Immunoaffinity purification, stabilization and comparative characterization of listeriolysin O from Listeria monocytogenes serotypes 12a and 4b

l~es. MicrobioL

© INS'nXUTPA.~'rEuR/ELsEVU~R Paris 1992

1992, 143, 489-498

Immunoaffinity purification, stabilization and comparative characterization of listeriolysin 0 from Listeria monocytogenes serotypes 1/2a and 4b G . M . M a t a r m W,F. Bibb, L. Helsel, W, Dewitt acid B. S w a m i n a t h a n

Meningitis and Special Pathogens Branch, Division of Bacterial and 34ycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control Atlanta, GA 30333 (USA)

SUMMARY We developed a simple and highly effective procedure for stabilizing the baemolytic activity of li,steriolysin 0 (LLO) from Listeria monocytogenes after irnmunoaffinity purification. The haemolyfic activity of LLO was stabilized by eluting it directly into tubes containing an alkaline buffer (5 mM ly~ine, 140 mM KCI, 50 % ethylene glycol, pH 11.5). The purified LLO retaipa;.t 1 0 0 % of it8 haemolytic activity after 6 weeks of storage at - 2 0 ° C , LLO purified from a strain of L, monocytoge~es serotype 1/2a (ATCC 432491 and LLO purified from a strain of L. mone~yt~;~res serotype 4b IF 23fl5) isolated from a Mexican-sty|e cheese, showed no aign;f!cant differences in pH and temperature stability. When in(;ubated in buffers at pH valaes from 4 to 12 at 4 ° C and 25 ° C, LLO from serotypes l12a and 4b retained maximal haemolytic activity at pH 8 after 4 h of incubation. LLO from both serotypes lost their haemolytic activity after incubation at 5 0 ° C for 25 rain.

Key-~'¢orcls: ListerioLysinO, Virulence, Listeria nlonocytoge~.~; Serotypes 1/2a and 4b, Haemo!ytic activities.

INTRODUCTION

Listeria monocytogenes, the aetioIogic agent o f listeriosis, caused four f o o d b o r n e o u t b r e a k s in N o r t h A m e r i c a a n d E u r o p e .in the 1980s (Gelfin a n d Broome, 1989). All four o u t b r e a k s were caused by L, monocytogenes serotype 4b. Listeriosis also occurs sporadically; the a m l u a l incidence in the U n i t e d States is estimated to be 1,600 cases with a morta.lity rate o f 25-30 %

( ~ h u c h a t eL aL, 1991). Serotypes l / 2 a , l / 2 b and 4b account for more than 90 v/0 o f sporadic cases o f listefiosis. Data from a recently cot, eluded active surveillance study indicate that a substantial proportio~l o f s p o r a d i c listeriosis is f o o d b o r n e a n d t h a t serotype 4b is more likely t h a n other serotypes to be associated with foodb o r n e h u m a n disease (Pirtner et aL, personal c o m m u n i c a t i o n ) . Those at high risk o f d~'ve!oping disease include i m m u n o c o m p r o m i s e d per-

Submkted November 18, 1991, accepted April 2, 1992. 1") Correspondingauthor: |600 Cfi|lOn Rd.. MS G06 Ce~ll¢l~for Dis.:a~cConlrt3i, Atlata~a,GA 30333.

