Quorum sensing Flashcards
1
Q
what are the two genes of quorum sensing
A
I gene encodes an autoinducer synthase and the R gene encodes a transcriptional activator protein (R-protein).
2
Q
how do the autoinducer synthase and R protein work together
A
- Autoinducer synthase is responsible for the synthesis of an autoinducer molecule (AI), which crosses the cell membrane.
- With increasing cell-density the intracellular concentration of AI reaches a threshold level, and the AI then binds to the transcriptional activator.
- The complex R-protein/AI activates the expression of specific target genes.
3
Q
what happens to polymorphous leukocytes when they approach the biofilm boundary?
A
Initially when polymorphos (leukocytes) come in they mop up the free living bacteria but become passive even necrotic on the boundary of the colony (where the shield is being established)
PMN attaction and destrucction causes collateral damage to the tissue and inflammation develops
4
Q
what steps of biofilm formation are under cyclic-di-GMP control
A
Reversible attachment, irreversible attachment, cell proliferation
5
Q
what steps in biofilm formation are under control of QS factors?
A
Biofilm maturation, dissolution
6
Q
What are the steps in biofilm formation?
A
- Reversible attachment
- Irreversible attachment
- Cell proliferation
- Biofilm maturation
- Dissolution
7
Q
what happens when polymorphonuclear leukocytes approach a biofilm
A
THey are able to mop up the free living bacteria that having joined the biofilm yet
At the boundary of the biofilm there is a rhamnolipid sheild that causes them to become necrotic. this cause collateral damage to the tissue as inflammation develops
the exacerbates wounds and infections
8
Q
what are three major quorum sensing systems in Pseudomonas Aeruginosa
A
Las
RHL
PQS
9
Q
describe the las operon produces QS molecule
A
LasR = regulator and LasI = synthase
LasI codes for synthesis of OdDHL (C12 homoserine lactone)
At low concentration OdDHL doesnt do much, when cell density increase, OdDHL conc increases which feeds back to the cell to upregulate the LasR regulator therefore producing more OdDHL
LasR also induces RHL system to produce BHL
10
Q
A
11
Q
what is OdDHL?
A
N-(3-oxododecanoyl)-L-homoserine lactone
A Qs molecule produced by the Las operon in Pseudomonas aeruginosa
12
Q
A
13
Q
What is BHL?
A
N-butyoyl-1-homoserine lactone (C4)
A QS molecule produced by the RHL operon in pseudomonas aeruginosa
14
Q
hows does the RHL system produce Qs molecule?
A
rhlR regulator and rhlI synthase
rhlI produce BHL (c4)
BHL increases in concentration and positively upregulates rhlR in an induction loop
15
Q
what virulence factors does OdDHL upregulate
A
elastase, LasA protease, alkaline protease, exotoxin A, protein secretion
16
Q
what virulence factors does BHL upregulate
A
elastase
alkaline production
chitinase
lipase
rhamnolipids
cyanide
pyocyanin
rpoS
pilin export and adhesion
17
Q
A
18
Q
how does pseudomnas immunomodulation effect t helper cells
A
the virulence factor upregulated by the QS molecules disrupt the baclance of Th1 and Th2 cells
tipping the balance from th1 to th2 causes and imbalance that results in an inflammatory response
19
Q
what major factors are controlled by the QS systems in P.aeruginosa
4
A
biofilm development, antibiotic tolerance, virulence and immune shielding
20
Q
what molecule is responsible for the PMN leukocyte sheild
A
rhamnolipid
21
Q
what is one (immune related) caveat of laboratory biofilm research
A
In vitro biofilms seems to produce only minor amounts of rhamnolipids
(bcos PMN leukocytes arent present, rhamnolipid production seen when they are added)
22
Q
what do you see upregulation of when you had PMN leukocytes to P.aeruginosa biofilms grown in flow cells
A
You see profound upregulation of the PQS system, pyocynin and rhamnolipid.
