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IInntteerrnnaattiioonnaall JJoouurrnnaall ooff BBiioollooggiiccaall SScciieenncceess 2011; 7(3):333-346

Research Paper

Site-Directed Mutations of Thermostable Direct Hemolysin from Grimontia hollisae Alter Its Arrhenius Effect and Biophysical Properties Yu-Kuo Wang1*, Sheng-Cih Huang1*, Yi-Fang Wu1, Yu-Ching Chen1, Yen-Ling Lin1, Manoswini Nayak1, Yan Ren Lin1, Wen-Hung Chen1, Yi-Rong Chiu1, Thomas Tien-Hsiung Li2 , Bo-Sou Yeh3, and Tung-Kung Wu1

1. Department of Biological Science and Technology, National Chiao Tung University, 30068, Hsin-Chu, Taiwan, Republic of China

2. Institute of Biochemistry, National Chung Hsing University, 40227, Taichung, Taiwan, Republic of China 3. Hsin Chu General Hospital, Department of Health, Executive Yuan, Taiwan, Republic of China

* These authors contributed equally to this work.

Corresponding author: T. K. Wu, Department of Biological Science and Technology, National Chiao Tung University, 30068, Hsin-Chu, Taiwan, Republic of China. Tel: +886-3-5729287; Fax: +886-3-5725700; E-mail: tkwmll@mail.nctu.edu.tw. T. Li, Institute of Biochemistry, National Chung Hsing University, 40227, Taichung, Taiwan, Republic of China. Tel: +886-4-22840468; E-mail: lithomas@dragon.nchc.edu.tw

© Ivyspring International Publisher. This is an open-access article distributed under the terms of the Creative Commons License (http://creativecommons.org/ licenses/by-nc-nd/3.0/). Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited.

Received: 2011.02.13; Accepted: 2011.03.23; Published: 2011.03.31

Abstract

Recombinant thermostable direct hemolysin from Grimontia hollisae (Gh-rTDH) exhibits paradoxical Arrhenius effect, where the hemolytic activity is inactivated by heating at 60 oC but is reactivated by additional heating above 80 oC. This study investigated individual or collective mutational effect of Tyr53, Thr59, and Ser63 positions of Gh-rTDH on hemolytic activity, Arrhenius effect, and biophysical properties. In contrast to the Gh-rTDH wild-type (Gh-rTDHWT) protein, a 2-fold decrease of hemolytic activity and alteration of Arrhenius effect could be detected from the Gh-rTDHY53H/T59I and Gh-rTDHT59I/S63T double-mutants and the Gh-rTDHY53H/T59I/S63T triple-mutant. Differential scanning calorimetry results showed that the Arrhenius effect-loss and -retaining mutants consistently exhibited higher and lower endothermic transition temperatures, respectively, than that of the Gh-rTDHWT. Circular dichroism measurements of Gh-rTDHWT and Gh-rTDHmut showed a conspicuous change from a β-sheet to α-helix structure around the endothermic transition temperature. Con-sistent with the observation is the conformational change of the proteins from native globular form into fibrillar form, as determined by Congo red experiments and transmission electron microscopy.

Key words: Grimontia hollisae, thermostable direct hemolysin, Arrhenius effect, Circular Dichro-ism, virulence factor

Introduction Grimontia hollisae, formerly described as Vibrio

hollisae, usually causes moderate gastroenteritis in humans by ingestion of contaminated raw seafood, or contact with the environmental reservoir [1-6]. The pathogen, along with V. vulnificus, has been suggested to have a predilection for bloodstream invasion in

people with liver abnormalities [2,7]. In addition, the organism can adhere to and invade tissue culture cells [8]. Recently, patients with severe gastroenteritis, hypovolemic shock, bacteremia, and septicemia have been identified with G. hollisae infection alone, sug-gesting a likely underestimation of the incidence of

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the G. hollisae infection as an invasive disease [8-13]. Thermostable direct hemolysin (TDH) of Vibrio