G.M. M A T A R E T AL,

490

sons, pregnant women, elderly persons and newborns (Schuch~t etal., 1991). The high mortality rate of listeriosis and the implication of foods in sporadic and epidemic listeriosis have caused the regulatory agencies and the food industry to focus on I,. monocyrogenes as a major foodborne pathogen and have stinm]ated research on the pathogenicity of this organism. The pathogenic mechanisms of L. rnonocytogenes are not fully understood; however, listeriolysin O (LLO), a beta-haemolytie eytolysin produced by most L. monocytogenes strains, is an essential virulence factor. LLO is a sulphydryl-dependent haemolysin which is inactivated by cholesterol and has a single cysteine residue located near the C-terminal region of the molecule similarly to pneumolysin and streptolysin O (Walker et at., 1987). After phagocytosis, LLO allows L. monocytogenes to escape the phagosome (Chakraborty and Goebel, 1988; Berehe e t a L , 1988; Cossart etal., 1989; Mengaud e t a L , 1989; Kuhn et al., 1990), multiply in the cytoplasm and invade adjacent cells without ever encountering the intercellular space (Tilney and Portnoy, 1989). Despite the importance of LLO in the pathogenicity of L. monocytogenes, the protein has not been well characterized. Early studies (Siddique et eL, 1974) directed at the purification of the beta-haemolysin by column chromatography with cross-linked polydextran and carboxymethyl cellulose indicated that the molecular size of the haemolysin was less than 10,000 kDa. Mengaud et aL (1989) convincingly demonstrated the role of a 58-kDa betahaemolysin (which they termed listeriolysin O) in the pathogenicity of L. monocytogenes. Geoffroy et aL (1987) and Krcft etal. (1989) purified LLO using a combination of thioldisulphide exchange chromatography and gel

t3SA CAPS DTT HU LLO mAb

= = = =

~,~'.;neserum albumin. 3-(cyc[ohexylamino)-l-propane-sulplnonicacid. diethiethteitol. haemolyticunit. li~icriGlysinO. monodonalantibody.

filtration, but the yield of the purified LLO was very low ( < 2 07o). Helsel et al, (submitted for publication) have purified LLO by immunoaffinity chromatography, greatly surpassing the 2 07o yield obtained by Geoffroy et al, The haemolytic activity o f the affinity-purified LLO, however, was lost within 24 h. The objectives of this study were to affinity purify LLO from L. monocytogenes strains of serotypes 1/2a and 4b, stabilize the haemolytic activity of immunopurified LLO, determine the optimal pH for its haemolytic activity and determine if the L L O from the two serotypes show differences in their pH and thermal stability. This work was undertaken as part of a project designed to evaluate the use of preformed LLO as a diagnostic indicator for the presence of L. monocytogenes in food products.

MATERIALS AND METHODS Source of isolates, culture and harvest conditions

L. monocytogenes strain ATCC 4324'3..,erotype l/2a, and F2365 serotype 4b isolated from a Mexican-style cheese were cultured in brain heart infusion broth (1 I) (Difeo, Detroit, MI) at 37°C with shaking (I00 rpm) for 30 h. Cells were harvested by eentrifugmion at 14,000 g for 30 rain at 4°C. Supernatant fluids were fi!tered through a 0.45-ttm membrane filter (Nalgene, Rochester, I,IY). Partial purification of LLO by ammonium sulphate precipitation

Proteins were precipitated from the culture supernatants with 50 % ammonium sulphate (313 mg/I). After adding the ammonium sulphate, suspensions were mixed by gentle inversion and stored overnight at 4°C. The mixture was centrifuged at 14,000 g for 30 min at 4°C. After discarding the supernatant fluids, the pellet was dissolved in 10 ml dislilled water and stored at -70°C.

PBS = phosphate-bufferedsalinn. RFLP = reslriclionfragment length polymorphism. SDS-PAGE sodiumdodecyl~ulphatc/polyacrylamid¢gel ele¢trophoresis. SLO = :streptolysinO. SRBC = sheepred blood cell.

LISTERIOL YSIN 0 F R O M S E R O T Y P E S l/2a A N D 4b

Purification of LLO by affinity chromatography 1) Preparation o f the affinity column MonocIonal antibody (mAb) HeI4D4D6C9 (lgG2a) prepared against LLO from L. monoeytogenes serotype 1/2b was used for affinity purification of LLO. The development and characterization of this mAb is described elsewhere (Helsel etaL, submitted for publication). Ten milligrams of the mAb (affinity-purified) were coupled to 1 g of cyanogen-bromide-activatedSepharose-4B (Pharmaeia, Uppsala, Sweden) and equilibrated with 0.01 M phosphate-buffered saline (PBS), 0.5 M NaCI, pH 8.0, according to the manufacturer's instructions. The gel (17.5 ml swollen particles) was transferred to a column, packed in 0.1 M NaHCO3, pH 8.3, and washed with 0.2 M borate buffer, pH 8.0, until absorbanee at 280 nm (A2~0) of the etuate was below 0.02. 2) Affinity purification Five to ten ml of the partially purified LLO were loaded on the affinity column, and the column was washed with 0.2 M borate buffer pH 8.0 until the Az~o of the eluate was less than 0.02. Bouud LLO was eluted from the column using 0.1 M acetic acid, 0.85 % NaCI, pH 2.g. Fractions o f the eluate (0.5 ml) were collected in 4 ml of alkaline buffer (5 mM lysine, 140 mM KCI, 50 % ethylene glycol, pH ! 1.5), assayed for haemolytie activity and stored at - 20°C.

