Biofilms and bacterial toxin production: Coordinated metabolic control

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Biofilms and bacterial toxin production
Coordinated metabolic control

• Linnea Ista
• Biology of Toxins
• Spring 2010

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What is a biofilm?
Collection of microbes, small organics and
metabolites growing on a surface
Ista, 1999 Appl. Environ, Microbiol. 651591
http//www.bact.wisc.edu/themicrobialworld/Intro.h
tml
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Why we care about biofilms- scientific curiosity
they are everywhere!

• Most abundant form of life on earth
• Total number of prokaryotic cells on earth at any
  given time 5x1036
• Most bacteria grow in biofilms.
• We have 10x more bacterial cells living in and on
  us than human cells.

Human intestines
Deep ocean hydrothermal vent
http//eager2009.files.wordpress.com/2009/06/schre
nkbiofilm.png?w500h373
Glacier
http//www.morning-earth.org/graphic-E/BIOSPHERE/B
ios-Microbe-Image/M-Fileum.jpg
http//www.uni-due.de/imperia/md/images/biofilm-ce
ntre/solfatare.jpg
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Why we care about biofilms- scientific curiosity
-they are old life!

• They are the oldest identified form of life on
  earth
• Fossils of stromatolites date to 3.8 Gya
• Stromatolites are layered formations of minerals
  and bacteria
• Formed at intersection between ocean and land

http//www.photosynthesisresearch.org/
Modern stromotalite (Australia)
Fossil stromatolites (New York State)
http//cas.bellarmine.edu/tietjen/Evolution/
http//www.petrifiedseagardens.org/
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They are the workshop for biochemical evolution!

• Most of the compounds we know today , with the
  exception of some plant toxins have evolutionary
  roots in bacteria
• Bacteria in biofilms exchange genes like mad!
• Biofilms might also have been the place where
  endosymbiosis occurred.
• Bacteria in ancient biofilms significantly
  altered the atmostphere of ancient earth
• They continue to fuel biogeochemical cycles today

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They were probably the prototype for
multicellarity
Sea lettuce development
Pseudomonas biofilm development
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Negative Significances of Biofilms -Medical

• Device failure
• Catheters
• Heart valves
• Contact lenses
• Stents

• Disease
• Otitis media
• Cystic fibrosis

• Nosocomial infections
• Biofilms serve as reservoirs
• Increase chance of antibiotic resistance

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Negative Significances of Biofilms -Industrial
Corrosion- lithotrophic bacteria oxidize the
metal or sulfate reducing bacteria produce
sulfuric acid! Oil platforms are vulnerable
Biofilms on ship hulls increase drag, can cause
corrosion and recruit macrofoulers such as
barnacles
Heat exchangers demonstrate up to 75 decreased
efficiency with a monolayer of bacteria
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Biofilm development

• Most of what we know is based on biofilm
  development in Pseudomonas aeruginosa strain PAO1
• P. aeruginosa is a ubiquitous organism
• Soil
• Water
• Anywhere there are humans
• Sometimes indicative of human contamination- for
  example in Tokyo Harbor
• Very adaptive
• Can grow aerobically, through anaerobic
  respiration and fermentatively
• Uses many carbon sources
• Is a very good bioredediator
• Major opportunistic pathogen
• Otitis media (ear infection)
• Burns if you survive a motorcycle crash, your
  next biggest danger is P. aeruginosa infection
  of your road rash
• Lung infections- most cystic fibrosis patients
  die of P. aeruginosa pneumonia
• Major contaminant of medical devices, especially
  contact lenses and catheters

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How do biofilms form?

• Stages
• Attachment
• Adhesion
• Propagation
• Maturation
• Dispersal

Annu. Rev. Microbiol. 2002. 56187209
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How do biofilms form?

• Attachment
• Planktonic (cells growing in liquid) cells become
  associated with surface
• Cells lose their flagella (if they have them)
• In pathogens and commensal organgisms, this is
  often mediated by host cell receptors
• In environmental biofilms, this process is
  thought to be mediated by the surface tensions of
  the attachment substratum, the bacterial surface
  (or part of the bacterial surface) and the
  surrounding liquid (usually water)
• Depending on the interactions between these
  surface tensions, attachment can be irreversible
  within minutes or it may take hours.