species has long been suspected in virulence for bac-terial pathogenesis. A major virulence factor of Vibrio parahaemolyticus is TDH (Vp-TDH), which is com-posed of 189 amino acids containing a 24 amino acid signal peptide in the N-terminal region and a 165 residue matured peptide in the C-terminus region. The TDH of G. hollisae (Gh-TDH) is antigenetically and genetically related to that of Vp-TDH [5,14-20]. Previous studies suggested that the Gh-TDH exhib-ited different heat stability of hemolytic activity compared to that observed for the Vp-TDH, in that Gh-TDH is heat labile, while the Vp-TDH is thermo-stable after heating for 10 min at 70 or 100 oC [16]. Moreover, a paradoxical phenomenon known as the Arrhenius effect, whereby the hemolytic activity is inactivated by heating at 60 oC but is reactivated by additional heating above 80 oC, was observed for Vp-TDH but not reported for Gh-TDH [21]. These results provoked us to investigate whether the Gh-TDH exhibits a similar Arrhenius effect as that of Vp-TDH. Detailed comparison of the TDH protein sequences from various G. hollisae strains revealed a few residues that may be involved in increasing hy-drogen bonding, electrostatic interactions, and/or secondary structure and contribute to the enhanced thermostability [22]. In this study, we report the indi-vidual or collective mutational effect at positions 53, 59, and 63 on the Arrhenius effect, hemolytic activity, and biophysical properties, based on the sequence differences among various TDHs and their heat sta-bility relative to Vp-TDH [19,21,23]. Our results indi-cated that amino acid mutations at these positions can alter the protein’s Arrhenius effect and hemolytic ac-tivity. Furthermore, results from circular dichroism (CD) and differential scanning calorimetry (DSC) ex-periments showed consistent correlation between conformational change and endothermic transition temperature. Finally, data from transmission electron microscopy and Congo red experiments also supports a model in which conformational changes trap the protein into an aggregated fibrillar form.

Results Molecular cloning, site-directed mutagenesis, and recombinant production of G. hollisae thermostable direct hemolysin protein

The Gh-tdh gene was amplified from G. hollisae ATCC 33564 genomic DNA and subcloned into the plasmid pCR®2.1-TOPO to generate the

pTOPO-Gh.tdh recombinant plasmid. The recombi-nant pTOPO-Gh.tdh plasmid was subjected to protein over-expression and subsequent site-directed muta-genesis. The amino acid residues at positions Tyr53, Thr59, and Ser63 of Gh-TDH were individually or collectively mutated to His53, Ile59, and Thr63, to construct single-, double-, and triple-mutants. The wild-type and mutated Gh-tdh genes were separately subcloned into the plasmid pCR®2.1-TOPO, and sub-sequently transformed into Escherichia coli BL21(DE3)(pLysS) cells for proteins over-expression. The PCR®2.1-TOPO plasmid itself, which contained no tdh gene insert, was used as a negative control. Following incubation for 16 h at 37 °C, the produced recombinant wild-type and mutated proteins (Gh-rTDHs) were collected, extracted, and subjected to protein purification methods repeatedly using Phenyl-Sepharose 6 Fast Flow columns. Electropho-resis of the purified Gh-rTDHs revealed homogene-ous bands at approximate 22 kDa, as determined by sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE) (Fig. 1A). The protein identities of the Gh-rTDHs were also confirmed by MALDI/TOF/TOF mass spectrometry (data not shown). The immunoblot analysis also revealed that both Gh-rTDH wild-type (Gh-rTDHWT) and mutants (Gh-rTDHmut) produced single bands (Fig. 1B). To determine the protein’s native state, the Gh-rTDHWT and Gh-rTDHmut proteins were examined using non-denaturing PAGE, showing a single band of ap-proximately 90 kDa (Fig. 1C). Interestingly, the Gh-rTDHY53H/T59I and Gh-rTDHT59I/S63T dou-ble-mutants and the Gh-rTDHY53H/T59I/S63T tri-ple-mutant migrated slightly faster on the non-denaturing PAGE gel than that of the Gh-rTDHWT and other Gh-rTDHmut proteins. In par-allel, the hemolytic activities of Gh-rTDHWT and var-ious Gh-rTDHmut proteins were detected when these proteins were embedded in a blood agar plate (Fig. 1D). For the Gh-rTDHWT, the molecular mass was also detected from the sedimentation coefficient (s) of an-alytical ultracentrifugation and gel filtration chroma-tography as 71.3±10.8 and 75 kDa, respectively (Fig. 2A and 2B). Finally, transmission electron microscopy (TEM) analysis of negatively stained Gh-rTDHWT ol-igomers revealed the presence of particles organized into a square configuration comprised of four smaller particles (Fig. 2C). These results indicated that the Gh-rTDHWT protein exists as a monomer under de-natured condition and associates as a homotetramer in solution.

Int. J. Biol. Sci.

