Biofilm and Quarum Sensing

1. Biofilm and Quarum Sensing M. Sc (P) Biotechnology

2. Biofilms are organised microbial systems consisting of cells associated with surfaces • - Likely the most wide-spread mode of growth for bacteria in nature Biofilms

3. Anton van Leeuwenhook used a primitive microscope to look at biofilms in 1684.

4. Biofilms In the natural world most bacteria aggregate as biofilms - they form when bacteria adhere to surfaces in aqueous environments and begin to excrete a polysaccharide that can anchor them to all kinds of material. The biofilm is held together and protected by the polysaccharide matrix. This matrix protects the cells within it and facilitates communication among them through biochemical signals. Bacteria living in a biofilm usually have significantly different properties from free-floating bacteria of the same species, as the dense and protected environment of the film allows them to cooperate and interact in various ways.

5. Biofilms • A biofilm can be formed by a single bacterial species, but more often biofilms consist of many species of bacteria, as well as fungi, algae, protozoa, debris and corrosion products. • Once anchored to a surface, biofilm microorganisms carry out a variety of detrimental or beneficial reactions (by human standards), depending on the surrounding environmental conditions.

6. Advantages for Bacteria ♦ Creation of habitable niches ♦ Protection against: - Physical forces (e.g. in flowing systems) - Phagocytosis by immune cells - Grazers (e.g. ciliates, amoeba) - Viruses ♦ Barrier against toxic substances ♦ Facilitates intercellular communication ♦ Close proximity of cells enables genetic exchange -

7. Disadvantages for mankind: ♦ Immune system can not attack biofilms ♦ Antibiotics/antimicrobial agents fail ♦ Slow the flow of liquids or clog pipelines ♦ Accelerate corrosion of pipelines ♦ Risk for drinking water supply via pipes

8. Effects of Biofilms • Microbial biofilms on surfaces result in billions of dollars in losses yearly due to equipment damage, product contamination, energy losses and medical infections. • Conventional methods of killing bacteria (such as antibiotics, and disinfection) are often ineffective with biofilm bacteria. • The huge doses of antimicrobials required to rid systems of biofilm bacteria are environmentally undesirable  and medically impractical. • Conversely, microbial processes at surfaces also offer opportunities for positive industrial and environmental effects, such as bioremediating hazardous waste sites, biofiltering industrial water, and forming biobarriers to protect soil and groundwater from contamination. 

9. Pseudomonas strain S61 biofilms on glass slides Staining technique (Congo red) in which the bacterial cells stain dark red and the exopolysaccharide stains orange-pink

10. Biofilms grown in soil 14 day old biofilm 7 day old biofilm

11. Structure of Biofilms • Although bacteria can grow in a free-living or “planktonic state” it is common for them to adhere to surface by producing extracellular polysaccharides. • The adherent bacteria produce microcolonies leading to an intricate three-dimensional structure. • Biofilms survive so well because they have channels, like aqueducts, that transport water, oxygen, and nutrients to all the bacteria of the community. • These channels also get rid of the bacterial wastes, making these biofilms seem almost as complex as a city. • The complexity goes so deep that even the different regions of the biofilm have bacterial cells with different genetic information, physical characteristics, and “duties” for the community.

12. Close-up of a microcolony

13. Stages in biofilm formation • Attachment of bacteria • Irreversible binding by bacteria • Formation of microcolonies • Maturation of microcolonies • Dispersal

14. biofilm attachment Maturation

16. Biofilm-forming Bacteria

17. Campylobacter jejuni • Gram-negative bacteria • genus was first discovered in the 1970’s • most common cause of gastroenteritis • Microbial coloniser of surface waters • Incidence in the U.S. is estimated at 30 to 60% per 100,000 of the population.

18. Legionellapneumophila • Gram-negative bacteria • Causes legionellosis, commonly known as Legionnaire’s disease • Transmitted to host via aerosolisation and ingestion. • Found in domestic households and large municipal buildings: plumbing, air conditioning systems, etc. • Occurs in biofilms where symbiotic relationships with other heterotrophs are evident

19. Salmonella enteritidis • Gram-negative bacteria • Anaerobic • Present predominantly in raw water and occssionally in potable water. • Chlorine is effective in destroying this organism in the planktonic state. • Forms dense, metabolically active biofilms. • Often develop on stainless steel.