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How do biofilms form?

• Adhesion
• Attached bacteria start making exopolymeric
  substances (EPS)
• EPS consists mostly of water
• Organic compounds, made by the organism include
• Polysaccharide (in P. aerugonisa-the most famous
  type is alginate- which is what clogs the lungs
  of CF patients)
• Glycoproteins
• Nucleic acid
• Functions
• Keeps cells on surface under flow (e.g. blood
  flow, peristalsis in intestines, air flow in
  lungs, water flow in streams and soil
• Prevents desiccation of underlying cells
• In opportunistic pathogens, provides protection
  from white blood cells and antibiotics
• In environmental organisms, can serve as a
  storage source of organic carbon
• In pathogens, is considered a pathogenic
  mechanism
• Can itself cause immune response

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How do biofilms form?

• Propagation- cells accumulate on surface
• Under high nutrient conditions, this can actually
  be growth
• Recruitment to surface from liquid medium by
  soluble factors (quorum sensing-see below)
• Dispersed cells on surface gather by surface
  motility mechanisms which are actually super cool
• Gliding
• Twitching motility (literally they walk on
  pili)
• Rolling motility (held to the surface by EPS,
  they roll along the surface

Annu. Rev. Microbiol. 2002. 56187209
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How do biofilms form?

• Maturation
• Biofilm starts building in Z-direction
• Mutant studies and gene expression studies
  indicate that cells in different part of the
  structure are metabolically and phenotypically
  different- i,.e., there is differentiation
  occuring
• Processes that make this happen-
• Cell surface motility over existing cells (mostly
  twitching type motility)
• Programmed cell death
• Cell division in certain parts of biofilm
• Cells on can be dead , thus protecting
  underlying structures
• Leads to a very tissue- like structure with
  liquid channels

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How do biofilms form?

• An important part of the biofilm life cycle that
  the canonical view leaves out Persistance
• Biofilms can last for years
• In CF patients, 25-30 years
• Some biofilms on rocks at the deep subsurface are
  estimated to be thousands to millions of years
  old
• During this time, biofilm structure can still be
  dynamic.
• This is the point at which biofilms can exchange
  a lot of genetic info!
• This is the least well studied, but probably most
  important part of biofilm development,
  particularly in terms of human disease.

Annu. Rev. Microbiol. 2002. 56187209
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How do biofilms form?

• Dispersal
• Seems to start with a hollowing of the core of
  the biofilm
• This part reminds me of blastocyst formation in
  human development, but I may have a vivid
  imagination
• Cells in the center of the hollow revert to
  planktonic growth phenotype
• Regain flagella (if present)
• Lose surface motility structures
• Reactive oxygen and nitrogen species (especially
  nitrous oxide) may play a role in triggering the
  metabollic conversion
• EPS breaks down in part of the biofilm and
  planktonic cells are released.

Annu. Rev. Microbiol. 2002. 56187209
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So how is this all regulated?

• Two main mechanisms currently recognized
• Quorum sensing- bacterial hormones
• Involvement in every step of biofilm development
• Seems very evolutionarily conserved
• Both big control and fine tuning
• Internal second messengers
• Controls whether cells are planktonic or biofilm
  form
• Similar to cAMP signalling in eukaryotes
• Genetic studies indicate that it has evolutionary
  relationship with cAMP functioning in eukaryotes

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First regulatory pathway discovered quorum
sensing in Vibrio fischeri
Vibrio fischeri on a squid

• Produces bioluminescence but only when
• Attached to surface
• Enough bacteria are there
• Called quorum sensing because the mechanism
  only operates when a threshold level of cells are
  present
• Found to be controlled by acculmulation of an
  acyl-homoserine lactone, 3-oxohexanoyl-homoserine
  lactone

http//keck.bioimaging.wisc.edu/mcfall-lecture.jpg
Lux protein
http//www.pnas.org/content/102/33/11882/F3.large.
jpg
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LuxR operon as a model for quorum sensing