23
Q
how do we measure the physiological status of PMN leukocytes at the sheilf
A
Measure lactate dehydrogenase activity showing potential leakage from polymorphs
24
Q
what do rhamnolipids do to pmn leukocytes at the sheild
A
cause necrosis
25
what causes rhamnolipid production?
PMNs produce and release dynorphin at sites of inflammation
Dynorphin A induce PQS and rhamnolipids
26
what is dynorphin A
## Footnote
5
* Dynorphin A induces PQS and rhamnolipids
* Endogenous κ-agonists belonging to the opioid peptides
* Modulates pain and stress signals in the body
* Found in Central Nervous System
* Contained in various immune cells, including polymorphs
27
describe the molecule cascade that leads to pmn necrosis
Biofilm produces virulence factors that cause inflammation. Polymorphs are attracted and produce and secrete this molecule dynorphin A which causes pseudomonas puts up the shield (produces rhamnolipids) in response to this subsequently causing lysis of pmns
28
how does pqs link the las and rhl systems
las upregulates pqs which upregulates rhl; pqs links las and rhl systems
29
which qs system is responsible for the rhamnolipid
pqs
30
how is PQS synthesised
PQs is synthesized through the PQS operon, A B C D E
PQS A – autoinducer synthase, leads to the production of HHQ a precursor of PQS.
PQS positively upregulates either directly PQS A or indirectly though another molecule PQS R which also positively upregulates the PQS A system
31
what is PQS
2-heptyl-3-hydroxy-4-quinolone
the 2-heptyl-3-hydroxy-4-quinolone (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,
PQS promotes 2-alkyl-4-quinolones (Aqs) biosynthesis, the expression of genes involved in the iron-starvation response and virulence factor production via PqsR-dependent and PqsR-independent pathways,
32
what is the precursor molecule to PQS
2-heptyl-4-hydroxyquinoline (HHQ)
33
what does HHQ do
| in the PQS pathway
triggers a PqsR-dependent positive feedback loop that leads to the increased expression of only the pqsABCDE operon,
## Footnote
serves as substrate for PQS synthesis
promotes PpqsA activity via PqsR to increase its own synethsis and PqsE expression
34
what does PqsE do
**represses PqsA activity**
PQS E negatively regulates PQS A if too much produced
**increases expression of virulence determinants**
involved in the regulation of diverse genes coding for key virulence determinants and biofilm development,
35
how can PMN function be restored?
By a QS inhibitor
Small molecule signal blockers are introduced which do not kill bacteria but block their QS system. The dynorphin signal is not perceived. The shield does not form and the immune cells can actively break down the bacterial biofilm
36
what is quorum quenching?
the inhibition of QS using chemical or enzymatic means to counteract behaviors regulated by QS.
37
give an example of quroum quencing
Bacillus produce lactonase that cleaves the lactone ring so you get a diol that can no longer function as a QS molecule
## Footnote
also
Acylase and Oxireductase
38
what is biofouling
the accumulation of microorganisms, plants, algae, or small animals on wet surfaces that have a mechanical function, causing structural or other functional deficiencies
39
why is Delisea pulchra rarely fouled?
* 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
40
give two synthetic analogs of halogenated furanones
C30 and C56
41
what happens when you add furanone to mouse lung infected with p.aeruginosa
Add low furanone treatment not much effect, as increase furanone conc you see a dramatic decrease in CFUs
The furanone is inhibiting the pseudomnas aeruginosa QS defense mechanism so now the the PMN are now able to clear the infection
42
give three examples of bacterial species that dont respond to homoserine lactones (AI1)
E.coli
H.pylori
Salmonella
43
how is autoinducer 2 produced
methyltransferases act upon S-adenosylmethionine to make S-adenosylhomocysteine
the adenine is removed to make S-ribosylhomocysteine
LuxS removes the homocysteine catalysing the production of DPD, 4,5-dihydroxy-2,3-pentanadione
this acts a precursor which can cyclise into a moelcule called proautoinducer 2, which turns into autoinducer 2 when in complex with LuxP, some bacteria require borate also at this step
44
why called lux genes?
when studied in vibrio, large colonies luminesce
45
how is the activated methyl cycle (AMC) involved in AI2 production
The AMC is responsible for the generation of the major methyl donor in the cell, S-adenosyl-L-methionine (SAM), and recycling of methionine by detoxification of S-adenosyl-L-homocysteine (SAH).
