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Bacteria as communities sociomicrobiology

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Title: Bacteria as communities sociomicrobiology


1
Bacteria as communities sociomicrobiology
  • Biofilms
  • Quorum sensing

2
Biofilms Community 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

3
Components
  • Microorganisms
  • Single or heterogeneous
  • Sessile (attached) vs planktonic (unattached)
  • Glycocalyx
  • Glue containing polysaccharide
  • Traps/concentrates nutrients
  • Surface
  • Abiotic (inert)
  • Biotic (living)

4
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

5
Bacterial species associated with biofilms
  • P. aeruginosa
  • S. epidermidis
  • S. aureus
  • E. coli
  • Lactobacillus spp.
  • Streptococcal spp.
  • …and many more…..including candida albicans

6
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
7
3-day old sock
Image courtesy of Eric Goulbourne, Proctor and
Gamble
8
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

9
Multiple steps and gene products required for
biofilm formation
10
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
11
Initial clonal growth 11 mix of red and green WT
bacteria
12
Initial clonal growth followed by migration
Tolker-Nielson, 2003
13
PilA mutants fail to climb the mushroom stalk
  • pilA
  • WT

Tolker-Nielson, 2003
14
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
15
Biofilms are antiobiotic resistant
Tobramycin Gentamycin Ciprofloxacin
Otoole et al 2003
16
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

17
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
18
Biofilms mutant that is antiobiotic sensitive
Tobramycin Gentamycin Ciprofloxacin
No difference for some antibiotics
Otoole et al 2003
19
-abx
abx
1000 fold difference
Otoole et al 2003
20
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
21
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
22
  • 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
23
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

24
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!

25
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

26
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
27
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
28
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)
29
Larger pro-peptide processed during secretion
Acyl side chain varies
30
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?

31
(No Transcript)
32
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

33
V. Harveyi QS private vs public
conversations multilingual 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

34
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

35
P. aeruginosa QS cascades in series that
control virulence factor production
  • Amplification LasR positively regulates LasI
  • Temporal control LasR regulates Rhl

36
Biofilm maturation QS
37
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

38
QS in S. aureus
Time
39
Inhibitory QS AIP from one strain inhibits agr
response of other strains
40
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?
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