20. Biofilm Interactions • Quorum sensing • Interspecies interactions • Symbiosis • Population relationships • Spatial • Temporal • Metabolic • Genetic

21. Warning: Biofilms present! • Biofilms may be 50 to 500 times more resistant to chemotherapy than planktonic bacteria of the same strain. • Cause cosmetic degradation in toilet bowls • Are the cause of flawed prints and malfunctioning machines during photo processing • Infect implanted devices such as: contact lens, catheters, prosthetic heart valves, and cardiac pacemakers. • Cause such chronic infections as:cystic fibrosis, pneumonia, biliary tract infections, osteomyelitis, and bacterial prostatitis. • They are also the cause of dental plaque!!!

22. Biofilms to the Rescue! • Play an essential role in the processing of sewage water prior to its discharge into rivers • Bioremediation • Clean up groundwater • Oil recovery • Mine remediation

23. How do biofilms form? • The formation of a biofilm requires coordinated chemical signalling between cells. • Unless an adequate number of neighbouring cells are present, the costs of biofilm production to an individual bacterium outweigh the benefits. • A signalling process benefits the bacteria by allowing them to sense the presence of neighbouring bacteria and respond to varying conditions. • The process by which a bacterium does this is called quorum sensing.

24. Quorum sensing Biofilm formation involves more than just bacteria attaching to a solid surface; individual organisms aggregate with their kin and often congregate with members of other species. Bacteria accomplish this through chemical signaling mechanisms. When the local extracellular concentration of the chemical signal reaches a threshold level, indicating that the population of microbes has reached a minimum density—a quorum—the community of organisms undergoes phenotypic changes. The process of chemically sensing the population density is called quorum sensing.

25. Signal molecules produced by individual cells do not have an effect until the bacterial population density is sufficient to provide a concentration of molecules, which then cross cell membranes and activate the manufacture of such cellular products as toxins, enzymes or surfactants.

26. Quorum sensing • The term 'Quorum Sensing' (QS) is used to describe the phenomenon whereby the accumulation of signalling molecules enable a single cell to sense the number of bacteria (cell density). • In the natural environment, there are many different bacteria living together which use various classes of signalling molecules.

27. Introduction • Quorum sensing is cell to cell signaling mechanism that enables the bacteria to collectively control gene expression. • This type of bacterial communication is achieved only at higher cell densities. • Bacteria release various types of molecules called as autoinducers in the extracellular medium, these molecules are mediators of quorum sensing. • When concentration of these signaling molecules exceed a particular threshold value, these molecules are internalized in the cell and activate particular set of genes in all bacterial population, such as genes responsible for virulence, competence, stationary phase etc .

28. Cell density and quorum sensing R gene I gene R protein I protein AHL diffuse in Cell density R gene I gene R protein I protein + AHL diffuse out AHL diffuse out Time

29. Quorum sensing controlled processes It occurs in various marine bacteria such as Vibrio harveyi and Vibrio fischeri. Takes place at high cell density. • Bioluminescence • Biofilm formation • Virulence gene expression • Sporulation • Competence It iscompact mass of differentiated microbial cells, enclosed in a matrix of polysaccharides. Biofilm resident bacteria are antibiotic resistant. Quorum sensing is responsible for development of thick layered biofilm. QS upregulates virulence gene expression • Virulence gene expression QS upregulates spore-forming genes in Bacillus subtilis It is ability to take up exogenous DNA QS Increase competence in Bacillus subtilis

30. Quorum sensing molecules Three types of molecules : 1: Acyl-homoserine lactones (AHLs) 2: Autoinducer peptides (AIPs) 3: Autoinducer-2 (AI-2)

31. Acyl-homoserine lactones (AHLs) • Mediate quorum sensing in Gram-negative bacteria. • Mediate exclusively intracellular communication. • These are of several types depending on their length of acyl side chain. • Able to diffuse through membrane. • These are synthesized by an autoinducer synthase LuxI and recognized by a autoinducer receptor/DNA binding transcriptional activator protein LuxR. AHL core molecule

32. Acyl-homoserine lactones (AHLs) cont….AHL mediated quorum sensing cycle LuxI AI AI LuxR + RNA polymerase Transcription promoter target genes

33. Autoinducer peptides • These are small peptides, regulate gene expression in Gram-positive bacteria such as Bacillus subtilis, Staphylococcus aureuas etc. • Recognized by membrane bound histidine kinase as receptor. • Regulates competence and sporulating gene expressions.

