Seminar at IBBR

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



Diagnostics of bacterial infections

• !atients" disease phenotype #symptoms$

• !athogen isolation and detection #microscopy$

•

Biological samples" molecular diagnostics#immunology% biochemical analysis% D&A$



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 (



,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



(ain technological development

areas

5

*ample environment6measurement device

*ensitivity6reproducibility

6standardi7ation of tB'( sensors

(easurements and data analysis8lectrochemical impedance spectroscopy$



*ample environments

0

'aboratory device"9/vial electrochemical cell

A/ 3or:ing electrodeB ; reference

< ; auxiliary electrode

aboratory device"

/electrode electrochemical cell



=lo3 through measurement cell

>

#1 x number of channels$ measurements



*ensitivity of tB'(s

9

?esidual defect density is one of the ma@or factors determiningsensitivity of tB'( device



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



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



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



&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



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 $



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



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)



?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

$tanari:ation of t4M eice$;



?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



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



<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



<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.



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



!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)



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



<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



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



'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



8lectrochemical impedance is capable ofdetecting smallest lesions in bilayers

>

Adapted from" 'eung < et al% 8life. #14$O)"e44>

2emolysis

=luorophore eRux

Ionpermeation



<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



composition modulate activityof <D<s



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



<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.

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Seminar at IBBR

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