• Acyl homoserine lactones have been found in many
  Gram negative organisms.
• Control secreted compounds (e.g. toxins) and
  biofilm formation.
• All AHL systems found so far have a component
  that evolutionarily related to Lux.

http//gcat.davidson.edu/GcatWiki/index.php/Davids
on/Missouri_Western_iGEM2008
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Generalized AHL pathway
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How does QS regulate biofilm formation? An
example from Pseudomonas aeruginosa PAO1

• Has two known quorum sensing systems
• Las- which is analogous to Lux
• Rhl- controls production of a surfactant called
  rhamnolipid.
• Las and Rhl are currently targets of antibiofilm
  therapy development
• A third AHL has been discovered but its role is
  uncertain
• Las is thought to be the pathway that controls
  biofilm formation while Rhl controls soluble
  secretion
• But these two pathways interact, so it is unclear
  how this can be stated definitively.

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So how do Las and Rhl interact?

• Activating the Las pathway produces AHL
  (3-oxo-C12-HSL)
• AHL triggers more production of AHL
• AHL also actviates Rhl pathway which feeds back
  to the Las pathway
• Notice that in addition to inducing biofilm
  differentiation, Las also releases compounds that
  modify the immune system.
• Rhl induces the production of pyocyanin (a toxin)
  and cyanide.

http//www.cdc.gov/ncidod/eid/vol4no4/vandel4b.gif
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How do quorum sensing molecules control biofilm
development in P. aeruginosa?

• Attachment-
• 3-oxo-12-HSL may recruit more bacteria to the
  surface
• Promotes loss of flagella
• Adhesion
• 3-oxo-12-HSL directly activates alginate
  production pathway
• Maturation
• Production of a 3 dimensional biofilm requires
  periodic release of bacterial DNA to form a
  scaffold for bacterial cells- 3-oxo-12-HSL
  promotes cell death in certain cells
• Rhl pathway produces rhamnolipid, which is
  important in surface motility.
• Persistance presence of 3-oxo-12-HSL helps
  maintain the structure
• A gradient of AHL is found in the biofilm
  structure.
• Reliease- lack of AHL seems to result in release

TRENDS in MicrobiologyVol.13 No.1 January 2005
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Peptide hormones in Gram positive organisms
Agr Accessory gene regulation Agr D is a
prepeptide that is cyclized and transported by
AgrB transmembrane protein to form AIP (auto
inducer protein).
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Quorum sensing is common in prokaryotes

• Present in both eubacteria and archaebacteria
• General wisdom, based mostly on pathogens,
  suggests that quorum sensing in Gram negative
  organisms procedes through acyl homoserine
  lactones and in Gram positive organisms through
  peptides
• It seems though that Actinobacteria use AHLs as
  well
• Recent research shows that quorum sensing can
  also effect eukaryotic hosts
• Legume-nitrogen fixing bacteria
• Maybe even human intestinal flora

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Intercellular regulation cDGMP

• Cyclic di-guanidine monophosphate
• Promoters binding proteins with GGDEF turn on
  cgGMP production
• Promoter sequences are similar to those turning
  on cAMP in eukaryotes
• Promotoers binding EAF turn on stuff that chops
  up cDGMP.
• Activation of GGDEF promoters results in biofilm
  development/maintenance
• Activation of EAF promoters results in release of
  biofilm/production of planktonic cells

NATUREVol 44118 May 2006
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A-factor and antibiotic production in streptomyces

• In Streptopmyces griseus sporulation and
  antibiotic production are controlled by
  A-factor
• Some sporulation and antibiotic mutants can be
  cured by adding A factor into a culture.
• A-factor is a homoserine lactone.
• Other antibiotic producing streptomycetes have
  similar factors.
• The general wisdom is that only G- organisms
  use quorum sensing for metabolic control. I
  would argue that it this might not be true for
  non-pathogens.