46
how does the AI2 production process in Vibrioanceae differ from that of other species
DPD undergoes further reactions to form distinct biologically active signal molecules generically termed AI-2. (2S,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuryl borate (S-THMF-borate), the AI-2 signal of Vibrionaceae, is produced without the help on any known enzyme in the presence of boric acid, whereas in other bacteria (e.g., S. Typhimurium) DPD rearranges spontaneously to form (2R,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran (R-THMF) as AI-2 signal
## Footnote
the virbio uses borate to cyclise the diol (THMF borate) whereas in the other you dont need the borate
47
why does vibrio use borate to make AI2
because it has evolved this way as it is foudn in the ocean where the borate concentration is about 4mM
48
how is the AI2 signal transduced in enterobacteriaceae
Enterobacteriaceae, the AI-2 signal R-THMF is imported by the means of the Lsr ABC transporter (using atp to facilitate uptake) in the cytoplasm of the cell, where is phosphorylated by LsrK. AI-2-P binds the repressor LsrR which is then released from the lsr promoter to allow the expression of the autoinducer operon.
49
50
how does vibrio transduce the AI2 signal
In the presence of S-THMF-borate, AI- 2 receptor LuxP converts LuxQ from kinase to phosphatase, reversing flow of phosphate through the pathway and hence allowing expression of lux operon.
S-THMF-borate binds LuxP causing phosphorylation of LuxQ, then LuxU, then LuxO causing expression of LuxR
51
what does LuxQ do without AI2 present
The histidine/aspartate (H/D) hybrid kinase LuxQ autophosporylates in the absence of autoinducers in Vibrionaceae. The phosporylation signal is transmitted to the histidine phosphorelay LuxU that conveys it to the σ54-dependent response regulator LuxO, which activates the expression of small regulatory RNAs (sRNAs). The complexes of these sRNAs and chaperone protein Hfq destabilizes the mRNA of master regulator LuxR, thereby repressing the transcription of the lux operon.
52
why is Ruminococcus obeum is considered a probiotic?
in terms of virbio cholera
Ruminococcus obeum restricts Vibrio cholerae gut colonisation by downregulating autoinducer2 therefore downregulating virulence genes helping to resist the cholera infection
53
what are the four QS systems of Burkholderia cenocepacia
* Synthase (I) and a receptor (R): CepIR; CciIR systems - 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
* Burkholderia Diffusible Signal Factor (BDSF)-based system RpfFBC
* Non-ribosomal peptide synthetase-like cluster ham
* diketopiperazines (DKP) inhibit CepI in vitro, impairing the ability of B. cenocepacia to produce proteases, siderophores, and to form biofilm
54
what is DSF
Diffusible 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
55
what diffusible signal factor is in xanthomonas campestris?
DSF
11-me-c12
56
what diffusible signal factor is in burkholderia cenocapecia
BDSF
57
what diffusible signal factor is in pseudomonas aeruginosa
Cis decenoic acid (CDA)
58
what diffusible signal factor is in streptomonas mutans
SDSF
## Footnote
trans decenoic acid
59
60
what happens to pseudomnas aeruginosa biofilms when you apply CDA along with tobramycin
removes the biofilm
61
what molecules does DSF synthesis involve?
