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7/25/2019 Seminar at IBBR
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1
Application of tethered bilayers fordetection of bacterial toxins
Dr. Gintaras Valincius
Institute of BiochemistryVilnius University
7/25/2019 Seminar at IBBR
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Diagnostics of bacterial infections
• !atients" disease phenotype #symptoms$
• !athogen isolation and detection #microscopy$
•
Biological samples" molecular diagnostics#immunology% biochemical analysis% D&A$
7/25/2019 Seminar at IBBR
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tB'( based solution
)
!atient *ample
!uri+cation
tB'( device (easurement Data analysis
,ypical - D values for antibodies
(ost 1/0 to 1/ (2igh sensitivity 1/0 to 1/ (
Ultra high sensitivity 1/ to 1/1
*ource" 333.abcam.com
tB'( responsedetected at"
1/ to 1/11 (
7/25/2019 Seminar at IBBR
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,et ere ayermembranes
4
M c G i l l i v r a y e t a l . B i o i n t e r p h a s e s .
2 ( 2 0 0 7 ) 2 1 - 3 3
7/25/2019 Seminar at IBBR
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(ain technological development
areas
5
*ample environment6measurement device
*ensitivity6reproducibility
6standardi7ation of tB'( sensors
(easurements and data analysis8lectrochemical impedance spectroscopy$
7/25/2019 Seminar at IBBR
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*ample environments
0
'aboratory device"9/vial electrochemical cell
A/ 3or:ing electrodeB ; reference
< ; auxiliary electrode
aboratory device"
/electrode electrochemical cell
7/25/2019 Seminar at IBBR
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=lo3 through measurement cell
>
#1 x number of channels$ measurements
7/25/2019 Seminar at IBBR
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*ensitivity of tB'(s
9
?esidual defect density is one of the ma@or factors determiningsensitivity of tB'( device
7/25/2019 Seminar at IBBR
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Vesicle fusion
Allo3s real time monitoring of formation #surface plasmon resonance$
2ybrid bilayer on D,one phospholipid layer
*upported bilayer on β/(8% ,3o phospholipid layers
Dierent 2<19 anchor density
>C
5C
)C
1C
Vesicle fusion dynamics sho3s that tB'( forms very uic:ly% E .5 hrs
7/25/2019 Seminar at IBBR
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Vesicle fusion*!? allo3s to uantitatively estimate tB'( parameters
1
Molarfraction of(HC18S)2 in
SAMformationmixture
10% 30% 50% 70%
Xtether
(tethere!"#C
chain$)
.)1 .5 .0 .>
X!"#C
(exchana&le li'i in'roximalleaet)
.0 .41 .)9 .)
raction ofol
$urfaceoccu'ie &*
(HC18S)2
anchor+Xanchor
>.5C
14.)C
#5"1$
15.C
).5C
7/25/2019 Seminar at IBBR
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Vesicle fusion
11
eutron reectometry attest for the structure and integrity of tB'(s
utron reectometry reveals formation of lipid overlayer that can be removed bsing 3ith 5C/)C ethanol solution in 3ater.ometry parameters of V= tB'( are the same as tB'(s obtained by ?*8
7/25/2019 Seminar at IBBR
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&eutron reectometry revelsstructural parameters of vesicle fused tB'(s
1
-ether com'o$ition (mol%) i.HC18/ .merca'toethanol (30/70)
Molar fraction of tether h*rocar&on chain$in $u&$trate.'roximal li'i leaet
.>5 H .19
um&er of .merca'toethanol 'er anchor )"1 #>5C$
-hicne$$ of $u&.mem&rane $'ace 11.9 H .4
-hicne$$ of h*rocar&on chain$ in$u&$trate.'roximal li'i leaet /
14.4 H .>
-hicne$$ of h*rocar&on chain$ in$u&$trate.i$tal li'i leaet /
1>.0 H .4
Molar fraction of chole$terol in non.tetherli'i com'onent of t4M
.) H .
ila*er com'letene$$ 1. H .1
7/25/2019 Seminar at IBBR
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8I* spectroscopy provides highest resolutiondata on defect density in tB'(s
1)
Metho 4ateral en$it*re$olution
*urface plasmon .1!(/?AI?* .5
&eutron reectometry .1
8lectrochemicalimpedance
spectroscopy
1/> ; .1
?esolution $
7/25/2019 Seminar at IBBR
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8lectrochemical impedancespectroscopy
14
*i6Au
Alternating electric current methodology"
J K U6I K LJL exp#iωt M φ$
LJL is the impedance magnitude #modulus$φ is the impedance phaseBoth are freuency dependent #1/ to 15 2
7/25/2019 Seminar at IBBR
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15
1$ Impedance magnitudevariation is anindicator of an extentof dielectric damage" N K J /1 at f K f min
$ f min position of the
freuency scale is auantitative measure ofdefect density"
Valincius et al. 'angmuir 1% 9% >>/OValincius and (ic:evicius% 15% Advances in !lanar 'ipidBilayers and 'iposomes% Vol. 1% p.>/01.
