Mutation And Diversification In Biofilms Flashcards
Biofilms are highly heterogenous structures
Eg Stalk and cap
Microcolonies in cystic fibrosis the same not just in the lab
Aggregates and microcolonies structure
Antimicrobial tolerance in bacterial biofilms
Matrix impendence eg gent
Enzymes eg beta lactemase
Biochemical changes eg periplasmic glucans
Metabolic gradients eg ciprofloxacin and tobramycin only fill cells in metabolically active top layer
Physiological sun pops eg persisters
What causes a subset of cells within the biofilm pop to develop into microcolonies?
Challenges of biofilm antirestance and tolerance
Understanding biofilm development
Identification of gene systems / mechanisms involved
Possible to manipulate physiological status
Biofilm flow cell
Medium
Pump
Flow cell
Waste
“Continuous culture” and can image and watch biofilm grow, nutrient medium flowing, bacteria can washed through so only watching behaviour of biofilm and not planktonic
Medium, bacteria, surface seeking and attach to cover slip, biofilm formation
Biofilm structure development
2 Fluorescent protein reporters
Same organism but 2 pops one blue one yellow
Mixed population
Separated itself
Why? Global structure from single cell and grown into colony
Phenotypic variation in biofilm derived bacteria
Bacteria develop phenotypic differences from each colony
Eg morphology, small phenotypes, wrinkled, different pigmentation, motility
Doesn’t happen in planktonic cells so biofilm specific phenomena
Phenotype arrays show different substrate - utilisation profiles of CF p.aeruginosa isolates
Purple - uses carbon source
Wt and SC 1 biofilm derived, lost ability to use some carbon sources and gained use of others
Metabolic and phenotypic change
Is there a role for mutation and genetic variation in biofilm structure?
Maybe
Mutations
Driving phenotypic variations and structures of biofilms
Is there a role for mutation and genetic variation in microcolony growth
P. Aeruginosa DNA mismatch repair deficient strains
-WT
-MutS
-mutL
Mut strains frequency 100x greater than WT
P. Aeruginosa mutator strain exhibit enhanced microcolony development
Biofilm bio volume increased in mutator strains
Increased size of microcolonies
Microcolony initiation
PAO1 DNA repair mutants
Mutation frequency increases and increases microcolony formation (hetergenous micro colonies)
Deep sequencing of p. Aeruginosa biofilm pop - observation of genome evolution
Genomic sequencing of pop in biofilm
Which genes changed by biofilm
Grow biofilm, extract DNA, sequencing, compare to WT strain, mutation discovery
115 SNPs
Encodes bacteriophage region, lots of mutations (not key)
Lots of mutations across whole genome
Single nucleotide polymorphisms I’m coding and no coding regions
Which genes affected by nonsyn mutations
PA4341
pA263
Mutation detection in situ
GFP +1 reversion system pMDGFP
Green fluorescent protein gene, frame shift mutation, can’t express functional so can’t flow, place into p. Aeruginosa biofilm
Mutation and glows green again if mutation happened
Some did this
Increased 100-1000 fold higher in microcolony cells than non
Darwinian processes in biofilm development
Microcolonies foci for genetic mutation and evolution
Microcolony growth may involve mutation selection
Model for microcolony develop
Primary mutation
Clinal expansion
Secondary mutation
Clinal selection
Analgy: clinal succession and solid tumour development
Evolution of cancer as multiple mutations
Proliferation of beneficial (selective)
Lots of commonality of the selective ones even tho random
Active dispersal in biofilms
Some cells can do this
Intrinsic mechanisms - bacteria escape and colonise new sites, hollowing out of colonies
Gaps and pores and so escape
Happens in multi species dental biofilms & Waste water treatment granules
Understanding genetics and physiology of biofilm dispersal
