Biofulm Physiology, Quorum Sensing, Antibiotic Resistance Flashcards
(111 cards)
1
Q
Psuedomonas aeruginosa
A
Chronic wounds
Cystic fibrosis - green lumps
With aggressive antibiotic regime the bacteria are suppressed but not eradicated in conductive zone. Respiratory zone is protected
2
Q
Does Psuedomonas aeruginosa cause monocultures?
A
No
Mixed culture with other bacteria
3
Q
PMN leukocytes versus biofilm bacteria
A
Polymorphs sit on top. Can’t penetrate biofilm
Bacteria use quorum sensing (cell cell signalling to coordinate expression of virulence in density related processes)
4
Q
Principle of quorum sensing
A
Increasing cell density (non virulent)
Quorum size reached
Culture makes collective decision and express QS controlled target genes (virulent)
5
Q
What stages of biofilm formation is quorum sensing present
A
Biofilm maturation
Dissolution
6
Q
What does PMN attraction and destruction cause?
A
Collateral damage to tissue
Inflammation
7
Q
Specifics of quorum sensing (summary)
A
Cell to cell signalling system of 2 genes
/ Gene encodes an autoinducer synthase and R gene encodes transcriptional activator protein (r-protein)
Autoinducer synthase - responsible for synthesis of autoinducer molecules (AI) which cross cell membrane
Increasing cell density, [intracellular AI] reaches threshold level and AI bind to transcriptional activator
Complex activated expression of target genes
8
Q
Pathogens is of p. aeruginosa
A
Associated with expression of virulence factors eg proteases, pigments, haemolysins, polysaccharides and toxins regulated by quorum sensing
9
Q
Quorum sensing cascade in p. aeruginosa
A
Regulator lasR, Synthase lasI switched on OdDHL (c12) (master regulator)
10
Q
Role of OdDHL
A
Induce production of:
Elastase
LasA protease
Alkaline protease
Exotoxin A
Protein secretion
Acts as inducer on rhlR system
11
Q
Rhl system
A
Induces production of: elastase
Alkaline protease
Chitinase
Lipase
Rhamnolipids
Cyanide
Pyocyanin
RpoS
Pilin export and adhesion
12
Q
What stage of biofilm formation does the Las system impact?
A
Biofilm differentiation
13
Q
What stage of biofilm production does the rhl system impact
A
Biofilm maturation and dispersal
14
Q
Las R
A
Induces PQS system and produces quinolone which switches on genes for elastase, exotoxins and bio surfactants
Involved in iron production (low during infection)
Induces rhl system which also responds to pqs system
TH1/TH2 balance
15
Q
How can biofilm development, antibiotic tolerance, virulence and immune shielding be controlled in p. aeruginosa?
A
Through QS system
16
Q
Complex communication networks in biofilms
A
Same species can receive and respond to signals
Other species can also pick up signals and respond
17
Q
Why does QS deficiency attenuate p aeruginosa in various infectious animal models?
A
Polymorphs can fight their way in
Don’t have discrete bacterial colonies, Polymorphs take over
18
Q
PMN leukocyte shield
A
Constituent: rhamnolipid
When cell produce this they can resist attack by Polymorphs
When not produces, Polymorphs mop up the bacteria
19
Q
Rhamnolipids in vitro
A
Biofilms only produce small amounts
Issue in researching in lab
20
Q
Lactate dehydrogenase activity in high control condition
A
High lactate dehydrogenase activity
Polymorphs cannot attack the biofilm
Biofilm inhibiting and killing the Polymorphs
21
Q
What is the trigger to make the Rhamnolipids?
A
Dynorhipin A produced by Polymorphs induces PQS and rhamnolipids
22
Q
Dynorhipin A roles
A
Endogenous k-agonist belonging to opioid peptides
Modulates pain and stress signal
Found in CNS
Contained in various immune cells
PMNs produce and release dynorhipin at sites of inflammation
Induces PQS and Rhamnolipids
23
Q
PqsA mutant
A
Cannot up reg Rhamnolipid production in response to PMN exposure
24
Q
What is pqs originally synthesised from
A
Chorionic acid, phnA and B (genes), anthranilic acid, HHQ (precursor molecule or pqs)
25
Enzymatic quorum quenching in biofilms
Oxidireductase (side chains)
Acylase (double bond o)
Lactonase (ring)
26
Why is there enzymatic quorum quenching in biofilms
Switch off competitors QS or use QS as carbon nitrogen source for growth
27
Biofouling
Major challenge for marine eukaryotes and bacteria can be highly detrimental to marine algae and other eukaryotes
28
Delisea pulchra
Rarely fouled in battier due to production of secondary metabolites (halongenated furanone compounds)
29
halongenated furanone compounds
Strong biological activity eg anti OS and anti microbial properties
30
Why is seaweed clean and healthy?