Fig. 1. Purificof Gh-rTDHW

proteins with proteins with in a blood agarlane 4: Gh-rTD

Fig. 2. PrelimGh-rTDHWT p71,255.72 ± 1units, calculatGh-rTDHWT pthe standard pkDa), γ-globulribonuclease Aimage of negat

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ation and identWT and Gh-rTDantiserum agaistandards. (D)r plate. Lane 1:DHS63T; lane 5:

minary structuprotein. The c0.83 g/mol. Ploted from the protein (open cproteins used tlin (158 kDa), A (14 kDa). Thtively stained n

tification of theDHmut proteins w

nst Gh-rTDH ) Hemolytic act: Phenyl SepharGh-rTDHY53H/T

ure of native alculated moleot of the distr

concentratiocircle). The figuto calibrate thealbumin (66 kDe inset shows tnative form of

e Gh-rTDHWT

with standardsyielded a singletivity detectedrose 6 Fast FloT59I; lane 6: Gh-

Gh-rTDHWT

ecular mass of ibution of sedin profiles. (Bure shows the e column. CloseDa), ovalbuminthe gel filtratioGh-rTDHWT. T

and Gh-rTDHs. (B) Immunobe band. (C) No when the Gh-w purified Gh--rTDHY53H/S63T;

protein. (A) Af Gh-rTDHWT

imentation coe) Gel filtratiomigration posied diamonds inn (44 kDa), chn analysis of GhThe inset show

Hmut proteins. (Ablot analysis of on-denaturing P-rTDHWT and G-rTDHWT; lanelane 7: Gh-rTD

Analytical ultrfrom sedimentefficients (c(s))on chromatogtions of the Gh

ndicate molecuhymotrypsinogeh-rTDHWT. (C

ws the image o

A) Coomassie Gh-rTDHWT a

PAGE of Gh-rTGh-rTDHmut p

e 2: Gh-rTDHY5

DHT59I/S63T; lane

racentrifugationtation coefficie) vs. S, where graphy estimath-rTDHWT proular mass standen A (25 kDa)) Representativ

obtained by sing

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blue-stained Sand various GhTDHWT and Ghroteins were e53H; lane 3: Gh-e 8: Gh-rTDHY

n determinatioent (s) is apprS is plotted in ted molecular otein in compardards: thyroglob, myoglobulin ve electron migle-particle ana

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DS-PAGE h-rTDHmut h-rTDHmut embedded -rTDHT59I; 53H/T59I/S63T.

on of the oximately Svedberg mass of

rison with bulin (670 (17 kDa), croscopic alysis.

Int. J. Biol. Sci.

Hemolytic amination ofproteins

To invetive amino a63 on the Areach of the eferent tempemin. Followitures, the prby two diffemolytic activserting the rewas by gradting the react

We firGh-rTDH prFigure 3 showtion of the Gh-rTDHWT at 2 μg/mL dividual or Thr59, and SInterestinglyilar activity atein, while Gh-rTDHY53H

mately 2-4-fo

Fig. 3. Timolysis was ahemolysis wasindependent e

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activity and f Gh-rTDHW

estigate whethacid substitutirrhenius effecexpressed proeratures ranging heat incuotein solution

erent means fvity. The firseaction tube iually decreastion tube in thst analyzed roteins at 37ws the percen

Gh-rTDH pprotein exhibconcentrationcollective m

Ser63 of Gh-ry, the Gh-rTDas that observ

the Gh-rTH/S63T mutanolds increased

itration of hemassessed by tus calculated as dexperiments.

thermostabWT and Gh-r

her the indivions at positict for the hemoteins was in

ging from 4 toubation at diffn was cooledfollowed by st was rapid into an ice ba

sing the temphe air.

the mutati7 oC for hemntage of hemoprotein concbited 50% hemn. We then amutational efrTDH for hemHY53H mutantved for the G

TDHT59I, Gh-nts demonstrd activity. Al

molytic activityrbidity at roomdescribed in th

bility deter-rTDHmut

vidual or colleions 53, 59, anmolytic activincubated at do 100 oC for ferent temper down to 37 analysis of hcooling by i

ath. The seconperature by pu

ional effect molytic activiolysis as a funcentration. Tmolytic activnalyzed the iffect of Tyr5

molytic activit exhibited sim

Gh-rTDHWT pr-rTDHS63T anrated approlternatively, t

y of human redm temperaturehe experimenta

ec-nd ty,

dif-30 ra-oC

he-in-nd ut-

of ty. nc-

The ity in-53, ty. m-ro-nd xi-

the

Gh-rTDGh-rTDtion inGh-rTD

WGh-rTDtures amolytiincubaappare3-10). H60-80 °°C resu(Fig. 4incubathe Ghhemolycontrar7.4±1.3°C folshownincubarapid cdetermabovem30 min4B).

d blood cells be using an autal procedures s

DHY53H/T59I, DHY53H/T59I/S6

n hemolytic DHWT.

