sociomicrobiology Biofilms Quorum sensing Biofilms

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Bacteria as communitiessociomicrobiology

• Biofilms
• Quorum sensing

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BiofilmsCommunity of microbes embedded in an
organic polymer matrix that adheres to a surface

• 3D structure containing 1 or more microbial
  species
• Forms at interfaces
• Solid/liquid, liquid/air, air/solid
• Spacial heterogeneity
• Exopolysaccharide glue
• Water channels
• Important in human infections
• Decreased antibiotic susceptibility
• Less conspicuous to immune system
• Targets organisms to favorable niches
• Protection from physical injury
• Promote horizontal gene transfer
• Enhance cell-cell communication

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Components

• Microorganisms
• Single or heterogeneous
• Sessile (attached) vs planktonic (unattached)
• Glycocalyx
• Glue containing polysaccharide
• Traps/concentrates nutrients
• Surface
• Abiotic (inert)
• Biotic (living)

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Biofilms

• Abundant in aquatic ecosystems
• Targets specialized microbes to specific locales
• Nutrionally favorable, abundant nutrients
• Symbiosis
• Rhizobium/legumes
• Ruminant digestive tracts degrade/recycle insol
  materials
• Non-hostile environment, Protection from
  predators
• Insurance hypothesis
• Presence of diverse subpopulations increases
  survival of community as a whole

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Bacterial species associated with biofilms

• P. aeruginosa
• S. epidermidis
• S. aureus
• E. coli
• Lactobacillus spp.
• Streptococcal spp.
• and many more..including candida albicans

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Biofilms are important in human infections

• Dental biofilms
• gt500 species mixed aerobes/anaerobes
• Caries, gigivitis, periodontitis
• Pneumonia in CF patients
• Endocarditis, osteomyelitis
• Artificial surfaces
• Catheter-related infections
• Contact lens infections
• Prosthetic joints
• Difficult to eradicate
• Antibiotic resistant
• Resist phagocytosis

Suture
Urinary catheter
Contact lens
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3-day old sock
Image courtesy of Eric Goulbourne, Proctor and
Gamble
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Technological advances

• Early steps mimicked on plastic surfaces
• Flow cells
• Confocal scanning laser microscopy
• Allows visualization of hydrated state
• 3D reconstruction
• Spatial temporal heterogeneity
• GFP labeled bacteria
• reporter constructs

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Multiple steps and gene products required for
biofilm formation
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Pseudomonas aeruginosa biofilm formation
Detachment
Planktonic cells
LasI dependent QS signalling, turn off TFP
type IV fimbriae twitching motility
flagella
Clonal cell proliferation
Mature biofilm
Attached monolayer
Microcolony
Attachment
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Initial clonal growth 11 mix of red and green WT
bacteria
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Initial clonal growth followed by migration
Tolker-Nielson, 2003
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PilA mutants fail to climb the mushroom stalk

• pilA
• WT

Tolker-Nielson, 2003
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Acute and chronic infection are mutually
exclusive states
sad-28
sadB
sadARS retS
OR
Reversible Attachment
Irreversible Attachment
Microcolony Formation
Monolayer Formation
TTSS
Acute Infection
Otoole et al
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Biofilms are antiobiotic resistant
Tobramycin Gentamycin Ciprofloxacin
Otoole et al 2003
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Why are they abx resistant?

• Physical or chemical diffusion barriers
• Bind to Glycocalyx
• Slow growth 2 to nutrient deprivation
• Altered microenvironment
• pH, reduced O2
• Activation of stress response
• E. coli RpoS mutants cannot make biofilms
• Biofilm-specific state
• Induction of efflux pumps
• Phase variation

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Molecular elucidation of a mechanism of abx
resistance

• Screened tranposon library for mutants with
  increased antibiotic sensitivity when grown as
  biofilms but not when grown planktonically
• Secondary screen for nl biofilm formation

Otoole et al 2003
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Biofilms mutant that is antiobiotic sensitive
Tobramycin Gentamycin Ciprofloxacin
No difference for some antibiotics
Otoole et al 2003
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-abx
abx
1000 fold difference
Otoole et al 2003
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NvdB encodes a glucosyl transferase required for
cyclic glucan synthesis

• Mutation in NvdB gene glucosyltransferase
  required for synthesis of cyclic b (1,3) glucans
  (normally found in periplasm)
• Mutant is defective in periplasmic glycans
• Binds to tobramycin
• Gene is expressed during biofilm growth
• Likely binds other antibiotics?