DSF synthesis involves RpfF, an enoyl coenzyme A (CoA) hydratase and RpfB, a long-chain fatty acyl CoA ligase
62
what initial molecule is required for the biosynthesis of DSF
leucine
63
what initial molecule is required for the biosynthesis of BDSF
carbohydrates
64
what initial molecule is required for the biosynthesis of IDSF
isoleucine
65
how many sensory domains are in DSF receptor, RpfC
5
66
how many sensory domains are in the bdsf receptor
2
67
P.aeruginosa responds to what diffusible signal factors?
its own DSF, CDA
and also the DSF from x.campestris
68
what happens when you phosphorylate RpfG
you switch on the phosphodiesterase activity and block cycic di gmp accumulation and biofilm formation – one way DSF can effect how biofilms are released (dispersal)
69
how does DSF signalling control motility
promotes RpfG binding to two proteins with a GGDEF diguanylate cyclase domain.
70
how does DSF signalling lead to inhibition of biofilm formation
Perception of DSF leads to autophosphorylation and phosphotransfer to the REC domain of RpfG (shown by the red arrow). This leads to activation of RpfG as a cyclic di-GMP phosphodiesterase, an activity associated with the HD-GYP domain. The consequent reduction of cyclic di-GMP levels promotes synthesis of extracellular enzymes and EPS but leads to inhibition of biofilm formation.
71
how do Stenotrophomonas maltophilia and burkholderia cenacepacia influence beahviour of p.aeruginosa and candida albicans
via DSF
72
how is RpfB-dependent DSF signal turnover in X.campetris regulated?
* Pre QS, DSF sensor RpfC forms a complex with DSF synthase RpfF, limits DSF biosynthesis at a basal level.
High c-di-GMP binds to transcription factor Clp. Clp complex binds to rpfB promoter to inhibit its transcription. Bound Clp fails to bind to promoter region virulence genes engXCA.
* QS phase, RpfC autophosphorylates upon sensing high levels of extracellular DSF signals. Through the conserved phosphorelay mechanism, RpfG is then phosphorylated, leading to activation of its c-di-GMP phosphodiesterase activity.
Clp is freed from c-di-GMP and binds to promoter of virulence genes engXCA, initiate transcription. Clp is also released from rpfB promoter enabling its transcription.
* Post-QS, extracellular DSF drop and dephosphorylated RpfC and RpfF reform a complex.
Dephosphorylation of RpfG leads to inactivation of its c-di-GMP phosphodiesterase activity. The intracellular levels of c-di-GMP return to a high level, enabling c-di-GMP-bound Clp to bind to the promoter region of rpfB therefore repressing the transcription of this gene.
73
what are Diketopiperazine inhibitors?
(3S)-3-Benzyl-6-(3,6-dioxocyclohexa-1,4-dien-1-yl)piperazine-2,5-dione (8b) **CepI synthase inhibitor**
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.
74
what is gram positive bacteria QS system controlled by ?
* 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
75
what does AgrD encode?
encodes a precursor of AIP
76
what does AgrB encode?
a transmembrane protein responsible for processing and secretion of the AIP.
77
what is AgrC?
a membrane sensor, its N-terminal region is the sensor domain containing a binding site for AIP
78
what is AgrA
the response regulator is phosphorylated by AgrC causing up regulation of the P2 and P3 promoters (auto induction)
79
how do AgrA and AgrC interact
* AgrC and AgrA form structures homologous to a classical two component signal transduction system.
80
how is promoter P3 involved in gram positive QS
* 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
81
what is the structure of AIPs?
* 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.
## Footnote
* AIP’s are highly polymorphic and fall into 4 major groups depending on their activation with AgrC.
82
What are autoinducer 3 moeclules?
Ecoli respond to adrenaline and noradrenaline. Receptor QseC triggers phosphorelay, regulatign trascnription of virulence genes
83
how do AI1 inhibitors work?
Inhibitors of acyl homoserine lactone (AHL) and Pseudomonas quinolone signal (PQS) bacterial cytoplasmic receptors (LasR, RhlR and PqsR) in Pseudomonas aeruginosa. The inhibitors are thought to bind to these receptors and prevent recognition of the bacterial signal that would activate virulence gene expression.