Identify gene systems involved in dispersal for targeting
Possibility to manipulate physiological status of biofilm to enhance/inhibit dispersal
Pseudoalteromonas tunicata (D2)
Obligate Marine bacteria
Colonised and biofilms on marine plants eg ulva lactuca
Produces bio active compounds that inhibits marine fouling organisms eg algal spores
Forms microcolony based biofilms in lab and vivo
Pseudoalteromonas tunicata biofilm
Biofilm lysis, detachment and dispersal
Inside colonies die
Biofilm parts detach
P. Tunicata auto lytic protein AlpP
Detected in waste effluent of biofilms ~ 3 days old
Autolytic, produced in biofilm
AlpP mediates cell death in P. Tunicata biofilms
Knock out gene
Don’t see lysis and death
Remains viable
AlpP mode of action
Production of hydrogen peroxide from oxidation of L-lysine
Lysine + 02+ H20 = 6 amino 2 oxo hexanoate + NH3 + H2o2
Amplex red reagent react with H2O2 to produce red fluorescent oxidation product resuforin
Hydrogen peroxide detected in p. Tunicata biofilms
AlpP is required for biofilm dispersal in p. Tunicata
Facilitates detachment of subset of survival and dispersal of some cells
Kills others during that break up
Mutant - biofilm just sits there, no dispersal
AlpP occurs in several gram negative organisms
Marinomonas meditteranea
AlpP and it’s homologues produce hydrogen peroxide and induce lysis, dispersal and phenotypic variation
Paudononas aeruginosa
Live dead stain
Similar lysis of subset of cells as biofilm develops like P. Tunicata
Microcolony differentiation linked to production of reactive oxygen species
Dihydromine- ROS detective
Production of peroxynitrite (ONOO) in microcolonies 
Supraoxide and nitric oxide
NirS expression in mature p. Aeruginosa biofilms
Reporter on NirS via transcriptional fusion
Bright fluorescent
So expressed in some colonies
Involvement of nitric oxide in cell death and dispersal events in p. Aeruginosa
Add SNP (nitric oxide) (low doses) causes dispersal when added to the biofilm. Makes rest of biofilm more sensitive to antimicrobials
Nitric oxide induced dispersal of p. Aeruginosa biofilms
SNP added at low dose diapers
Too much then reverse effect and even more biofilm formation (protect themselves?)
Microarracy analyses: p. Aeruginosa responds to low lvls NO
Increased motility genes eg flgG
Less adhesion and biofilm relevant genes eg cupP, C, EPS Pel genes, alg genes
Genes containing GGDEF or EAL domains - cyclic-di-GMP turnover? Eg PA1181, bdlA
Cyclic- di- GMP regulates biofilm vs planktonic lifestyle across broads range of bacteria
Increase cyclic-di-GMP = biofilm formation
Reduce cyclic-di-GMP = planktonic
INTRACELLULAR
Cyclic-di-GMP turnover
Conserved domains
GGDEF - guanylate Cyclades
EAL - phosphodiesterase (breaks down to GMP)
PAS or Nhox- bind and respond to NO in both eukaryote and promaryites
Influence NO on c-di-GMP
Reduced c-di-GMP
Causes dispersal in p. Aeruginosa
Turns off biofilm traits
Cystic fibrosis
Lethal hereditary disease
Autosomal recessive mutations in CF transmembrane conductance regulator (CFTR)
Increase mucous production so bacterial colonisation, poor ciliary clearance
Chronic P.aeruginosa key factor in death
Adjunctive NO in cystic fibrosis - hypothesis
Low dose NO to lung
Reduc pseudomonas aeruginosa by reducing antibiotic tolerance of biofilms
Enhance efficacy of antibiotics
Reduce ineffective treatment and burden
Improve respiratory function
Improve quality of life
Dispersal of p. Aeruginosa clinical isolates in CF- sputum
NO = break up colonies
Phase 2 pilot study - reducing antibiotic tolerance with nitric oxide (RATNO)
Placebo and NO
Patients didn’t know which
Reductions with NO adjunctive so proof of concept
Seen through FISH (targeted probe to p. Aeruginosa biofilm fluorescing green)
Can’t remove completely but proof of concept so promising