Delisea compound makes furinome with bromine side chain so halogenated
Inhibits quorum sensing of bacteria
c2 and c4
31
Furanone c30
Synthesised
Antimicrobial agent - promotes bacterial clearance by inhibiting QS so Polymorphs can do their job
32
AI-1
Autoinducer 1
Homoserine lactomes
33
Inter kingdom communication
AI2 luxS/luxP system
Importance of boron in secondary metabolite QS to establish QS/biofilm phenotype and virulence
34
Lux genes
Light - glowing bacteria (vibrio) usually symbiosis
35
Relationship between activated methyl cycle (AMC) and AI2
AMC is responsible for the generation of the major methyl donor in SAM and recycling of methionine by SAH
LuxS salvages homocysteine moiety from SRH and DPD byproduct made.
DPD becomes S-THMF-borate the AI2 signal of vibrionacea but in other bacteria DPD rearranges spontaneously to form R-THMF
36
Regulation of transduction of AI2 signal and autoinducer genes in enterobacteriaceae
RTHMF in through Lsr ABC transporter
Phosphorylase’s but LsrK
AI-2-P binds repressor LsrR
Released from promoter
Allow expression of autoinducer operon
37
Regulation of transduction of AI2 signal and autoinducer genes in vibrionaceae
In presence of S-THMF-borate
LuxP concerts LuxQ from kinase to phosphotase
Reverse flow of phosphate expression so expression of Lux operon
38
Ruminococcus obeum restricts vibrio cholerea gut colonisation
Uses AI2 and downtegulates virulence genes
39
Bacteria species found in CF patients
B. Cenocepacia
Synthase (I) and receptor (R)
CepIR;CciiR system
Burkholderia diffusable signal factor (BDSF) based system RpfF bc
Diketopiperazines (DKP) inhibit CepI
40
What is CepI involved in
biofilm formation
protease production
virulence
Acyl homoserine Lacton (AHL) systems CepIR, CciIR & BDSF based system
41
Diffusable signal factors (DSF)
Cis unsaturated fatty acid
Burkholderia (BDSF)
P. Aeruginosa (CDA)
Regulate virulence, biofilm formation, antibiotic tolerance
Autoinducer of biofilm dispersion
42
What does DSF synthesis involve
RpfF - coA hydratase,
RpfB - CoA ligase
43
Model for biosynthesis of DSF, BDSF and IDSF
Leucine to DSF
Carbohydrates to BDSF
Isoleucine to IDSF
RpfF has role in all these paghwYs
44
Different sensor kinases involved in DSF or BDSF perception in diverse bacteria
DSF - 5 intermembrane domains
BDSF - 2 intermemvrand domains
45
Multiple signal transduction pathways leads to expression of different DSF-regulated functions in x.campestris
Perception of DSF, autophosphorylation and phosphotrandfer to REC domain of RpfG
Activation of RpfG as a cyclic di-GMP phosphodiesterase
Reduction of cyclic di-GMP levels
Promotes synthesis of extracellular enzymes and EPS but inhibits biofilm formation
Also results in binding to GGDEF domain proteins - motility but nothing to do with biofilms
46
Signals of the DSF family play a role in inter species and interkingdom signalling
Molecules of DSF family made by s. Maltiphilia and b. Cenocepia influence p. Aeruginosa (also between eachother?)