84
how do inhibitors of the AGR system work
Autoinducing peptide (AIP) mimics bind to AgrC, preventing recognition of the S. aureus AIP. This inhibits AgrC autophosphorylation and the subsequent transfer of this phosphate group to the AgrA transcription factor, which would otherwise activate a multitude of virulence genes. Therefore, blocking AgrC by AIP inhibitors will prevent activation of virulence signalling.
85
how do QseC inhibitors work?
## Footnote
give an example
LED209 binds to QseC, preventing it from binding to its cognate signals to activate the expression of virulence genes.
86
how is the AI3 signal transducedin ecoli
QseC senses the bacterial autoinducer 3 (AI-3) signal and the host adrenaline (ADR) and noradrenaline (NA) hormones. Upon sensing these signals, QseC increases its autophosphorylation and transfers this phosphate group to three response regulators (QseB, QseF and KdpE) that activate a complex virulence gene regulation cascade in enterohaemorrhagic Escherichia coli
87
what is LED209?
a QseC inhibitor
LED209 blocks the binding of AI-3, adrenaline, and noradrenaline to QseC.
LED209 also prevents QseC autophosphorylation and subsequent downstream activation of virulence genes in three different bacterial pathogens.
88
how does pyocyanin cause death in C. elegans?
By oxidative stress
89
90
what do PqsR do?
triggers transcription of the pqsABCDE-phnAB monoscistronic unit when bound to HHQ or PQS
91
what is the molecular name for PQS
2-heptyl-3-hydroxy-4-quinolone
92
whats the molecular name for HHQ
2-heptyl-4-hydroxyquinoline
93
what compounds block QS in P.aeruginosa?
2-heptylthioacetyl-homoserinelactone
Patulin
Penicillic acid
4-Nitropyridine-N-oxide
C-30
C-56
94
give three examples of bacterial species that do not respond to any of the homoserine lactones
E.coli
Helicobacter pylori
Salmonella
95
how is the activated methyl cycle and AI2 production different in Alphaproteobacteria?
SAH is converted directly to homocysteine by SAH hydrolase
96
what are the interspecies sensor and regulator in p.aeruginosa
PA1396–PA1397
97
how does RpfG have phosphodiesterase activity
via the HD-GYP domain
98
what species can influence the behaviour of p.aeruginosa via DSF
Stenotrophomonas maltophilia
Burkholderia cenocepacia
Candida albicans
99
DSF and BDSF in the CF lung have what effect when they interact with sensor kinase PA1396
increase persistence and polymyxin resistance
100
what molecules produced by p.aeruginosa can inflence C.albicans and B.cenocepacia
N-acyl homoserine lactones (N-AHL) such as 3-oxo-dodecanoyl-homoserine lactone (oxo-C12-HSL),
101
when can Clp bind to rpfB promoter?
when its bound by cyclic-di-gmp
102
what is CepI?
A quorum sensing system in Burkholderia cenocepacia
103
what are the smallest cyclic peptides frequently found in nature
diketopiperazines
(cyclic dipeptides)
## Footnote
seondary metabolites produced by bacteria fungi plants and animals
104
how do cyclic dipeptides, diketopiperazines form?
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.
104
105
whats the funciton of the pormoters in the Agr locus?
While P1 governs the expression of AgrA, P2 controls the expression of the entire agr operon. P3 dependent expression leads to up-regulation of the effector RNAIII thus providing an additional indirect mode of transcriptional re-engineering upon a quorum stimulus
106
what part of their structure do all AIPs share?
a common central cysteine located 5 amino acids from the c terminal
## Footnote
which forms a catalytic thioester bond with the sulfhydryl group of the conserved cysteine (macrocycle)
107
how many groups of AIPs are there and how are they distinguished?
fall into 4 major groups depending on their activation with AgrC
108
how many aminod acids do AIPs usually have?
7-9 amino acids
109
how do ecoli respond to adrenaline/ noradrenaline
Receptor QseC triggers phosphorelay (QseB, QseF, KdpE), regulating trascription of virulence genes
110
