM in size)
Antibacterial nanomaterial mechanism
- bactericidal
mechanism similar to conventional drugs:
- membrane damage
- ribosome damage
- RNA/DNA damage
- generating oxidative stress
Name the 4 antibacterial nanoparticles
- engineered antimicrobial peptides
- silver nanoparticles
- polymeric nanoparticles
- liposomal nanoparticles for drug delivery
What are antimicrobial peptides?
- naturally produced
- part of the innate immune response
- alpha helices and beta sheets that insert into the membrane
Natural AMPs MOA
- alpha helices and beta sheets that have AA that make them cationic and ampiphilic
- bind to negative charged cell membrane and insert
- induce destabilization (neutraliza charge)
SNAPPs MOA
- synthetic nanoengineered antimicrobial peptide polymer
- inserts into membrane and causes destabalization
- causes cell lysis
- bactericidal
- excellent against MDR
What are silver nanoparticles?
- manufactured using nanotechnology
- irradiation of aqueous silver salt with surfactant produce silver NPs
- silver is toxic to bacteria
Silver nanoparticles MOA
Mechanism of AuNP involves both whole particles and silver ions
A. whole particle
1. disrupt the membrane through adhesion
2. interact with biomolecules like ribosomes
3. generate ROS through disrupting the respiratory pathway
B. silver ion
1. interacts with sulfhydryl proteins
2. distrust DNA sugar backbone (but in humans too which is toxic)
Silver nanoparticle administration
- not orally bioavailable
- must be applied topically or injected
What are polymeric nanoparticles and how do they work?
- aggregates of cationic polymer (derived naturally or synthetically)
- standalone polymers have electrostatic interactions with negative charges in membranes and ESP matrix (extraceullarlar polymeric substances)
- behave similarly to AMPs
Chitosan
- polymeric nanoparticle
- has antimicrobial activty on its own
- can be used to encapsulate conventional antibiotics as a delivery agent
–> dual effect: chitosan on the outside and antibiotic on the inside
Outcomes of polymeric NPs on biofilm and fibroblast-biofilm co culture?
dual species biofilm –> biofilm reduction
fibroblast and dual species biofilm –> maintains fibroblast viability and reduces biofilm
What are liposomal nanoparticles used for?
- don’t have antimicrobial activity themselves
- delivery vehicles for antimicrobial agents
What do liposomal NPs improve?
- stability
- solubility
- biocompactability
- controlled drug release
- effectiveness - can bypass drug transporters, wall, membrane
- can add multi drugs with different MOAs
What can you add to a liposomal NP
- multiple drugs loaded onto liposome
- antibodies to make it targeted
- PEGylation
What times of lipids can be used to generate liposomes
usually cationic lipids
- DOTA
- helper phospholipids
- cholesterol
Considerations with nanoparticles
- immune reaction - against foreign AMPs, lipids and polymers
- toxicity - AgNP silver damages DNA in human cells
- production issues - AMPs are a labourous process and need purification to lower risk of contamination