DSF and BDSF produced in CF lung interact with sensor kinase PA1396 - increase persistence and polyminx resistance
p. Aeruginosa makes N-AHL and oxo-C12-HSL influence c.albicans and b. cenocepacia
47
Bacterial cells need to exit high energy demand of QS
Need to exit in post-quorum phase
QS signal turnover systems are one of QS exit mechanisms most identified in bacteria
Strains belonging to baccilus, paenivacillus, microbacterium, staphylococcus, pseudomonas, can degrade DSF rapidly
Use RpfB homologous to turnover DSF-type QS signals
48
Regulation of RpfB dependent DSF signal turnover in x. Campestris
(pre QS)
Pre QS - RpfC and RpfF complex, high c-di-GMP binds to TF CLP
CLP complex binds to RpfB
Inhibits transcription
CLP fails to bind to promoter region virulence genes engXCA
49
Regulation of RpfB dependent DSF signal turnover in x. Campestris (QS phase)
RpfC autophosphorylates when high levels of extracellular DSF signal present
Through conversed phosphorylation mechanism RpfG is phosphorylated
Activation of c-di-GMP phosphodirsterase activity
50
Regulation of RpfB dependent DSF signal turnover in x. Campestris (clp freed from c-di-GMP)
clp freed from c-di-GMP
Binds to promoter of engXCA and initiates transcription
Clp release from RpfB promoter enabling its transcription
51
Regulation of RpfB dependent DSF signal turnover in x. Campestris (post QS)
Extracellular DSF drop and dephosphportlated RpfC and RpfF reform complex
Dephosphorylation of RpfF = inactivation of c-di-GMP phosphodirsterase activity
Intracellular c-di-GMP return to high lvls
CLP binds to promoter region of rpfB so repressing transcription
52
Diketopiperizine inhibitors
Inhibit Cepl1 synthase
Cyclic dipeptides and smallest in nature
Secondary metabolites formed by bacteria, fungi, plants and animals
Cyclic organic compound where 2 nitrogen atom is of piperazine 6 membranes ring form amide linkages
Dipeptidyl peptidases cleave terminal ends of proteins (form dipeptides, cyclise to CDPs)
53
How is CDP synthesised
Self condensation of aa
Intramolecular cyclization if linear dipeptides
Biosynthesis by CDP synthases and tailoring by enzyme
54
Gram positive QS: peptides
Most are pathogens
QS system encoded by global regulatory locus (agr)
2 combined signalling pathway: 2 divergent operon controlled by promoters P2 and P3
Operon p2 encodes genes: AgrA, B, C, D
55
What do agr A, B, C, D encode
Agr D - precursor of AIP
Agr B - transmembrane protein responsible for processing and secretion of AIP
Agr c and a - form structures homologous to 2 component signal transduction system
Agr c - membrane sensor (n terminal has sensor domain)
Agr a - response regulation, phosphorylated by agrC causing regulation of p2 and p3
Promoter p3 regulates transcription of RNAiii and hemolysin
56
What does increase RNAiii levels lead to
Directly or indirectly ride in factors and induce expression of p2 promoter
57
AIP structure
Cyclic peptide
Side groups and chain differ
Typically 7 - 9 aa and share central cysteine located 5 aa from c terminal
C terminal aa forms catalytic thiorster bond with sulfhydryl group of conserved cysteine
2-4 aa from tail (exocycle) of peptide
AIPs are polymorphic and fall into 4 groups
58
What is being researched to block biofilms
Inhibitors of AHL and PQS
Inhibitors of Agr system
Inhibitors of QseC
59
AI-3
QseC senses AI-3 signal and ADR and noradrenaline (NA) hormones
Increases autophosphorylation and transfers phosphate group to response regulators in Ecoli (upregulate when host stressed?)