Protein and haemolysin assay Proiein concentrations were determined by using a modified Lowry assay (BCA protein assay, Pierce, Rockford, IL). Assays fcPrdetermination of haem~. lyric activity were performed using a modification of the method described by Geoffroy et aL (1987). Microtltre plates were used instead of tubes and diethiolhteitol (DTT) instead o f cysteine (Hclsel el aL, manuscript in preparation). Dilutions of LLO were made in 0.01 M PBS pH 6.0, containing 0 . 1 % bovine serum albumin (BSA). Equal volumes (0.1 ml) o f serially diluted LLO and PBS-BSA containing 0.01 M DTT were mixed in wells of a microtitre plate (Immulon 2, Dynatech Laboratories, Chantilly, VA) and incubated for 5 min. A 0.1-ml suspension of sheep red blood cells (SRBC) at a concentration of 1.8 x 10~ cells/ml in PBS-BSA pH 6.0 was added to each well. The plates were incubated at 37°C for 45 min, centrifuged for 10 min at 2,000 g in microtitre plate carrier, and sul~ernatants were transferred into a second microtitre plate. Absorbanees were measured at 570 nm and results ex-

491

pressed as haemolytic units (HU) per rag of protein. One HU is equivalent to the amount of LLO needed to cause 50 % haemolysis of the SRBC.

Determination of optimal pH Microassays for determination of optimal pH for the haemolytic activity of LLO were done in 0.01 M PBS pH 5.0 to 10.0, containing 0.1 070 BSA and 0.01 M DTT. The tests were performed as described above. Because of the stability problems encountered with LLO, streptolysin O (SLO, Sigma Chemical Co., St. Louis, MO) was used to determine if the presence o f ethylene glycol !n the assay buffer had any effect on the determination of optimal oH for haemolytic activity. Dilutions of SLO were made in 0.01 M PBS pH 5.0 to 10.0 and in alkaline buffer pH II.5. The haemolysin assay was done as described above on SLO diluted in PBS pH 5 to 10 and in alkaline buffer. Absorbances of supernatants at 570 nm were measured as before after 10, 25 and 45 rain of incubation at 37°C. Results are expressed as percent haemolytie activity of LLO or SIO.

Stability studies I ) p H stability studies To determine the stabiIity of LLO from serotypes l/2a and 4b at different pH values, we incubated LI.O in buffers containing 0.1 M DTT at pH values ranging from a, to 12 at 4~C and 25°C for l and 4 h, respectively, and then assayed for haemolytie activity at pH 6.0. Results were expressed as percent retention of haemolytic activity. Buffers consisted of 0.0l M solutions of the following: succinic acid pH 4 and 6; acetic acid pH 5; PBS pH 7; Hepes pH 8 tApg~/~C=. - 0.GI4}; boric acid pH 9; CAPS pH 10 and 11 ; ~odium phosphate pH 12. Stability studies were also done using 0.0t M PBS adjusted to different pH values in the range of 4 to 12. 2) Thermal stability studies LLO from serotypes 1/2a and 4b was incubated at remperatures ranging from 4°C to 65°C from 5 min to 4 h, and was assayed for tlaemolytic activity at pl--I 6.0. Results were expressed as percent retention of haemolytic activity. SDS-PAGE and immunoblotting SDS-PAGE was done using a modified Laemmli gel system (Laemlli, 1970; Carlone et aL f1985). A discontinuous buffer system was used with a 4 ~/0

492

G.M. MA TAR E T AL.