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Cellular regulation by acyl homoserine lactones
in Actinomycetes

• Even though Gram positive cells are not supposed
  to be regulated by acyl-homoserine lactones,
  actinomytes are.
• Actinomycetes are antibiotic producers
• Produce about 2/3 of all naturally occuring
  antibiotics.
• Produce more antibiotic when growing in a
  biofilm than in liquid (i.e. planktonic culture)
• Extra step in biofilm development- the formation
  of exospores.

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Streptomyces griseus life cycle

• Streptomycetes are soil organisms
• This entire cycle takes place on surfaces (i.e.
  there is usually not a planktonic state)
• Antibiotic production is concomitant with
  formation of aerial hyphae and sporulation
• Acyl homoserine lactones control both entry into
  secondary growth and antibiotic production
• AHL production was first disovered in Steptomyces
  griseus which makes streptomycin
• Mutants that were unable to make spores or
  streptomycin were restored upon addition of
  extracts or exudate from wild type colonies
• Compound was called A-factor
• Has a Las/Lux sort of control system
• For many years biofilm people and antibiotic
  people did not talk to each other so the
  similarities between biofilm development and
  secondary metabolism in actinomycetes.

http//www.bioscience.org/2002/v7/d/horinouc/fig1.
jpg
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Bacterial toxins in pathogens are, however,
downregulated by compounds involved in biofilm
development!

• I had assumed that since biofilms are considered
  a virulence factor for pathogens, that biofilm
  production and bacterial toxins would be
  co-regulated
• They are 10 points to Ravenclaw!
• Because they are both involved in virulence, I
  thought both biofilm formation and toxin
  production would be upregulated
• They are not- 20 points from Ravenclaw
• Why would this be?
• Pathogens that are making you sick probably want
  to have lots of copies of themselves- so they
  need to dividing rapidly which they dont in
  biofilms
• If bacteria are persisting in an infection (such
  as P. aeruginosa in cystic fibrosis patients)
  they probably dont want to be detected.
  Therefore production of things like toxins are
  not int their best interests.
• Also-toxins are expensive. Where are the cells
  in bacteria, which are not metabolizing rapidly,
  going to get the energy to make them?

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Example 1 Cholera toxin production in V. cholerae

• Near relative of V. fischerii in which quorum
  sensing was first discovered.
• Lux/Las sort of quorum sensing system
• Activation of Lux turns on biofilm formation
• Notice activation of GGDEF promoters!- QS
  interfacing with secondary messengers
• Deactivation of Lux system
• Shuts down biofilm formation
• Turns on virulence genes (HA pathway)

Microbiol Molecular Biol Revi, 2009, 73.
310347.
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Gram positive pathogens

• Quorum sensing in Gram positives is achieved
  mainly through peptide hormones called
  autoinducer proteins
• Interact through the agr (accessory gene
  regulation) pathway
• Some evidence exists that genes for Lux-type
  regulons are also present in many Gram positives,
  but are dormant
• Most of the Gram positive organisms studied are
  pathogens so there may be a bias

AIP production in Staphylococcus aureus. Anal
Bioanal Chem (2007) 387437444
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Staphylococcus aureus

• Once again, activation of quorum sensing
  associated with toxin production (a-hemolysin)
  turns off biofilm production
• Even better, a-hemolysin itself down-regulates
  biofilm formation.

Microbiol Molecular Biol Revi, 2009, 73.
310347.
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So how did regulation evolve?

• Both acyl homoserine lactone-based quorum sensing
  and antibiotic (halocin) production has been
  detected in archaebacteria,
• Halocins are similar to Gram positive peptide
  hormones
• Not much has been discovered about their
  production
• It has been recently shown that antibiotics in
  low concentration can function as signaling
  pathways between cells.
• It has also been shown that some quorum sensing
  molecules have antimicrobial activity.
• Both quorum sensing and bacterial toxins
  (particularly antibiotics) tend to be small
  molecules, as are some toxins such as a-hemolysin
• Computer models show that cooperation by
  cell-to-cell signaling probably evolved early as
  it confers a selective advantage on the
  population
• I suspect that antimicrobial production and
  quorum sensing might have coevolved from ancient
  mechanisms of cell-to-cell communication

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