LEF209 blocks QseC sensing system
60
Intrinsic resistance
Independent antibiotic selective pressure and horizontal gene transfer, result of inherent structural or functional characteristics
61
Acquired resistance
Mutations in drug targets or transfer of resistance genes through phage mediated transduction and mobile plasmids
62
Tolerance
Related to adaptations to environment eg planktonic or sessile biofilm growth or mutations in target genes
63
Horizontal gene transfer
Promoted in biofilms so antibiotic resistance spreads more easily than in planktonic cultures
1) transformation (dead cell lysis)
2) conjugation (physical junction)
3) transduction (viral delivery)
64
Intrinsic Transformation
Competent cells take up foreign DNA and incorporate into own genome by genetic recombination
Eg Virulent but killed streptococcus pneumonia cells added to living culture of non virulent s. Pneumoniae causes some to become virulent
Erythromycin reactance by addition of naked DNA or heat killed donor cells
Rates 10 to 600 x greater than those in planktonic culture
Biofilm matrix is not a berried to DNA penetration
65
Transformation - new mechanism
Membrane vesicles- released from cell surface by many gram -ve and some gram +ve bacteria and contain proteins, polysaccharides and (for microbial adaptation) DNA
66
What kind of resistance determinants can transformation (new mech) carry
B lactams
Enzymes eg protease, endopeptidase
67
How does transformation give survival advantage to bacteria
Antibiotic resistance uptake (can be from biofilms) via OMVs and transformation
68
Example of transformation
Virulence genes, plasmid located antibiotic resistance genes and gfp from e.Coli in vesicular transform salmonella
69
Experimental evidence of transformation and biofilms
Streptococcus mutants shown to release extracellular DNA via membrane vesicles into developing biofilm and provides source of genetic material (blebs)
70
Transduction examples
Streptococcal Phages have transferred resistance to tetracycline, chloramphenicol, macrolides, lincomycin, clindamysin and streptomycin probably via transduction of non phage encoded resistance genes
Similar in staphylococci
71
Phage mediated transduction in salmonella
Believe to evolved from plant phage
n-1: tc against phage p22
72
Gram negative conjugation
Pili
Link and exchange DNA
(Sex)
73
Fimbriae and pili
Fimbriae short and straight
Pili (sex) longer
Both used for attachment
Pili exchange DNA with other bacteria
Fimbriae attach to surfaces and other bacteria
74
Gram positive conjugation
Conjugational transfer in e. Faecalis
Intergeneric conjugation in b. Subtilus and staphylococcus species
75
Antibiotic resistance: prevention of access to target
1) reduced permeability if the cell envelop
2) increased efflux activity
3) mutation in antibiotic target
4) enzymatic modification or inactivation of the drug ( hydrolysis or transfer of chemical group)
5) ability to form biofilms greatly enhance antibiotic resistance traits
76
Hallmarks of biofilms
High level resistance to antibiotics and biocides
Could be 1000x more resistant
77
Resistance to antimicrobials
Genotyping eg tetracycline resistance
Phenotypic eg marRAB locus upregulwtes AcrAB-ToIC efflux pump and down regulates OmpF potion influx, quorum sensing communities , global stress response factors, surface attachment and biofilm formation, slow growth rates in vivo
Physical eg exopolysaccharide production (slime) by biofilms shields susceptible cells eg to aggressive oxidant biocides eg chlorine
78
Antibiotic resistance mechanisms
Antibiotic degrading enzymes
Antibiotic resistance genes
Efflux pumps
Antibiotic altering enzymes eg adding acetyl groups
79
Multi drug efflux transporters
Prove means bacteria can confer intrinsic, low level resistance to diver group of antibiotics
Stepping stone to high level biofilm resistance
Expression regulated by antibiotics they remove from cell
High expression of multi drug efflux transporters confers multi drug resistance (MDR) phenotype
80
Multi drug efflux transporters: 5 structural families
H+/drug anti porters
1) resistance modulation cell division (RND, gram negative bacteria)
2) major facilitator superfamily (MF[S])
3) small multi drug resistance (SMR)
Na+/drug anti porters
4) multi drug and toxic compound extrusion (MATE)
ATP hydrolysis linked drug transporters
5) ATP binding cassette (ABC)
1-4 known as secondary transporters, use pre stored energy of chemical gradients across the membrane
ABC transporters directly coupled with energy generation
81
Multi drug efflux transporters: main responsibilities
MATE - antibiotics
MFS - antibiocides
SMR - antibiocides
RND - multiple (gram negative, periplasm, so AcrA sleeve connect to TolC to pump out of outer membrane)
ABC - multiple
82
Under or over expression affects resistance
normally relatively low level resistance
Biofilms - over expressed RND mex efflux pump
83
Intuitively assume for a “film”: slow penetration?
