1. Biofilms and bacterial toxin production: Coordinated metabolic control Linnea Ista Biology of Toxins Spring 2010

2. What is a biofilm? http://www.bact.wisc.edu/themicrobialworld/Intro.html Collection of microbes, small organics and metabolites growing on a surface Ista, 1999 Appl. Environ, Microbiol. 65:1591

3. 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 ~5x10 36 Most bacteria grow in biofilms. We have 10x more bacterial cells living in and on us than human cells. Deep ocean hydrothermal vent http://eager2009.files.wordpress.com/2009/06/sc hrenkbiofilm.png?w=500&h=373 http://www.uni- due.de/imperia/md/images/biofilm- centre/solfatare.jpg Glacier http://www.morning- earth.org/graphic- E/BIOSPHERE/Bios-Microbe- Image/M-Fileum.jpg Human intestines

4. 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/ http://www.petrifiedseagardens.org/ http://cas.bellarmine.edu/tietjen/Evolution/ Modern stromotalite (Australia) Fossil stromatolites (New York State)

5. 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

6. They were probably the prototype for multicellarity Pseudomonas biofilm development Sea lettuce development

7. Negative Significances of Biofilms - Medical Disease Otitis media Cystic fibrosis Device failure Catheters Heart valves Contact lenses Stents Nosocomial infections Biofilms serve as reservoirs Increase chance of antibiotic resistance

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

9. 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

10. How do biofilms form? Stages: 1.Attachment 2.Adhesion 3.Propagation 4.Maturation 5.Dispersal Annu. Rev. Microbiol. 2002. 56:187–209

11. 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.

12. 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

13. How do biofilms form? Annu. Rev. Microbiol. 2002. 56:187–209 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

14. 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

15. How do biofilms form? Annu. Rev. Microbiol. 2002. 56:187–209 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.

16. How do biofilms form? Annu. Rev. Microbiol. 2002. 56:187–209 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.

17. 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

18. First regulatory pathway discovered: quorum sensing in Vibrio fischeri 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 Vibrio fischeri on a squid http://keck.bioimaging.wisc.edu/mcfall-lecture.jpg http://www.pnas.org/content/102/33/11882/F3.large.jpg Lux protein

19. LuxR operon as a model for quorum sensing http://gcat.davidson.edu/GcatWiki/index.php/Davidson/Missouri_West ern_iGEM2008 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.

20. Generalized AHL pathway

21. 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.

22. So how do Las and Rhl interact? http://www.cdc.gov/ncidod/eid/vol4no4/vandel4b.gif 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.

23. 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

24. 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).

25. 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

26. 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 NATURE|Vol 441|18 May 2006

27. 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.

28. 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.

29. 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

30. 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 don’t in biofilms  If bacteria are persisting in an infection (such as P. aeruginosa in cystic fibrosis patients) they probably don’t 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?

31. 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:. 310–347.

32. 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) 387:437–444

33. Staphylococcus aureus  Once again, activation of quorum sensing associated with toxin production (α- hemolysin) turns off biofilm production  Even better, α-hemolysin itself down-regulates biofilm formation. Microbiol Molecular Biol Revi, 2009, 73:. 310–347.

34. 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 α-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

35. References Brenner K, Karig D, Weiss R, Arnold FH. 2007. Engineered bidirectional communication mediates a consensus in a microbial biofilm consortium. Proc Natl Acad Sci USA 104: 17300–17304. Czárán T, Hoekstra RF. 2009. Microbial communication, cooperation and cheating: quorum sensing drives the evolution of cooperation in bacteria. PLOS One 4:e6655. Davies J. 2009. Everything depends on everything else. 2009. Clinical Microbiol Infect 15:1-4. de Klevit TR. 2009. Quorum sensing in Pseudomonas aeruginosa biofilms. Environ. Microbiol. 11: 279–288. Hall-Stoodley L, Costerton JW, Stoodley P. 2004. Bacterial biofilms: from the natural environment to infectious diseases. Nature Rev Micriobiol 2:95-108 Hammer BK, Bassle B. Regulatory small RNAs circumvent the conventional quorum sensing pathway in pandemic Vibrio cholerae. Proc Natl Acad Sci USA. 104:11145–11149. Hara H, Ohnishi Y, Horinouchi S. 2009. DNA microarray analysis of global gene regulation by A-factor in Streptomyces griseus. Microbiology 155: 2197–2210. Karatan E, Whatnick P. 2009. Signals, regulatory networks, and materials that build and break bacterial biofilms. Microbiol Molec Biol Rev 73: 310–347. Kleerebezem M, Quadri LE. 2001. Peptide pheromone-dependent regulation of antimicrobial peptide production in Gram positive bacteria: a case of multicellular behavior. Peptides 22: 1579-1596. Kolter R, Greenberg EP. 2006. The superficial life of biofilms. Nature 441:300-302. Lim B, Beyhan S, Yildiz FH. 2007. Regulation of Vibrio polysaccharide synthesis and virulence factor production by CDGC, a GGDEF- EAL domain protein in Vibrio cholerae. J Bacteriol. 189:717–729. O'Toole G, Kaplan H. B., Kolter, R. 2000. Biofilm formation as microbial development Annu Rev Microbio. 54:49-79. Paggi RA, Martone CB, Fuqua C, De Castro RE. 2003. Detection of quorum sensing signals in the haloalkaliphilic archaeon Natronococcus occultus. FEMS Microbiol Lett 221:49-52 Ryder C, Byrd M, Wozniak DJ. 2007. Role of polysaccharides in Pseudomonas aeruginosa biofilm development. Curr Opinion Microbiol 10:644–648. Tischler AD, Camilli A. 2004. Cyclic diguanylate (c-di-GMP) regulates Vibrio cholerae biofilm formation. Molec. Microbiol. 53: 857– 869. Torreblanca M, Meseguer I. Ventosa A. 1994. Production of halocin is a practically universal feature of archaeal halophilic rods. Lett Appl Microbiol. 19: 201-205.