Biofilm physiology and quorum sensing Flashcards
Chronic bacterial infections involve…
the biofilm mode of growth: bacteria withstand the action of the immune system and tolerate the highest deliverable doses of antibiotics.
Pseudomonas aeruginosa
16% of nosocomial pneumonia cases
12% of hospital-acquired urinary tract infections
8% of surgical wound infections
10% of bloodstream infections
30% deaths in immunocompromised patients
38% deaths in intubated patients
Associated with 60% of deaths under outbreaks in burn units
Associated with 50% of deaths in the expanding AIDS population
Cystic fibrosis patients are susceptible to chronic infection, which is responsible for high rates of illness and death.
CF is the most frequent severe genetic disease among Caucasians (1:4700).
Chronic wounds (1:100 in Denmark)
With the aggressive antibiotic regime in CF patients
the bacteria are suppressed, however not eradicated in the conductive zone, whereas the remaining respiratory zone is protected from massive biofilm infection for prolonged time.
A minimum of ____ of these problematic wounds are infected with P. aeruginosa.
50%
“Quorum Sensing” (QS)
a density related process uses cell-cell signaling to coordinate expression of virulence
Principle of QS
Bacterial cells increasing in cell density, due to this level of signals increases
Quorum size is reached
The culture makes “a collective decision” and expresses QS controlled target genes
Within the steps of Biofilm formation, when is the C-di-GMP and QS processes identified
1) Reversible attachment, 2) irriverable attachment, 3) cell proliferation - C-di-GMP identified
4) biofilm maturation, 5) dissolution - QS process identified
Explain PMN attraction in biofilm formation
during biofilm maturation, PMN leukocytes have PMN attraction to the biofilm
The biofilm is surrounded by a PMN shield and outside, necrotic PMN and virulence
When the PMN shield is broken, dissolution occurs
Overall PMN attraction and destruction causes…
collateral damage to the tissue. Inflammation develops.
Name some virulence factors involved in pathogenesis, and the genes that cause these to be upregulated
elastase
exotoxins
bio surfactants
caused - PqsR, PqsE, RhIR
How many genes are regulated by QS
200 - 400 depending on the growth conditions
What activity does OdDHL have
Immunomodulatory and vasodilatory activity
Quorum sensing controlled production of rhamnolipid by Pseudomonas aeruginosa leads to what?
Rapid necrotic killing of PMNs
What can be controlled through the QS systems in P. aeruginosa
biofilm development
antibiotic tolerance
virulence
immune shielding
In vitro biofilms seems to produce only minor amounts of what?
rhamnolipids
what are rhamnolipids?
PMN leukocyte shield constituent, able to cause rapid lysis in various eukaryotic cells
What is pyocyanin?
a virulence factor that is known to cause death in C. elegans by oxidative stress
what is PQS?
A branch of the QS, a regulatory system
genes encoding signalling molecules of the PQS are upregulated in transacriptome analysis of P. aeruginosa
LDH assay shows ____ caused by rhamnolipid
PMN leakage
What is Dynorphin A?
- Endogenous κ-agonists belonging to the opioid peptides
- Modulates pain and stress signals
- Found in Central Nervous System
- Contained in various immune cells
- PMNs produce and release dynorphin at sites of inflammation
- Found by others to activate pqs, rhamnolipids and pyocyanine
NAme a dynorphin agonist and what it induces
U-50488 -A drug which acts as a highly selective κ-opioid agonist
induces PQS and rhamnolipids
odDHL + PQS synthesis =
PMN signal
Describe the relationship of las, rhl and pqs
las upregulates pqs which upregulates rhl; pqs links las and rhl systems
pqsA mutant is not able to…
up-regulate rhamnolipid production in response to PMN exposure.
HHQ role in QS system
serves a a substrate for PQS sysnthesis
promotes PpqsA activity via PsqR to increase itrs own synthesis and PqsE expression
HQNO role in QS
Has no effect on P. aeruginosa transcriptome
is a cytochrome inhibator usedin environmental competition
PQS role in QS system
Chelates iron
increases expression of virulence determinants
has negative impact on dentrification
promotes PpqsA activity via both Pqs-R dependant and independant pathways to increase its own synthesis and PqsE expression
Contributions of Specific 2-Alkyl-4-Quinolones and PqsE to Quorum Sensing
the PQS biosynthetic precursor 2-heptyl-4-hydroxyquinoline (HHQ) triggers a PqsR-dependent positive feedback loop that leads to the increased expression of only the pqsABCDE operon
PqsE is involved in the regulation of diverse genes coding for keyvirulence determinants and biofilm development
PQS promotes2-alkyl-4-quinolones (Aqs) biosynthesis, the expression of genes involved in theiron-starvation response and **virulence factor production via PqsR-dependent and PqsR-independent** pathways
What is 2-heptyl-3-hydroxy-4-quinolone
PQS
does 2-heptyl-4-hydroxyquinoline N-oxide (HQNO) function as a QS signalling molecule
No - it doesnt in fluence transcription
Ability of PQS to contribute to gene regulation independent of
both its ability to activate PqsR and to induce the iron-starvation response
Sputum of CF patients contains what?
rhamnolipids
What are PMN leukocytes, how do Pseudomonas aeruginosa recognizes and respond to these
polymorphonuclear leukocytes
Pseudomonas aeruginosa recognizes and responds aggressively to their presence
response does not involve protective mechanisms, rather it is dominated by an up-regulation of virulence determinants all of which are able to cripple and eliminate eukaryotic cells.
The response is dependent on functional QS systems
QS inhibitor can..