We then anaDH proteins and subsequeic activity. Intated at a tempent activity loHowever, inc°C for 30 minulted in a heA, column 11

ation at 85-10h-rTDHWT recytic activity ry, the hem3% when the llowed by sln). Next, eachated at 37, 70 cooling, and

mination of tmentioned mn, no apparen

by the Gh-rTDtomated microsection. Data p

Gh-rTD63T mutants sh

activity tha

alyzed the mincubated a

ently cooled dterestingly, w

perature belowoss was obsercubation of G

n followed byemolytic activ1-15). Further

00 °C and rapcovered 64.7-7

(Fig. 4A, comolytic activi

Gh-rTDHWT

low cooling h of the Gh-Tand 90 oC forthen subjecte

the hemolyticmutants were i

nt activity los

DHWT and Ghoplate reader. points shown ar

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DHT59I/S63T, howed a 2-folan that obser

mutational eat different tdown to 37 oC

when Gh-rTDw 55 °C for 30rved (Fig. 4AGh-rTDHWT py rapidly coolvity of only rmore, after a

pidly cooled t72.6±5% of its

olumn 16-19).ity declined T was incubat

to 37 °C (dTDHmut mutar 30 min, folled to Arrhenic activity. Wincubated at 3ss was observ

-rTDHmut protThe final percre mean ± S.D.

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and ld reduc-rved for

effect of tempera-C for he-HWT was 0 min, no , column

protein at ing to 37 8.1±1.6% a 30 min to 37 °C, s original . On the

to only ted at 95 data not ants was lowed by ius effect

When the 37 °C for ved (Fig.

teins. He-centage of . for three

Int. J. Biol. Sci.

Fig. 4. Armeasured at 3(p<0.05). Stati(B) Relative htreatment at various mutanactivities were(0.1% Triton X

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rrhenius effect37oC after varistically significahemolytic activ37, 70, and 90nts protein in 9e measured aftX-100).

t assay of Gh-rTious heat treatant (p < 0.05 bvity of Gh-rTD0 oC for 30 mi90 oC). Not siter various tre

TDHWT and Ghtment from 4 etween 4-55, 6DHY53X/T59X/S63X

n followed by ignificant betw

eatments (n = 3

h-rTDHmut proto 100 oC for

60-80 and 85-1individual or rapid cooling

ween various m3 per group). N

oteins. (A) Relar 30 min. *: no00 oC). Not sicollective muto 37 oC. Stat

mutants proteinN, negative co

ative hemolyticot significant; **gnificant in 4-5

utants, measurtistically significn in 37 and 70ontrol (PBS buf

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activity of Gh-*: Statistically

55, 60-80 and 8red at 37 oC acant (p < 0.05

0 oC. Relative ffer); P, positiv

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-rTDHWT, significant

85-100 oC. after heat 5 between hemolytic ve control

Int. J. Biol. Sci.

Surprisiat 70 and 90 rapid coolingactivity for Gh-rTDHY53H

mutants retaity (Fig. 4BGh-rTDHWT Vp-TDH, whbut can be reoC and subsmore, althoGh-rTDH diArrhenius efand triple mof the hemolnius effect.

Differential Gh-rTDH h

In 2005and transforrich in β-stra

Fig. 5. Dcapacity (Cp) v

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ingly, incuba°C, respectiv

g to 37 °C resuGh-rTDHY53H

H/T59I/S63T muained approxiB). These rexhibits Arrhhich is inactiveactivated by equently rapugh a singid not affect ffect, collectivutants involvlytic activity

scanning caheat stability

, Fukui et al.rmation of Vpands by incub

ifferential scanvs. temperatur

tion of Gh-rTvely, for 30 multed in the loH/T59I, Gh-rTDtants, while

imately 70% hresults indichenius effect svated when hadditional heid cooled to le mutation the hemolyt

ve mutationsved Thr59 cauand alteratio

alorimetry asy

. reported thep-TDH into bation at 60 oC

ning calorimetre at a scan rat

TDHmut proteimin followed oss of hemolyDHT59I/S63T, anthe rest of t

hemolytic acticated that tsimilar to thatheated at 60 eating above 37 oC. Furthe

on Thr59 tic activity ans of the doubused the declin of the Arrh

ssay of

e detoxificatinontoxic fibrC [21]. We ne

try scans of Ghte of 0.5 °C/mi

ins by

ytic nd the iv-the t of

oC 80 er-of

nd ble ine he-

on rils ext

investieffect oscanninheats orespectCp vsGh-rTD58.4, 5heat ca18.16, Gh-rTDGh-rTDGh-rTDteins, rtransitis at 5effect bproperthese pthe Artrap at

-rTDHWT and Gn for Gh-rTDH

igated the inon protein thng calorimetrof the Gh-rTtively. Figure

s. temperaturDHmut protein8.0, 57.1, 56.6

apacities of 2322.45 kcal

DHY53H/T59I/S6

DHT59I/S63T, DHS63T, Gh-rrespectively.

tion temperat6.6 oC, confoby heating arties of the supositions are rrhenius effect the Gh-rTDH