stacking gel and an 8 e0 separating gel (pH 6.8 and 8.8, respectively). Partially purified and affinitypurified LLO from L. monocytogenes serotypes I/2a and 4b was diluted in distilled water (35 g.g of protein in a total volume of 25 el), and an equal volume of treatment buffer was added (20 o70glycerol, 10 % 2-mercaptoethanol, 4 alp SDS, in 0.125 hi Tris pH 6.8, and 10 izl of 0.05 % bromophenoi blue/ml of buffer). After a 5-rain treatment in a boiling water bath and cooling, 10 gl of the samples and 10 I~l of protein moleeular weight standards (Sigma) were loaded on the gel and eleetrophoresed in an "LKB model 2001" electrophoresis apparatus at a constant voltage of 450 V and a current of 60 m A using an "LKB model 2197" power supply for approximately 4 h. The gels and lower buffer were kept at 10°C by an "LKB model 22f9" multitempurature refrigeraled circulating water bath. Proteins were transferred to nitrocellulose filters by Western blotting (Hoefer Scientific, San Francisco, CA) at constant voltage (220 V) for 3 h at 4°C. One filter was stained with colloidal gold stain (BioRad, Melvitle, NY) for total proteins, according to the manufacturer's instructions. One filter was incubated for I h at room temperature with anti-LLO mAb against purified LLO l / 2 b (1/500) and one with rabbit anti-LLO poly¢lonal antibody (l/5130) prepared against partially purified LLO l/2b. Antibodies were prepared in our !aboretories. A fo~-tb filter was incubated with a purified rabbit polyclona] antibody against the 60-kDa protein (putative invasion-associated protein produced by L. monocytogenes). Filters were washed with PBS-Tween and treated with peroxidase-labelled goat anti-mouse antibody (1/1000) (BioRad, Richmond, CA). After repeated washing to remove the excess secondary antibody, the bands reacting with the antibody were vlsnalU_ed by treating the filter with freshly prepared disclosing reagent (I .4 diaminobenzidine-4 HCI (DAB) in 0,0l M PBS) and 30 % H202 until the bands were visible. Western blots of LLO along with molecular weight standards (Sigma) were scanned using a charge-coupled device (CCD) camera incorporated into an eleetrophoresis analysis system (Biolmage Products, Millipore, Ann Arbor, MI).

isoelectric point was determined by comparison with isoelectric focusing standards (Pharmaeia LKB, Piscaraway, N J).

RESULTS LLO produced by L1 monocytogenes serotype l / 2 a (LLO 1/2a) a n d by L. monocytogenes serotype 4b ( L L O 4b) r e t a i n e d 100 % o f t h e i r haemolytic activity after 6 weeks of storage at - 2 0 ° C w h e n eluted f r o m t h e affinity c o l u m n with 0.1 M acetic acid, 0.85 % t.qact, p H 218, a n d collected in tubes c o n t a i n i n g the alkaline b u f f e r . T h e same L L O p r e p a r a t i o n s lost their haemolytic activity within one week u p o n storage at 4°C. Figure 1 shows the percent r e t e n t i o n o f haemolytic activity o f L L O i / 2 a eluted f r o m the affinity c o l u m n with a n d w i t h o u t collection in the alkaline buffer after storage at 4 a n d - 20°C. T h e specific a~:tivities o f L L O 1 / 2 a a n d L L O 4b before a n d after i m m u n o a f f i n i t y purification are s h o w n in table I. T h e i m m u n o b l o t s o f the affinity-purified L L O using anti-LLO polyclonal a n t i b o d y (fig. 2a) a n d a n t i - L L O m A b (fig. 2b) showed t h a t i m m u n o p u r i f i e d L L O 1 / 2 a a n d L L O 4b reacted with these antibodies. The poly-

m

Isneleetde [oeusing LLO l/:2a and 4b were electrophoresed on horizontal isoeleetrie focusing gels (pH range 3.5-10.0; LKB ampholine, Pharmacia, Bromma, Sweden). Efeetrophoresis was done on "LKB 2117, Multiphor" at 1,500 volts, 50 mA and 30 watts for about 90 min using an "LKB model 2197" electrofocusing constant power supply. The gel was cooled with an " L K 8 model 2219 multitemp I1" thermostatic circulator. The location of LLO on the get was visualized by Coomassie blue staining, and its

Fig. 1, Stability o f L L O l / 2 a in alkaline b a f f e r p H I 1.5. l = L L O l / 2 a eluted with acetic acid a n d stored at 4~C and - 20~C; 2 = L L O 1/2a eluted with acetic acid, collected in alkaline buffer with 50 ~0 ethylene glycol pFl 11.5 and stored at 4~C ; 3 ~ L L O l / 2 a eluted with acetic acid, collected in alkaline buffer with 50 % ethylene glycol pFI 11.5 a n d stored at - 2 0 ° C ) .

