Biofilms

RhII deficient

Biofilm similar to wild type

LasI deficient

Defective biofilm - LasR / LasI critical in biofilm morphology

Inhibition of quorum sensing-potential clinical applications

If we can block quorum sensing in P. aeruginosa CF biofilms this may provide a novel treatment, Study by Hentzer et al (2003), Produced synthetic furanone compound C30, similar structure to D pulchra, Grew P. aeruginosa biofilms in vitro and added C30, Inhibited production of virulence factors, Increased susceptibility of biofilms to tobramycin, C30 + tobramycin treatment has potent anti-biofilm properties, C30 treatment inhibited quorum sensing

LasI/LasR system

LasR binds ODHL lactone, Transcriptionally activates a number of virulence genes that encode secreted virulence factors - LasB = ELASTASE = Enzyme which destroys elastin connective tissue, LasA = Protease that destroys lung cell proteins and ToxA = Exotoxin = Necrotizing activity., transcription of LasI to produce more lactone to bind to LasR, LasR with lactone can then activate transcription of RhIR gene

Interference of qs

Marine seaweed (algae) Delis pulchra, Bacteria cannot form biofilms on this seaweed, Secretes a number of compounds called halogenated furanones which inhibit qs pathway

3-4 hours

Monolayer of cells covers surface

Quorum sensing

involved in biofilm development

Maturation of P. aeruginosa biofilm

10 days, production of EPS, elevated resistance to antimicrobials and development of complex heterogeneous structures, the morphology of these structures is affected by the flow characteristics of the aqueous environment

infections

Biofilms account for as much as 80% of infections that occur in your body e.g. Chronic lung infections in CF Patients by Pseudomonas aeruginosa biofilms

30 min or less

Cells form monolayer, cell movement continues via twitching motility (pili mediated). Twitching motility is 100 times slower than flagella powered motility

Anti quorum sensing medical treatments

Compounds that can disrupt the bacteria's ability to communicate might disable or diminish the bacteria's ability to become pathogenic. This gives the body time to eradicate the bacteria naturally through normal immune system functions. Since the compounds kill none of the bacteria, any resistant mutations must compete with living, non-resistant individuals.

0-30 minutes

Flagella mediated movement along surface. Reversible and irreversible attachment

S . liquefaciens

In S. liquefaciens swarming motility is controlled by quorum sensing - 2HSL molecules involved, these display some structural similarity with furanones from D pulchra, add these to plates with S . liquefaciens, Furanones inhibit quorum sensing process, Furanones bind to the LuxR type proteins displacing HSL, LuxR/HSL complex activate quorum sensing dependent genes, LuxR/Furanone complex does not activate quorum sensing dependent genes

P. aeruginosa biofilm

P. aeruginosa biofilm development involves a programmed pathway. Under at least some conditions, the last step in the pathway involves the conversion of microcolonies on a surface into a mature biofilm with cells encased in a self-produced 'slime'. Quorum sensing has a role in this step. We do not yet know which quorum-sensing-controlled genes are involved in biofilm development. It is also clear that the requirement for quorum sensing during P. aeruginosa biofilm maturation can vary with different environmental conditions.

Why do bacteria talk to each other?

QS enables bacteria to co-ordinate their behaviour. As environmental conditions often change rapidly, bacteria need to respond quickly in order to survive - adaptation to availability of nutrients, defence against other microorganisms which may compete for the same nutrients and the avoidance of toxic compounds potentially dangerous for the bacteria. Also avoid immune response of the host to establish successful infection

P. aeruginosa intra-cellular signalling pathways

QS positively regulates the expression of the periplasmic tyrosine phosphatase TpbA. TpbA dephosphorylates the membrane-anchored GGDEF protein TpbB deactivating its DGC activity and thus reducing c-di-GMP levels in the cell. As a result, the c-di-GMP receptor PelD is not longer bound to c-di-GMP and PEL polysaccharide production is decreased. QS also promotes the synthesis of rhamnolipids whose overproduction results in biofilm detachment

Quorum sensing

QUORUM SENSING is the ability of bacteria to regulate gene expression in response to fluctuation in cell population density, Process of Intercellular or CELL-CELL communication via extracellular hormone like molecules, cell-density dependent gene expression