Gh-rTDHmut pHWT and Gh-rT

ndividual or hermal stabilry (DSC) to d

TDHWT and Ge 5 shows there for Gh-rTns. Endotherm6, 56.3, 55.3, 53.66, 36.53, 34l/K.mol, we63T,

Gh-rTDHWT

rTDHY53H, anSurprisingly

ture above thorm to alteratat 70 and 90 ubstituted ami

similar, the dct may be duH fibrillar stat

roteins. ShownTDHmut protei

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collective muity, using dif

determine theGh-rTDHmut

e plot of heat TDHWT and mic transition52.0, and 51.8.59, 52.61, 65.4ere determin

Gh-rTDHT, Gh-rTDHnd Gh-rTDHy, the mutanthe Gh-rTDHW

tion of the AoC. Since theino acid side difference obse to a conforte formation.

n are the profilns.

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utational fferential e specific proteins, capacity various

ns at Tm = 8 oC and 48, 65.71, ned for

HY53H/T59I, HY53H/S63T, HT59I pro-ts with a

WT, which Arrhenius e general chains at served in mational

es of heat

Int. J. Biol. Sci.

Circular Dicformational

To inveondary struc(190-250 nmGh-rTDHT59I

were determin Fig. 6 anbelow 55 o

Gh-rTDHT59I

showed simitical negativcharacteristicβ-sheet structure was inc

Fig. 6. TeGh-rTDHY53H/

at 195 and 218the inset show

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chroism of Gl change

estigate the mcture of the m) of Gh-r/S63T, and Gh

mined at differnd supplemenoC, the Gh-/S63T, and ilar far-UV Ce elliptical pec of protein ctures. Nevercreased from

emperature d/T59I and (D) Gh8 nm indicate tws the second

Gh-rTDHs re

mutational effprotein, far-UrTDHWT, Ghh-rTDHY53H/T5

rent temperatnt Table 1, a-rTDHWT, Gh

Gh-rTDHY5

CD spectra wieaks minimu

containing rtheless, whe55 to 80 oC,

dependence ofh-rTDHY53H/T59I/

the isodichroicisodichroic po

eveals con-

fect on the seUV CD specth-rTDHY53H/T

59I/S63T mutantures. As showat temperaturh-rTDHY53H/T

53H/T59I/S63T

ith nearly ideum at ~218 n

predominanten the temper

the far-UV C

f the far-UV /S63T proteins inc points and theoint of Gh-rTD

ec-tra

T59I, nts, wn res 59I, all

en-nm,

tly ra-

CD

spectraat 202 from βSpecifiat 55(Gh-rTGh-rTDwas indiffereteins fmore, (Gh-rTGh-rTDondary

CD spectra n 10 mM phospe negative max

DHWT protein.

a changed fronm, indicatin

β-sheet to α-ically, the CD5 oC whileTDHY53H/T59I, DHY53H/T59I/S6

ncreased to 6ence in the Gfor their seco

the Gh-rTTDHY53H/T59I, DHY53H/T59I/S6

y structure ch

a of (A) Gh-phate buffer (pHximal peak, res

om positive tong the second-helix for all

D spectrum ofe those of

Gh-rTD63T) changed w60 oC, indicaGh-rTDHWT andary structuTDHWT and

Gh-rTD63T) proteins hange by 70 an

-rTDHWT, (B) H 7.0) at 37, 55pectively. The

http://www.

o negative abdary structurl measured

f Gh-rTDHWT

f the Gh-rDHT59I/S63T,when the temating the proand Gh-rTDHure stability. d the Gh-DHT59I/S63T

completed nd 80 oC, resp

Gh-rTDHY53H

5, 60, 70 oC. Tharrow at 198 n

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sorbance e change proteins. changed

rTDHmuts and

mperature onounced Hmut pro-

Further--rTDHmut

and the sec-

pectively.