Oxidising biocides (eg chlorine) bind to other EPS layers and diffusion is limited
But has water channels and antibiotic diffusion onto the micro colonies is only partially reduced
84
Micro colonies in biofilms
Many structures - like mushrooms
Stalk and cap structure via imagining
85
P. Aeruginosa rhamnolipids surfactants
Mutants deficient in rhamnolipid synthesis do not maintain noncolonised channels surrounding colonies
Rhamnolipids not required for formation of macro colonies and channels but mainly channels
Surfactants May be able to maintain open channels by affecting cell cell interactions and attachment of cells to surfaces
Induced synthesis of rhamnolipids during later stages of biofilm formation (when cell density high) implies active mechanism where bacteria exploit intracellular interaction and communication to maintain these channels
High cell density of planktonic growth indices the synthesis of qs dependent rhamnolipid production and in biofilms
86
Diffusion of rhodamine B into a s.epidermidis microcolony
Red fluorescent intensity at the centre of cells after rhodamine B into the system
Tracer can access the centre of large dense staphyloccal cell cluster within 300s- 5 mins. In context of antibiotic therapy, duration which is typically measure in days, this is fast. Delayed few mins will not buy much protection
87
Diffusion summary
Biofilms are mostly water and solutes the size of most biocides and antibiotics can diffuse in the biofilm
Do not move as fast as they would in pure water because the cells, EPS, and other constituents of the biofilm hinder their mobility
But measurements of diffusion coefficient me suggest that these splits diffuse at rates ~20-50% of rate in water
So physiology?
88
Adaptive mechanisms of biofilm physiology
Cell interactions and biofilm formation (HACCP)
1) electrochemical - EPS and lectin formation, adhesion to substratum, bioelectric effects (DLVO, PMF, sigma factors)
2) co agregation - adhesion to pioneer species, structures consortia (lectin induction, stationary phase)
3) maturation - cell density dependent qs (homoserine lactomes, A1-2 peptides)
4) micro environment colonisation - by microaerophiles and anaerobes passive or chromotactile
5) predator grazing and chemotaxis - Protozoa, nematodes, macrophages
6) disaggregation - passive, sloughing; active, daughter cells, nutritional status, undocking
89
1) DLVO theory
Variation of free energy with particle separation.
Net energy is given by sun of double layer repulsion and van der waals attractive forces that the particles experience as they approach
Variation of free energy with particle separation at higher salt concentrations showing possibility of secondary minimum
90
Quorum sensing (summary)
Bacteria cell to cell communication
Sense state of population density
Characterised by accumulation if autoinducing signal molecules in and around high density colonies
Cell coordination of gene expression in cell density dependent manner
controls transcription of many virulence genes of pathogenic bacteria
Block qs = render bacteria a virulent and/or susceptible to host attack and antimicrobial agents
Most work done in homoserine lactomes (gram -ve)
91
Mosaic of micro environmental niches
Heterogeneity
O2, redox - heterotrophic activity may lower o2, facilitates anaerobes, microaerophiles
EPS and products, pH, electrical - corrosion, nutrient attraction, disinfectant repulsion (and charged antibiotics)
92
Altered physiology and resilience
Upregulation of sigma3, global stress response, hsp (chaperone), catalase - attachment/detachment, starvation, temperature, disinfection, oxidative stress
Qs- auto induced acyl HSLs, AI-2, peptides
U (growth rate) - slow so fewer RNA/ribosomes - slow cell wall turnover, cell wall structure, porins, binding proteins
INCREASE ANTIBIOTIC RESISTANCE
93
Summary of mechanisms
Alterations in nutritional availability
Stress response
Antimicrobial neutralisation
Persister formation
94
Planktonic-biofilms transition in a complex and highly regulated process: Surface attachment defective (sad) mutants
1) p. Aeruginosa flaggelar mediated motility - initial attachment
2) polar localised type iv pili - twitching motility, 3D
3) global virulence regulator GacA -3D
Within mins of attachment many operating genes down regulated while others (EPS production) up regulated
95
P. Aeruginosa biofilm antibiotic resistance
Double membrane so has periplasm
NdvB required for synthesis of periplasmic anionic glucans- highly glycerol-phosphorylated-beta-glucans which bind to aminoglycisides
May prevent antibiotics from reaching sites of action by sequestering in periplasm
Also effects 8 ethanol oxidation genes involved in to rant in resistance
96
RpoS stress response - p. Aeruginosa infections
Stress response = physiological changes that protect the cell from environmental stresses
Cells protected from effects of heat shock, cold shock, changes in pH and chemical agents
Central regulator of response is the alternate factor RpoS, originally thought to only be expressed in stationary phase
Induced by high cell density, general stress response produces trehalose and catalase
Cells in biofilms experience high cell density so express RpoS
Shown by RT- PCR that rpoS mRNA seen in CF pateiengs with chronic P. Aeruginosa biofilm infections
97
p. Aeruginosa microarrays
Only 73 genes changed in growth states
0.5% activated, 0.5% repressed
Activated filamentous bacteriophage Pf1- mediate gene transfer in biofilms?