LISTERIOL ]:SIN 0 F R O M S E R O T Y P E S I/2a A N D 4b

493

Table !. Specific activity o r L L O l / 2 a and L L O 4b before a n d after i m m u n o a f f i n l t y purification.

Strain

Serotype

ATCC 43249

I/2a

F 2365

4b

1

Totat cone. (10 ~ HU)

Protein activity (mg)

2560

2,56

24,5

5

2560

1.28

0.038

10

10240

10.24

8

2560

1.28

Purification step

Volume (ml)

A m m o n i u m sulp h a t e precipit. lmmunoaffinity chromatography

l0

A m m o n i u m sulphate precipit. Immunoaffinity chromatography

h:ctivity (HU)

Specific (HU/mg) 1045 336842

24

4266

0.017

1355294

2

1

1

2

2

3

) 60 k Da'--=

55.7 K Da , - ~

55.7 KDa .-.~.,,l~lP. ~

.,=_.~=

•I - 54.9 K D a

.,p__ 54.9 KDa

Fig. 2. [mmunobiots of immunoaffinity-purified LLO l/2a and LLO 4b. Left pone/: using rabbit pol:,,cionat anti'~ody prepared against partially purified L[.O 1/25 (lane 1 ~ LLO | / 2 a ; lane 2 = L L O 4b). Centre panel: using mAb against purified LLO t/2b (lane I = l_LO I/2a; lane 2 = LLO 4b). Right panel: immunoblots of LLO l/2a and LLO 4b using rabbit polycio~al antibody prepared against the 60-kDa putative invasion-associated protein of L. monoc>'logen~* (lane 1 = crude LLO 1/2a; lane 2= purified LLO I/2a; Ia.e 31purified LLO 4b; lane 4= Cl-ude LLO 4b).

4

494

G.M. M A T A R E T AL.

clonal •antibody against the 60-kDa putative invasion protein of 1,. monocytogenes reacted only with the crude LLO from serotypes 1/2a and 4b

(fig. 2c). The calculated molecular mass of LLO I/2a was 55.7 kDa and that of LLO 4b was 54.9 kDa (fig. 2a and 2b). LLO from both serotypes showed a pI of approximately 7.9 (fig. 3). The optimal pH for the haemolytic activity of LLO l/2a was determined to be between pH 6,0 and 7.0 after 25 min of incubation at 37°C (fig. 4). The haemolytic activity of LLO at pH 5.5 was only 12.5 o70 of the maximal haemolytic activity observed at pH 6.0 and 7.0,

SLO shifted from 7.0 to 6.5 in the presence of ethylene glycol (data not shown). Stability studies using buffers containing DTT in the pH range between 4 and 11 for f to 4 h exposure, at 4°C and 25°C, showed that LI.O 1/2a and LLO 4b were maximally stable at pH 8.0. However, some differences were seen between the haemolytic activity of LLO 1/2a and LLO 4b. LLO 4h lost all of its haemolytic activity on exposure to pH 5 and 11 for 1 h at 4°C. In contrast, LLO 1/2a retained 50 % of its haemolytic activity after exposure to pH 5 at 4°C for I h and 25 % of its haemolytic activity af-

The optimal pH for haemolytic activity of

100 90

1 ...........

2

~ :

3

.'"..

•

,

4 =

9.30

8.45 7.90 7.35

2a-/ +.<+

..~-k:;

++

...

. '

10

~.

.IL

L~

45

Time(rain)

6.55

,~

.. Fig. 4. D e t e r m i n a t i o n o f o p t i m a l p H for the h a e m o l y t i c aclivity o f L L O I / 2 a .