8I* allo3s estimation of defect density intB'(*
/log#&def $K/.0log#LJfminL$MAO AK
).40
log#&def $K/.0log#LJfminL$MBO BK
/.5)
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?eal/time monitoring of the vesiclefusion by 8I*
10
8I* on di/2<196dib/(8 anchor. 'ipid" D!<0C6<2'4C
6S $'ectral traectorie$ ma* $ere a$ 9ualit* te$t an
$tanari:ation of t4M eice$;
7/25/2019 Seminar at IBBR
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?esidual defect density may be ad@ustedby varying tether density
1>
HC18+ % f min1+& H: f min2+c H: ef1+ <m.2 ef2+e <m.2
100 E.1 / E. /80 E.1 / E. /50 .1 / .1 /30 .1 .9) .0 .
20 .0 4.14 .) .410 .> 05.5 .1 5.5
Denselytethered
*parselytethered
Upper limit densities of defects
7/25/2019 Seminar at IBBR
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Vesicle fusion may be used for repetitive tB'(formation although 3ith uality deterioration
19?a:ovs:a et al% 'angmuir 15% vol. )1% 940/95>
!efect en$it* increa$e$ &* t=o orer$ of a manitue from 0;1 m.2 to a''roximatel* 7;>5 m.2 oer the cour$e of 8 re'etitie formation$remoal$ of t4M
8I* on di/2<196dib/(8 anchor. 'ipid" D!<0C6<2'4C
7/25/2019 Seminar at IBBR
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<onclusions I
1
• ,ethers synthesi7ed at &I*,6IBB? allo3s achieving extremely lo3
residual defect densities amounting to less than 1 nanometer si7e defect p s. micronsO
• Vesicle fusion is an alternative techniue allo3ing real/time monitoringof formation of tB'( sensors
•
Density of tethers allo3 modulation of residual defect density
7/25/2019 Seminar at IBBR
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<holesterol dependent cytolysins
1$ <D<s are virulence factors secreted bygram/positive bacteriaO
$ It is a class of pore forming proteins
strictly dependent on cholesterol. Diameter ofthe pores may be as large as /5 nm.
)$ *ome of <D<s evolved to recruit the G!I/
lin:ed regulatory protein <D5 for poreformation. 4$ Because of reuirement of ligation to ahuman <D5% some <D<s exhibit speci+cityrestricting their host range.
7/25/2019 Seminar at IBBR
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ur eorts focused on the follo3ing<D<s #?is: Group pathogens$
1
6ntermeil*$in% 64?+ 58 !a+ Streptococcus intermedius ( brain and liver abscesses$ ;it is believed I'N is strictly dependent on <D5O
@ainol*$in + @4?+ 5 !a+ Gardnarella vaginalis, #bacterial vaginosis$ ; hemolyticactivity depends on <D5O
#neumol*$in+ #4?+ 55 !a Streptococcus pneumoniae #pneumonia% meningitis$%<D5 independentO
4*$teriol*$in+ 44" 58 !a Listeria monocytogenes, #infections of central nervoussystem% causing meningitis% meningoencephalitis% brain abscess% cerebritis% alsobacteremia% and gasteroentritis$% activated in fagosomes. Detected by microorganismcultivation from <*= samples.
#erfrinol*$in+ 44" 58 !a Listeria monocytogenes, #infections of central nervoussystem% causing meningitis% meningoencephalitis% brain abscess% cerebritis% alsobacteremia% and gasteroentritis$% activated in fagosomes% releases the bacteria intocytosol.
#iol*$in+ #4"+ 58 !a% Arcanobacterium (Actinomyces) pyogenes% an animalpathogen% resides in mucous membranes of cattle% sheep% s3ine and other
economically important animals% 3hich cause postpartum endometritis. ,he diagnosisof subclinically infected animals remains a challenge on farm and sub6infertile animals
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!o C!CB$ a$$em&le into 'ore$ in t4M$Dhat i$ the eEect of mem&rane
com'o$itionHo= much of the 'rotein can &e etecteHo= clo$e the etection limit i$ to a'h*$ioloical leel$
Puestions
(cGillivray et al.% Biophys.Q. %p. 154>/155)
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A=( allo3s direct visuali7ation of protein pores in tB'(s%&? revels insertion of the protein complex into tB'(
)
ginolysin V'N% at 1 n(% in D!<>C64C<2' tB'( on 2<19 ancho
7/25/2019 Seminar at IBBR
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<omplete incomplete pores" V'N vs. !'N
4
Under the same condition !'N typically results in more protein oligomers in tthan V'N produces. In both cases% tB'( D!< 0C6<2' 4C
7/25/2019 Seminar at IBBR
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5
8lectrochemical impedance spectroscopy Vesicle lysis #uorescence$
Dierent techniues yield dierent seuenceof toxin activities
,ethered bilayers Unilamellar vesicles #approx. 1 nm$
2ydrated sodium% chloride ions <alcein
' t ti
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'o3er concentrationslead to a larger fractionof incomplete pores
0
At 5 n(
#roteinmor'holo*
15 nM 50 nM
V'N pores 0C 9C
V'N arches 4C C
!'N pores C 5C
!'N arches 1C >5C 2 0 . 0 0 n m
0 . 0 0 n m
400nm
V'N
!'N
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8lectrochemical impedance is capable ofdetecting smallest lesions in bilayers
>
Adapted from" 'eung < et al% 8life. #14$O)"e44>
2emolysis
=luorophore eRux
Ionpermeation
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<holesterol state in tB'(s modulatesactivity of <D<s" Intermedilysin
9
A d m i t t a n
c e p h a s e
A d m i t t a n
c e p h a s e
ons in phospholipid bilayers are detected by 8I* do3n to SI'NT ≈ 1 p(. =ractioct pores E1C
2 0 . 0 0 n m
0 . 0 0 n m
200nm
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composition modulate activityof <D<s
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8lectrostatics matter
)
Proteins:
pIV! " #$#%& pII! " '$(1
)*olesterol +in,in- ,omains:
pIV'N K 1%49O pII'N K 5%>9
p2 >.
rane charge may be used to increase selectivity of tB'( devices as 3ell as sen
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<onclusions II
)1
• <D<s may be reconstituted into tB'(s in the form of pores orincomplete poresO
• In both cases damage is inicted on the dielectric properties oftB'(s and can be detected by 8I*
• *ensitivity as 3ell as selectivity of tB'( sensors can be ad@usted
to a speci+c <D< by changing composition and structure of tB'(s.