Otoole et al 2003
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Self generated diversity produces insurance
effects in biofilm communities

• Short term growth in biofilms generates extensive
  genetic diversity
• RecA-dependent

Biofilm
Planktonic
Boles et al, 2004
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• Increases ability of biofilm to withstand
  external stress
• WT biofilm-grown bacteria survive exposure to
  oxidative stress better than RecA mutant

Detaches better
Detaches less well
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Quorum sensing

• Intermicrobe communication
• Signaling (language) system that allows bacteria
  to sense cell density
• Inter- vs intra-species
• Early examples
• Vibrio harveyi-only produces light at high cell
  densities
• Can induce light by adding supernatants from
  stationary phase cultures
• Strep pneumonie-competence induced at late-log
  phase
• Produces competence factor at high cell
  densities
• Important in many processes
• Symbiosis
• Virulence
• Antibiotic production
• Biofilm formation
• Microbe-host communication
• Novel drug targets

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Why quorum sense?

• Bacterial population can coordinately control
  gene expression of entire community
• Allows bacteria to act as multicellular organisms
• Allow bacteria to obtain nutrients from breakdown
  of complex polymers
• Keep bacteria invisible to host defenses until
  enough bacteria present to overwhelm host
• Bacterial army does not display its weapons until
  troops have amassed and are prepared to attack!

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Quorum sensing in Vibrio harveyi

• Bacteria live in light organ of fish
• Need late log-phase bacteria in vitro to get
  light production
• Can recapitulate with late log-phase supernatants

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Gram negative QS

• Found in gt 70 GNs
• Players
• LuxI homolog acyl homoserine lactone (AHSL)
  synthetase
• AHSL membrane permeable signal
• Lux R homolog DNA binding transcription factor
  that is activated upon binding AHSL

Lux R
Lux I
Lux CDABEG
Op
Virulence factor
Lux R
Lux I
Virulence gene
Op
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LuxI LuxR

• LuxI

• LuxR
• Intraspecies communication
• C-terminus allows Multimerization
• In absence of HSL, N-terminus binds to C-terminus
  and inhibits DNA binding
• Lux box 20 bp palindrome at -40
• Specificity comes from exquisite specificity of
  HSL binding LuxR homologs recognize similar DNA
  sequences (Lux Box)

HSL
Lux box
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Gram positive QS
ABC transporter
Sensor Kinase
Modified peptide
Activate response regulator
Target gene
Target gene transcription
Peptide precursor
Gene encoding peptide precursor
Virulence factors (S. aureus) Competence (S.
pneumoniae) Plasmid transfer (Enterococcus)
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Larger pro-peptide processed during secretion
Acyl side chain varies
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Hybrid QS

• Two circuits in parallel
• Converge to regulate common set of target genes

• AI1 is a HSL but signals through 2 CS.
  Intraspecies
• AI2 furanosyl Borate diester. LuxS is
  widespread interspecies communication?

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(No Transcript)
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Combinatorial Diversity

• Series circuit
• P. aeruginosa virulence factor production

• Parallel circuit
• B. subtilis competence

• How to distinguish multiple temporally coincident
  chemical cues
• How to preserve info encoded in each signal
• How to integrate info from multiple signals

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V. Harveyi QS private vs public
conversationsmultilingual communication

• Hybrid system
• 2 QS
• Private vs public conversations
• Multilingual communication
• LuxS AI-2 present in many GNs

AI-1 intraspecies
AI-2 interspecies

• 4 possible input states
• AI-1
• AI-2
• AI-1 AI-2
• No inducer
• Alteration in gene expression requires both
  autoinducers concurrently

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B. subtilis either/or

• Signals have opposing effects on response
  regulator
• One causes phosphorylation
• Other causes dephosphorylation
• Allows choice of 2 mutually exclusive states
• Competence vs
• Sporulation

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P. aeruginosa QS cascades in series that
control virulence factor production

• Amplification LasR positively regulates LasI
• Temporal control LasR regulates Rhl

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Biofilm maturation QS
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Interspecies cell-cell communication

• Many bacteria encode LuxS and produce AI-2
• V. harvyei can respond to cell-free sups from
  many gram pos and gram neg bacteria
• Eavesdropping
• P. aeruginosa responds to AI-2 even though it
  lacks LuxS
• High levels of AI-2 in sputum from CF patients
• Induces QS-dependent virulence factors
• Eukaryotes may also respond to bacterial QS
  signals
• IL-8
• The literature is complicated because the
  presence of host-encoded QS-degrading enzymes has
  only recently been appreciated

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QS in S. aureus
Time
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Inhibitory QS AIP from one strain inhibits agr
response of other strains
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Quorum quenching
Prokaryote-prokaryote
Eukaryote-prokaryote

• Seaweed
• Halogenated furanones
• Inhibit Pseudomonas biofilm formation Serratia
  swarming
• Human airway epithelial cells
• membrane-associated activity that degrades
  Pseudomonas C12 HSL but not C4 HSL
• Synthetic halogenated furanones abrogate
  pneumonia in mouse model of acute Pseudomonas
  pneumonia

• Bacterial antagonists
• S. aureus
• Proteases
• Bacillus AiiA destroys AHSLs required for plant
  pathogen virulence factor production
• Allows Bacillis to compete with Erwinia
• Transgenic potato tobacco plants expressing
  AiiA are resistant to Erwinia infections
• Therapeutics?