Importance of qs

Quorum sensing is critical in the formation of Pseudomonas biofilms, Make mutants cells that lack LasI or Rhll i.e. unable to produce HSL signalling molecules, resulted in flat featureless biofilms, Loss of distinctive micro colonies, Cells more densely packed, Biofilm has 20% thickness of Wild type

RhII/RhIR system

RhIR binds BHL, results in increased expression of certain virulence factors, These include enzymes involved the production of Rhamnolipid which lyses red blood cells, Pyocyanin antibiotic that kills other bacteria allowing Pseudomonas to prosper, also transcriotipn of RhII to produce more lactone to bind to RhIR

Reduction of QS signal production

The RsaL protein represses transcription of the lasI signal synthase gene, and thus reduces QS signal production. RsaL binds simultaneously with LasR to the rsaL-lasI bidirectional promoter thereby preventing the LasR-dependent activation of both genes

stress signal

Upon sensing a stress signal, free-living cells will initiate attachment to a surface, leading to the formation of a biofilm with a greater ability to withstand environmental challenges, Once the mature biofilm has developed, some cells within the population start to dissociate, to disperse, from the sessile structure.

8 hours

Visualisation of microcolonies (clusters of cells). Formed by twitching motility movements of cells towards one another

polysaccharides important for biofilm formation

alginate, cellulose and PsI - produced by pseudomonas

Nature of biofilms

aqueous channels there to carry nutrients to the biofilms, usually develop in response to some environmental stress

Quorum sensing cell-density dependent gene expression

as concentration of cells increases, producing lots of pheromone molecule, lots with receptors, can interact with receptor, tell bacteria to switch on different set of genes, critical stage in population, reached quorum, results in chemical communication between cells

intra-cellular signalling pathways

c-di-GMP signalling molecule involved in attachment of biofilm and inhibits motility, produced by diguanylate cyclase DGC, phosphodiesterases break it down, c-di-GMP controls production of polysaccharides like alginate, pel and psl

Biofilm

describes microorganisms attached irreversibly to a surface, cells are imbedded in a matrix of extracellular polymeric substances (EPS) that they have produced, and exhibit an altered phenotype in respect to growth rate and gene transcription.

tpbA - phosphatase

down-regulates cellular cGMP-levels. Inactivation of of the tyrosine phosphatase tpbA increases biofilm formation.

biofilm formation

environmentally driven developmental process that increases resistance to exogenous stresses, enabling bacterial survival under unfavorable conditions.

Lactones

homoserine lactones - use as chemical messengers, different bacteria produce different homoserine lactones, gram negatives, can diffuse into neighbouring cells and interact with LuxR which is a transcription factor, switch on regulatory genes, LuxR will regulate expression of many genes e.g virulence genes to survive in host, alsocontrol expression of LuxI

Another control of biofilm development

intra-cellular signalling pathways

EPS - extracellular polymeric substances

polysaccharides, proteins and nucleic acids - acts as a barrier preventing antimicrobials gaining access to the cell, Acts as a glue or slime layer entrapping organism in the biofilm matrix

5 steps of biofilm development

surface will become coated with organic molecules, bacteria adhere to surface, begin to spread out using flagella, Twitching motility is a flagella-independent form of bacterial translocation over moist surfaces. It occurs by the extension, tethering, and then retraction of polar type IV pili, The biogenesis and function of type IV pili is controlled by a large number of genes, form a uniform coating, bacteria can start to move towards one another - pilli, clump together to form micro colony, these start to produce extracellular polysaccharide substance that binds them together, develop into biofilm with mushroom shape structures, some cells will be released from biofilm and dispersed back into environment to re start process

QS

therefore important in dispersion of biofilms by down regulation of cGMP

Transition to biofilm

transition from the planktonic state to biofilm growth occurs as a consequence of environmental changes that trigger the dysregulation of multiple regulatory networks, Biofilm development initiates when flagella-propelled planktonic cells receive a stress signal from the environment. This stress signal, possibly combined with surface adherence, initiates the biofilm developmental program leading to increased (adaptive) resistance, enabling cell survival

P. aeruginosa quorum sensing

two HSL signalling molecules - LasI that produces ODHL lactone and RhII that produces BHL lactone, Las I is involved in Las R system and RhII in the RhIR system

Mature biofilms

water channels between microcolonies