.#~4

5.85

.~

,

12'0

g

I

~n9

25~C, 1 h

252e, +.

5.20 4.55

4c.

4~

v

3.50

~

4o

g

~o

L a:

LLL J

e,

7

.~

9

10

12

pH

Fig. 3. |soelectric f o c u s i n g o f L L O

112a a n d L L O 4b.

Lane I ~ pl ~taadard, s; tane 2 = L L O 1/2a; lane 3 = L L O 4 b ; lane 4 = m i x t u r e o f L L O l / 2 a and 4 b .

Fig. ':;. p H s|abilit~.,"~." (3 +t3 I / 2 a e>:~r.-:--r-~l as !~ercent retention o f haemolyLi¢ acti~ity.

LISTERIOL YSIN 0 F R O M SEROTYPES l / 2 a A N D 4b

120

m

i

"'r'

i

i

495

i

1

o 100

::%

4C'C. 1

h

25°c, 4 h

'5-

+., (.I O

1

80

¢°C, 4 h

U

60

>., O

m

E ~D 40

C"

"6 E 'O

20

'C 0

0) Q:

-20

I

1

I

I

I

6

8

9

10

II

12

pH

Fig. 6. pH stability of LLI') 4b expressed as percenz retention of haernolytic activity.

r

i

i

i

1

| O serogroup

80-

I

4b

$or.ggroup 1/2a

- -

te.r exposure to pH 11 at 4°C for I h. Moreover, the haemolytic activity o f LLO 4b was less affected than that of LLO 1/2a after exposure to 25°C for 4 h at oH ranging from 6.0 to t0.0 (figs. 5 and 6). Thermal stability studies demonstrated no significant differences between LLO l / 2 a and L L O 4b in rates of inactivation, Both were inactivated when incubated for 25 min at 50°C (fig. 7).

4 deg C

i ,° 40

#

20

DISCUSSION The purification and stabilization of LLO

0 50

100 Time

150

200

250

(mln)

Fig. 7. Thermal stability of LLO I/2a and LLO 4b ex pressed as percent haemolytic activity of untreate~i LLO. Percent haemolytic activity is calculated separately for haemolysin from serotype I/2a and ab with each expressed as percclnalgeof maximal activity obtained ~ith the rcspecfive r~r=para'~i.on.

were two major objectives of this study. Other investigators have purified L L O 1/2a and LLO 4b by thiol-disulphide exchange affinity chro-

matography on thiopropyl/Sephar0se-6B (Geoffrey et aL, 1987; Kreft et aL, t989) or by immunoaffinity ,:hromatography (Helsel el aL, 1990). The procedure o f Geoffrey et aL (1987), included rhiopropyl/Sepharose-6B chroma~og-

496

G,M, M A T A R E T AL.

raphy followed by 3 gel filtrations. Approximately 90 °70 of LLO was lost in the thiopropyl/Sepharose 6B chromatography and tile final recovery was only 1.3 070.The authors did not provide any information oa the retention of haemolytic activity by the purified protein. Helsel et aL (submitted for publication) used the same LLO-specific mAb utilized in this investigation and 0.1 M acetic acid, 0.85 °70 NaCI, pH 2,8, for the one-step immunoaffinity purification of the protein from ammoniumsulphate-precipitated culture supernatants. Their yields were acceptable (40-60 %}, but the purified LLO lost its haemo!ytic activity within 20, h of purification. Using the modified procedure described here, we were able to stabilize the haemolytic activity of the LLO while maintaining ;he same recovery achieved by Helsel et aL (1990). Inclusion of organic solvents ir.. elution buffers provides a convenient means of weakening the hydrophobic interactions in immune complexes (Anderson et aL, 1978). As much as 50 07a ethylene glycol was found to favour conditions which lower the polarity of ~he eluent, and hence promote its desorption without inactivating it (Anderson et al., 1979). initially we attempted to elute the LLO from the immunoaffinity column directly with the alkaline buffer. The LLO purified in this manner retained 40 07~ of its haemolytic activity for 6 weeks at 4°C. However, the immobilized antibody in the affinity column was inactivated by the alkaline Duffer. To maintain the activity of LLO and to preserve the affinity column, we used 0.1 M acetic acid, 0.85 °70 NaCI, pH 2,8, to elute the LLO from the column into tubes containing the alkaline buffer. When purified in this manner and stored at - 20°C, LLO r~i.ained 100 % of itg haemclytic activity when tested after 6 weeks, and the affinity column could be reused. Most of the work on the pathogenic mechanisms of L. m o n o c y t o g e n e s has been done using only serotypes 1/2a and 1/2c (Chakraborty and Goebel, 1988; Berche el ai-, 19~,8; Cossart et al., 1989; Mengaud et al., 1989; K uhn et M,, 1990). Recently, data obtained in different laboratories using ribosomal DNA analysis (Graves el al.,