H/T59I, (C) he arrows nm within

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Consistent with the observation is that no further change at 201 nm could be detected for Gh-rTDHWT protein at 70 oC, while a further increase of the nega-tive ellipticity at 201 nm absorbance could be detected for those of Gh-rTDHY53H/T59I, Gh-rTDHT59I/S63T, and Gh-rTDHY53H/T59I/S63T mutants at temperatures above 70 oC. In parallel, an isodichroic point at 195 nm was observed for both the Gh-rTDHWT and all the Gh-rTDHmut proteins, supporting a two-state transi-tion for all measured proteins between 55 and 80 oC. Further increasing the temperature from 70 to 95 oC, a decrease of the absolute ellipticity in the far-UV re-gion was observed for Gh-rTDHWT, characteristic of the secondary structure change into an unfolded state. However, at temperatures above 80 oC, the Gh-rTDHY53H/T59I, Gh-rTDHT59I/S63T and Gh-rTDHY53H/T59I/S63T proteins exhibited distinct CD spectra from that of the Gh-rTDHWT, which no ap-parent change of the absolute ellipticity could be ob-served for Gh-rTDHY53H/T59I, Gh-rTDHT59I/S63T and Gh-rTDHY53H/T59I/S63T. A second isodichroic point at 198 nm could be detected for the Gh-rTDHWT but not for Gh-rTDHmut proteins, indicating a second two-state transition occurred specifically for Gh-rTDHWT (Fig. 6A inset). These results suggest that the Gh-rTDH proteins exhibit the first secondary structure change generally for both the Gh-rTDHWT and the Gh-rTDHY53H/T59I, Gh-rTDHT59I/S63T and Gh-rTDHY53H/T59I/S63T proteins around the endother-mic transition temperature, and exhibit the second secondary structure change specifically for the Gh-rTDHWT at temperature above 70 oC. Therefore, at least three states, a native state below the endothermic transition temperature, an intermediate state at 55-70 oC, and an unfolded state above 80 oC, could be de-tected for the heat-induced transitions of the Gh-rTDHWT protein. Conversely, perhaps a two-state but not a three-state transition occurred for the Gh-rTDHY53H/T59I, Gh-rTDHT59I/S63T and Gh-rTDHY53H/T59I/S63T proteins, which the amino acid mutation within the Gh-rTDHWT that causes the loss of the Arrhenius effect.

Inhibitory effects of Congo red on Gh-rTDHWT hemolytic activity and fibril formation

Hemolytic activity analysis revealed that the paradoxical Arrhenius effect of the Vp-TDH protein is related to structural changes in the protein that pro-duce fibrils [21]. Previously, inhibitory effect on red blood cell hemolysis and a characteristic red shift in absorbance spectrum were observed when Congo red bound to native amyloidogenic proteins and amyloid fibrils, respectively [28,29]. A dose-dependent inhibi-tory effect of Congo red was also observed on

Vp-TDH hemolysis [21]. We then investigated the effect of accumulation of Congo red to the inhibition of Gh-rTDHWT hemolytic activity and its absorbance spectrum on binding to Gh-rTDHWT fibrils. The re-sults showed that Gh-rTDHWT hemolysis is inhibited by Congo red in a dose-dependent manner (Fig. 7A). The IC50 for Congo red inhibition on Gh-rTDH he-molysis was approximately 11 μM. To investigate the effect of Congo red binding on absorbance wave-length shift, apo-myoglobin (apo-Mb), native Gh-rTDHWT and heat-treated Gh-rTDHWT, respec-tively, were incubated with Congo red and the cor-responding absorbance spectra were measured. The negative control, the α-helix rich apo-Mb, exhibited a characteristic absorbance increase with only a slight red shift. In addition, the native Gh-rTDHWT also ex-hibited an absorbance increase but not a red shift. On the contrary, a characteristic absorbance increase with a clear red shift (495 to 527 nm) could be detected for the heat-treated Gh-rTDHWT (Fig. 7B). Consistent with the observation is the detection of Gh-rTDHWT fibrils from the TEM assay after heat treatment (Fig. 7C). These results indicated that Congo red can bind to the native Gh-rTDHWT and inhibit the hemolytic activity as well as cause a red shift in the absorbance spectrum when the protein produces fibrils.

Discussion The number of reports on the connection be-

tween G. hollisae infection and patients with severe gastroenteritis diseases is increasing. In addition, the characterization of virulence factors responsible for pathogenesis is a prerequisite for future development of targeted drug therapy. In this study herein, we have characterized the mutational effect of Gh-rTDH on the Arrhenius effect, hemolytic activity and bio-physical properties. Recently, Hamada et al. reported that the Vp-TDH protein possesses a tetrameric and a monomeric structure in aqueous solvents and high salt concentrations, respectively; whereas the protein transforms into nontoxic fibrils rich in β-strands by incubations at 60 oC [21,40]. Similarly, the expressed Gh-TDHWT protein exists as a monomer under dena-tured condition and associates into a homotetramer in solution. In addition, the Gh-rTDHWT protein can be transformed into nontoxic fibrils when incubated between 60 and 80 oC, as shown by the Congo red and TEM results. The Gh-rTDHWT fibrils incubated above 80 oC dissociate into an unfolded state, which can re-fold into the toxic Gh-rTDHWT native form upon rapid cooling to 37 oC. These phenomena are consistent with the Arrhenius effect, in which the protein is detoxified by heating at 60-80 oC but reactivated by additional heating above 85 oC.

Int. J. Biol. Sci.