Restore PMN function
Describe the shield model
- Bacteria emerge in a harmless state (no expression of virulence factors)
- Subsequently they increase in numbers and build biofilms.
- When quorum size has been attained, the communication system signals and the biofilm microcolonies launch a cocktail of virulence factors including a rhamnolipid based shield which obstructs the function of cellular components of the innate immune system (the PMN Leukocytes)
- These are killed but bacteria persist.
What is biofouling?
a major challenge for marine eukaryotes and bacteria can be highly detrimental to marine algae and other eukaryotes
Delisea pulchra is rarely fouled in nature due to the production of secondary metabolites (halogenated furanone compounds).
These compounds have strong biological activity, including anti-QS and antimicrobial properties
Secondary metabolites such as the _____ are produces and liberated from specialized vehicles present in the algae Delisea pulchra
halogenated furanones
Name three compounds that block QS in P. aeruginosa
Furanone C-30
Furanone C-56
OdDHL
Furanone 30 promotes…
Bacterial clearance
QS in other Gram-ve bacteria:
CF patient Burkholderia cenocepacia: 4 QS systems
Synthase (I) and a receptor (R): CepIR; CciIR systems
Burkholderia Diffusible Signal Factor (BDSF)-based system RpfFBC,
non-ribosomal peptide synthetase-like cluster ham
CepI involved in biofilm formation, protease production, and virulence, as well as an interplay among the Acyl Homoserine Lactone (AHL) systems CepIR and CciIR and the BDSF-based system
diketopiperazines (DKP) inhibit CepI in vitro, impairing the ability of B. cenocepacia to produce proteases, siderophores, and to form biofilm
What does DSF stand for and features
Diffusable signal factors
DSF is a cis-unsaturated fatty acid first described in plant pathogen Xanthomonas campestris. cis-11-methyl-dodecenoic acid
Produced by bacteria Burkholderia cenocepacia (BDSF), P. aeruginosa (CDA): regulate virulence, biofilm formation, antibiotic tolerance
Autoinducer of biofilm dispersion of P. aeruginosa and other species
Involved in interspecies signalling that modulates bacterial behaviour
Removal of established P. aeruginosa biofilms can be done using what?
cis-decenoic acid (CDA) combined by antimicrobial treatments
DSF synthesis involved what?
RpfF: an enoyl coenzyme A (CoA) hydratase and
RpfB: a long-chain fatty acyl CoA ligase
Different sensor kinases involved in DSF or BDSF perception in diverse bacteria
two-component signal transduction system:
RpfC- sensor kinase
RpfG - response regulator
Signals of the DSF family play a role in..
interspecies and inter-kingdom signalling.
Bacteria need to exit the highly energy-demanding QS maximal activation phase during _____
the post-quorum phase
QS signal turnover systems are…
one of the QS exit mechanisms most frequently identified in bacteria
Strains belonging to _______ can rapidly degrade DSF
Bacillus, Paenibacillus, Microbacterium, Staphylococcus and Pseudomonas
Use _____ to turnover DSF-type QS signals
RpfB homologs (fatty acyl CoA ligase)
Diketopiperazine inhibitors
Inhibit CepI synthase
Cyclic dipeptides also called 2,5-diketopiperazines, are the smallest cyclic peptides frequently found in nature
Secondary metabolites produced by bacteria (90%); fungi, plants and animals
cyclic organic compounds in which the two nitrogen atoms of a piperazine 6-membered ring form amide linkages.
dipeptidyl peptidases, cleave terminal ends of proteins to generate dipeptides, naturally cyclize to form CDPs.
Gram Positive QS: Peptides
Gram positive QS is exciting because most are pathogens
The QS system is encoded by the global regulatory locus Agr (accessory gene regulator)
The Agr locus encodes a two-component signalling pathway, consisting of two divergent operons – controlled by the promoters P2 and P3
Operon P2 encodes 4 genes; AgrA, AgrB, AgrC and AgrD
AgrD encodes
a precursor of the AIP
AgrB encodes
a transmembrane protein responsible for processing and secretion of the AIP.
AgrC and AgrA…
form structures homologous to a classical two component signal transduction system
AgrC is a…
membrane sensor, its N-terminal region is the sensor domain containing a binding site for AIP
AgrA the response regulator is phosphorylated by ____ causing up regulation of the _____
AgrC
P2 and P3 promoters (auto induction)
Promoter P3 regulates
the transcription of RNAIII and δ hemolysin
An increase in RNAIII levels leads directly or indirectly to…
…a rise in numerous factors and induces the expression of the P2 promoter
AIP Structure
AIP’s have between 7 and 9 amino acids, they all share a common central cysteine located 5 amino acids from the C terminal.
The C terminal amino acid forms a catalytic thioester bond with the sulfhydryl group of the conserved cysteine (macrocycle)
2 to 4 (depending on species) amino acids typically form the tail (exocycle) of the peptide.
AIP’s are highly polymorphic and fall into 4 major groups depending on their activation with AgrC.
QS principles
Cell-to-cell signalling systems composed of 2 genes,
I gene encodes an autoinducer synthase and ER gene encodes a transcriptional activator protein (R-protein)
AI synthase = responsible for synthesis of AI molecule, which crosses the cell membrane
With inceasing cell density intracellular AI conc reaches threshold and AI binds to transcriptional activator
The complex R-protein/AI activates the expression of spoecific target genes
What is the master QS regulator?
LasR
What 2 systems do you need foe the rhll system to be activated
The Las and the Pqs system
What else is the Pqs system involved in?
Iron metabolism