1991) and multilocus enzyme electrophoresis (Bibb et aL, t989; Piffarette et aL, 1989), clearly show the existence of the two subgroups within L. m o n o c y t o g e n e s : one group (subgroup A) includes serotypes l/2a, 1/2¢ and 3a, and the other (subgroup B) includes serotypes l / 2 b and 4b (Swaminathan el al., 1989; Johnson et al., 1990). Furthermore, structural differences at the nucleotide sequence level exist between LLO from L. m o n o c y t o g e n e s serotypes 1/2a and 4b (Vines and Swamiaathan, manuscript in preparation). Because serotype 4b caused all of four foodborne disease outbreaks in North America and Europe in the 1980s and may be strongly associated with sporadic human listeriosis, it was included in the present experiments. Our data indicate a difference in molecular mass of 800 daltons between LLO l/2a and LLO 4b. These differences in molecular mass would translate to a difference of up to 11 amino acids. Hehel ei ai. observed a similar difference in molecular size between LLO l / 2 a (55.755.9 kDa) and LLO 4b (54,9-55,1 kDa) (submitted for publication). The observed molecular mass of LLO l/2a is in agreement with that calculated from the nucleotide sequence of the structural gene coding for LLO I/2a reported by Leimeister-Wachter et aL (1989). The pI values of LLO 1/2a and LLO 4b were slightly different when LLO !/2a and LLO 4b were run on different lanes on the pH gradient polyacrylam:,de gel. However, when a mixture of L L O l/2a and LLO 4b was subjected to isoelectric focusing, a single band migrating at a pI of 7.9 was observed. There may possibly be a non-covalent interaction between LLO l/2a and LLO 4b when they are mixed, which may affect their individual mobilities. The pI values for LLO 1/2a and LLO 4b reported above are not in agreement with the pl for LLO 4b reported by Kreft et el. (1989). The difference in pl values of LLO may be due to different purification methods. The observed differences in molecular size between LLO 1/2a and LLO 4b are consister,,,t with the structural differences observed in the nucieotide sequences of the genes coding for L L O in the two subgroups of L. monacytogenes, as evidenced by restriction frag-