Fig. 7. EfGh-rTDHWT hsequently incuhemolytic actia Congo red sapo-Mb was uabsence of Ghresentative tra

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ffect of Congohemolysis. Varubated with huvity assay. Theolution in the a

used as a negatih-rTDHWT fibransmission ele

o red on the ious concentrauman 4% (v/v) e data are meanabsence or preive control. Thrils from the cctron microsco

Gh-rTDHWT

ations of Congerythrocytes

ns ± S.D. from sence of native

he difference sporrected Congopic image of t

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Int. J. Biol. Sci.

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biolsci.org

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Int. J. Biol. Sci. 2011, 7

http://www.biolsci.org

343

prediction, individual substitution of Tyr53 to His, Thr59 to Ile, or Ser63 to Thr does affect the β-sheet probability [38]. However, collective mutations at positions 53 and 59, 59 and 63, or simultaneously in-cluded 53, 59 and 63, may disrupt the secondary structure or tertiary structure. Consistent with the prediction is the difference observed in far-UV CD spectra for Gh-rTDHY53H/T59I and Gh-rTDHT59I/S63T double-mutants and the Gh-rTDHY53H/T59I/S63T tri-ple-mutant (Fig. 6).

The CD properties difference of Gh-rTDHWT, Gh-rTDHY53H/T59I and Gh-rTDHT59I/S63T, and Gh-rTDHY53H/T59I/S63T can potentially explain the al-teration of the Arrhenius effect. CD analysis showed that Gh-rTDHWT could interconvert among at least three conformational states, depending on the tem-perature; while two states were identified for Gh-rTDHY53H/T59I, Gh-rTDHT59I/S63T and Gh-rTDHY53H/T59I/S63T mutants. A decrease of CD spectrum at 201 nm was observed when the temper-ature was increased above 60 oC, indicating the de-struction of β-sheet and the accumulation of α-helical intermediates. Similarly, Hamada et al. also reported accumulation of α-helical intermediates during the unfolding of β-lactoglobulin, a predominantly β-sheet protein, supporting our observations [39]. Subse-quently, Gh-rTDHWT fibrils incubated over 80 oC dis-sociate into unfolded states, which refold into native form upon rapid cooling to 37 oC. Conversely, the Gh-rTDHY53H/T59I, Gh-rTDHT59I/S63T and Gh-rTDHY53H/T59I/S63T fibrils retained the secondary structures when incubated over 80 oC and showed no hemolytic activity when cooled to 37 oC, consistent with an alteration of the Arrhenius effect. Further-more, CD and DSC results of Gh-rTDHWT, Gh-rTDHY53H/T59I, Gh-rTDHT59I/S63T, and Gh-rTDHY53H/T59I/S63T showed close correlation be-tween conformational change and their endothermic transition temperatures. Consistent with this obser-vation is that a mutationally-induced increase of the endothermic transition temperature caused the alter-ation of the Arrhenius effect and the heat-induced fibril development of Gh-rTDHWT and Gh-rTDHY53H/T59I/S63T when incubated around 55 oC and 60 oC, respectively.

In summary, a TDH protein from G. hollisae was successfully produced, and amino acid residues pu-tatively involved in affecting Arrhenius effect, hemo-lytic activity, and biophysical properties were identi-fied. The Gh-rTDHY53H/T59I, Gh-rTDHT59I/S63T and Gh-rTDHY53H/T59I/S63T mutants can alter the protein’s Arrhenius effect and hemolytic activity. Furthermore, consistent correlation of conformational changes from β-sheet to α-helix and subsequent aggregation into

fibrillar form to the endothermic transition tempera-ture were also observed from CD, DSC, TEM and Congo red experiments. Further studies to elucidate the physiological and structural characteristics of Gh-rTDHWT and related mutants are warranted.

Materials and methods Bacterial strains and materials

The G. hollisae strain ATCC 33564 was obtained in a freeze-dried form from the Culture Collection and Research Center (Hsin-Chu, Taiwan). This bacterium showed hemolysis on tryptic soy broth (TSB) agar plates containing 1.5% NaCl and 5% sheep blood. Phenyl Sepharose 6 Fast Flow and protein molecular weight standards were purchased from GE Healthcare (Piscataway, NJ). The protein assay kit was obtained from Bio-Rad (Hercules, CA). Protein purification chemicals were obtained from Calbio-chem (La Jolla, CA).