34. Autoinducer peptides cont…AIPs signaling mechanism in Bacillus subtilis In Bacillus subtilis QS is mediated by two AIPs : 1: ComX: involve in competence development 2: CSF (competence and sporulation factor): regulates spore formation Christopher et al.,2005 Figure: ComX and CSF pathway in Bacillus subtilis

35. Autoinducer-2 (AI-2) • Involve in interspecies communication among bacteria. • Present in both Gram (+) and Gram (-) bacteria. • Chemically these are furanosylborate diester. S-ribosyl-homocysteine (SRH) LuxS 4,5-dihydroxyl-2,3 pentanedione (DPD) Cyclization Autoinducer-2 (AI-2)

36. Autoinducer-2 (AI-2) cont…AI-2 controlled processes • Induces mini cell formation • Induces expression of stationary phase genes • Inhibition of initiation of DNA replication Figure: AI-2 signaling in E. coli

37. Quorum sensing in bacterial pathogenesis • QS is involved in expression of virulence genes in various bacteria, indicating the possible role of quorum sensing as a drug target. • Several QS system mutant bacteria show the heavily reduced pathogenicity. • Pseudomonas aeruginosa mutant in synthesis of autoinducer molecules shows heavy reduction in pathogenesis.

38. Quorum sensing in bacterial pathogenesis cont…Quorum sensing in P. aeruginosa • In P. aeruginosa QS molecules are synthesized by two autoinducer synthase; LasI and RhlI LasI 3-O-C12 -HSL (AI) AI LasR + Transcription RNA polymerase promoter target virulence genes AI RhIR + RNA polymerase C4-HSL(AI) RhlI

39. Quorum sensing in P. aeruginosa cont.. • In an in-vivo study, using two strains P. aeruginosa;PAO1 (virulent), and PAOR (lasI and rhII double mutant, avirulent), it was seen that rats infected with PAOR are much immunologically active and number of P. aeruginosa also reduced. POA1 POAR Wu et al., 2001

40. Quorum Sensing AIs are sensed by two major mechanisms: 1. AI diffuses into cytosol and is bound by a cytosolic regulatory proteins (eg. LuxR); once bound the regulator changes conformation and either activates or represses genes (eg., Vibrio fischeri) 2. Sensor kinase on cytoplasmic membrane senses AI and transmits the signal to a response regulator through a phosphorylation cascade (Two-component signal transduction system) (eg., Vibrio harveyi)

41. Quorum Sensing Mechanisms • Autoinducer diffusion and binding to LuxR • homolog directly

42. Quorum sensing in bioluminescent bacteria

43. Bioluminescence: first quorum sensing system discovered light Vibrio fischeri lux operon Luciferase Divergent transcription Homoserine lactone (Autoinducer) Provided by J. Foster

45. C4 RhlI C12 LasI http://www.apsnet.org/education/AdvancedPlantPath/ LabExercises/BacteriaSignaling/Images/fig1.gif

46. Quorum Sensing Mechanisms • Autoinducer sensed by sensor kinase and signal • relayed to response regulator by phosphorylation • (two-component system)

47. Two-component signal transduction systems can activate or repress gene transcription

48. Two-component systems Transmitter Sensor kinases often exist as dimers and are often also phosphatases

49. No recognition of AI in cytoplasm (phosp form represses) From B. Bassler

50. P LuxO Low Cell Density LuxQ LuxN LuxU P P σ54 sRNAs/Hfq LuxR No Light Production Provided by J. Nordstrom