Genes for pili and flagellum repressed in biofilms
Analysis
Different activities of cells, different patterns of gene expression
98
p. Aeruginosa microarrays - antibiotic resistance
Cationic anyibiotics eg tobramycin and gentimicin bind to negatively charged LPS of outer membrane
Transport into cell correlated with lvl of transmembrane electrical potential
TolA gene effects LPS structure, decreased aminoglyciside affinity for outer membrane
Repression of cytochrome c oxidase should decrease sensitivity
Tobramycin induce stress response eg activation of dnaK and groES and 2 probable efflux systems (non RND and P type ATPase)
99
Comparison wild type and RpoS mutant vs tobramycin
Cannot survive as well
100
Persisters and antibiotic tolerance
Small subpop of dormant persister cell that highly tolerate killing by antibiotics
[Antibiotiv] drops, persisters reesyablish the pop so chronic infection
Significant when pathogen shielded from immune system by biofilms or in sites where immune components are limited in NS, stomach or macrophages
Toxin-antitoxin modules key for persister formation eg RelE, MazF toxins cause dormancy by cleaving mRNA, hipA inhibits translation by phosphorylation elongation factor Eg-Tu, TisB toxin forms membrane pore, decrease in pmf and ATP
101
Types of toxin-antitoxin systems (TAS)
5 types
All proteins
1 and 3 antisense RNA
2,4,5,6 protein
1 antitoxin blocks mRNA if toxin
2 direct protein protein interaction
3 direct RNA- protein interaction
4 blockage of toxins effect on cellular target
5 RNAase if antitoxin degrades mRNA of toxin
6 degredation of toxin by ClpXP Seine protease
102
Toxin antitoxin modules and persister formation in e.coli
Obg hokB system and poly phosphate long system
Controlled by PGpp or pppgpp
Alarmones - accumalate when cell starved of amino acids or nitrogen source
Antitoxin eg sokB inactivated and hokB is release which incorporates into cell membrane and blocks protein motor force
In poly phosphate - block ppx and phosphorylate lon and binds to messenger and shut down protein production
Rec, tisB, pore, disrupts protein motor force and so inhibition of DNA replication/translation/transcription
103
How to fight antibiotic resistance
Attachment and detachment phases?
104
What are the conditions inducing differentiation and dispersal in p. Aeruginosa?
Accumulation of perixynitrite (ONOO) in micro colonies
High concentration kills bacteria in middle of colony but also encouraged survivors to swim away
105
Nitric oxide in bacteria
Important signal molecule
Linked with iron acquisition, anaerobic growth, qs
Produced by nitrite reductive, nirS during anaerobic respiration
Removed by nitric oxide reductase, norB
Add back of No to p. Aeruginosa = decreased biofilm/planktonic ratio with increased SNP. Can use others to eg GSNO and SNAP but SNP best
106
Add vack of NO enhanced biofilm removal using antimicrobials
With SNP makes completely susceptible
So NO increases biofilm susceptibility to antimicrobials
107
MRSA
Gram positive staphylococcus aureus
Methicillin resistance staphylococcus aureus
Cases increased
Last standby = vancomycin but intermediate and full resistance strains appearing so new therapies needed
108
Biofilm physical barrier and phenotype resists antimicrobials
Add SNP on resistance if immature and mature MRSA biofilms
500nM drop in viable cell count but staph not detached
But increase and resistance increased
109
Conclusion
Treat biofilm before it’s mature
NO donors and antimicrobials
110
Why is there sucseptible in presence of NOs?
NO activation of PDD reduces c-di-GMP so less sessility, less biofilm formation and cell cycle progression
111
Future studies
Test existing antimicrobials and emerging compounds in presence of NO donors
Compare SNP with other donors
Investigate other physiological release agents eg vis-2 decenoic acid
Synergy with qs inhibitors