Grimontia hollisae thermostable direct hemoly-sin: Cloning, expression, purification, and prep-aration of monoclonal antibodies

The cloning, expression, purification, prepara-tion and characterization of monoclonal antibodies of Gh-rTDHWT were performed according to the previ-ous report [24,25]. Site-directed mutations of Tyr53, Thr59, and Ser63 in the G. hollisae tdh wild-type gene were performed using the QuikChange site-directed mutagenesis kit (Stratagene Inc., La Jolla, CA). The oligonucleotide primers used are shown in Table 1. Mutations were confirmed by DNA sequencing. The recombinant plasmids were electroporated into the E. coli BL21(DE3)(pLysS) cells, and read for loss or de-crease of hemolytic activity for growth on 5% sheep blood agar plates.

Assay for hemolytic activity and thermostability of the G. hollisae TDH

Hemolytic activity was assayed according to a previously described method with rabbit erythrocytes that were washed three times with the 100 mM phosphate-buffered saline (PBS) (pH 7.6) and resus-pended at a concentration of 4% (v/v) [25]. For the hemolytic activity assays, 0.1 mL of 0.1% Triton X-100, which causes complete release of hemoglobin from erythrocytes and results in the maximum change in absorbance at 540 nm, was used as a positive control. The elution buffers, which caused negligible erythro-cyte hemolysis compared with sample fractions, were used as negative controls. The effect of temperature on the hemolytic activity of purified TDH was deter-mined by incubating 1 μM of the purified protein in PBS for 30 min at different temperatures (4, 16, 25, 30,

Int. J. Biol. Sci.

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. 2011, 7

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biolsci.org

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Int. J. Biol. Sci.

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. 2011, 7

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apacity (Cp) iper mole, whwere done in d

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corded with akyo, Japan) eqrature cononal transitiouring the ellips were taken f 1 mm, scannn. The data fr

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(50-90 oC). temperatures

The raw CD verted into mn deg cm2 dm

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terest e

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gerwrio h1982

2. MorHickrio d

3. Lowsept730-

4. Kellyand Diag

5. Nishwataof phem1988

ctivity assaysured as aboveperformed as ns of CR (5-5L of Gh-rTDHwere incubato spectral a

300 to 700 nmre, as well as ate concentrahe PBS bufferate.

owledgemeWe thank Nattry of Educa(MOE ATU cil (NS99-2113-M-009g this researc

for his comr for High-Perrotein mode

Ching Chang ent of BiologChiao Tung U

eviations Gh-TDH: Grim

ysin; Vp-TDHdirect hemo

metry; CD: cir

lict of IntereThe authors haexists.

rences kman FW, Farmwalt AG, Weaverhollisae sp. nov. fr2; 15: 395-401 rris JG Jr., Millekman FW, Weavdamsela and Vibriwry PW, McFarlaticemia after cons-731 y MT, and Strohcultivated oyst

gn. Microbiol. Inhibuchi M, Dokeani T. Isolation f

producing a hemmolysin of Vibrio 8; 54: 2144-2146

s, UV absorbe-mentioned.

following: fi50 μM) were

H in fibril formated at room analysis. Them were recor

the CR and ations. The insr. All reaction

ents tional Chiao

ation, AimingPlan), and tSC-97-2627-M9 -004 -MY2)ch. We are gmments. We rformance Co

eling jobs. Wg and Mr. Hsgical Science University) fo

montia hollisaeH: Vibrio paraolysin; DSC: rcular dichroi

ests ave declared

mer JJ 3rd, Hollr RE, and Brenne

rom patients with

er HG, Wilson ver RE, and Blakeo hollisae. Lancet and LM, and Thsumption of catf

h EM. Occurrencter populations

nfect. Dis. 1988; 9e S, Toizumi S, Ufrom a coastal fismolysin similar parahaemolyticus

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bance at 540 Binding exp

irstly, variouse incubated wm. Mixtures o

temperaturee absorbancerded for the fibril alone astrument wasns were carrie

Tung Univerg for Top Uthe National

M-009-003 ) for financiagrateful to M

thank the omputing for

We also thansueh-Liang Cand Technolo

or their assista

e thermostabahaemolyticusdifferential s

ism.

that no confl

lis DG, Fanninger DJ. Identificath diarrhea. J Clin

R, Tacket CO, He PA. Illness caust 1982; 1: 1294-129hreefoot HK. Vifish. J. Infect. Dis

ce of Vibrionaceaein the Pacific N

: 1-5 Umeda T, Yoh Msh of Vibrio hollito the thermost. Appl. Environ.

biolsci.org

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nm was periments s concen-with 100

of CR and e for 1 h e spectra CR-fibril

at the ap-s blanked ed out in

rsity and University

l Science and

ally sup-Mr. Justin

National r running nk Prof.

Chu (De-ogy, Na-ance.

ble direct thermo-

scanning

lict of in-

g GR, Stei-tion of Vib-

n Microbiol.

Hollis DG, sed by Vib-97 ibro hollisae s. 1986; 154:

e in natural Northwest.

M, and Mi-sae capable

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