LISTERIOL YSIN 0 F R O M SERO'I'YPES 1/2a A N D 4b m e n t length p o l y m o r p h i s m ( R F L P ) (Vines a n d S w a m i n a t h a n , manuscript in press) a n d by a c o m p a r a t i v e study o f the L L O nucleotide seq u e n c e f r o m L, m o n o e y t o g e n e s serotypes 1/2a a n d 4b (Rasmussen et aL, 1991). G e o f f r o y et aL (1987) showed t h a t the opt i m a l p H for haemolytic activity o f L L O l / 2 a is 5.5. O u r resul!s indicate that p H 6-7 is optimal foc the haemolytic activity o f L L O l / 2 a . This difference in o p t i m a l p H could be due in part to the difference in p u r i f i c a t i o n p r o c e d u r e a n d to the use of 50 % ethylene glycol in the alkaline buffer, Because haemolytie activity of LLO is not m a i n t a i n e d without ethylene glycol, we determined the effect o f ethylene glycol o n optimal p H for haemolytic activity using streptolysin O. O u r results showed t h a t in the presence o f ethylene glycol, the o p t i m a l p H for haemolytic activity o f S L O ghifted f r o m 7.9 to 6,5. A l t h o u g h b o t h L L G I / 2 a a n d 4b showed m a x i m a l stability at p H 8.0, L L O ~ was m o r e sensitive to p H 5 a n d 11 a n d less affected t h a n L L O l / 2 a u p o n increasing the time o f incubation to 4 h in p [ l 6-10 at 25°C. W h e t h e r these differences a l o n g witl~ differences observed in their specific activity significantly affect the p a t h o g e n i c i t y o f t h e two serotypes of L. m o n o c y t o g e n e s is not k n o w n . Vines a n d S w a m i n a t h a u (in press) have observed R F L P in the genes c~ding for L L O in L. m o n a c y t o g e n e s serotypes 1/2a m~d 4b, a n d their observation suggests possible structural differences between the two L L O . R a s m u s s e n et al. (lq91), however, c o m p a r e d the nueleotide sequence o f the gene c o d i n g for L L O f r o m a clinical strain o f L. monocytogenes serotype 4b isolated in Denmark with t h a t o f the nueleotide sequence o f the L L O gene in L. m o n o c y t o g e n e s serotype l / 2 a a n d f o u n d only m i n o r differences at the level o f the p r i m a r y structure o f the two proteins. W h e t h e r the nucleotide sequence o f the gene coding for L L O 4b determined by Rasmussen et al. is representative o f L L O 4b gene sequence from L, monocytogenes 4b strains associated with epidemic o u t b r e a k s needs to be determined. In conclusion, using the two-step imm u n o a f f i n i t y c h r o m a t o g r a p h y procedure (elution o f L L O with acetic acid b u f f e r p H 2.8 a n d

497

collection o f eluates in alkaline buffer containing 50 07o ethylene glycol, p H 11,5), LLO can be easily purified while preserving its haemolytic activity. Moreover. t h o u g h L L O l / 2 a a n d L L O 4b showed slight differences in molecular size, they b o t h have similar haemolytic activities and are maximally stable at p H 8.0, These findings should facilitate functional studies o n characteristics o f I.I.O a n d its role in the pathogenicity o f L, m o n o c y t o g e n e s , a n d o n the differences in pathogenic mechanisms between the two subgroups o f L. m o n o c y t o g e n e s . F u r t h e r m o r e , we are using i n f o r m a t i o n o b t a i n e d from this investigation o n the characteristics o f L L O ( p H a n d thermal stabilities) to develop a rapid immtmoassay for L L O in e n r i c h m e n t cultures o f foods.

Ackaowiedgements The authors w~uld like to thank Drs. Karen Gutekunst ~nd l en Pine for the gift of polyclonal antibody to the 60-kDa putative invasion-a~sociatcd protein of L. monocytogenes. This investigatio~ was sugported in part by a grant from the Nafionai Dairy Promotion ann Research Boa~'d' G.M.M., was a post~tocloral fellowsupported by this grant.

Purii'icafion par immunoaffinit~, stahilis-qtion et caract6risation de la list~riolysine 0 de Listeria monoo,togenes s~rotypes 1/2a el 41)

Un pent,de simple et ires efficient a ere mis an point pour stabiliser I'activite h~molytiqu¢ de la list6riolysine O (LLO) de Listeria monocytogenes ape, s purification par irnmunoaffinitd. Cette stabilisation est obtenue apres ~lution direete de la LLO dans de~ tubes contenant un tampon alealin (lysine 5 raM, KCI 140 mM, ethy|¢ne glycol 50 070, pH 11,5). La LLO purifi6e conserve 100 % de son aetivit& hdmo!ytique apr6s 6 semaines de conservation h - 20~C. La LLO purifi6e de sdrotype l/2a (ATCC 43249) et la LLO obtenue d'une souche de serotype 4b (F 2365) iso16e d'un fromage de type mexicain n'ont pus montr~ de dirf6rence significative de la stabilit6 A l'dgard des variations de pH et de temp6rature. L'iucubation darts des tampons dc pH allan~ d~ 4 a 12 eta des ~emp~ratures de 4 h 25°C a montr,~ que |es LLO de serotype l/2a et 4b conservent un maximum d'activii6 hdmolylique a pH 8 duns one limite de 4 h d'incubation, kes deux LLO perdent Ieur activit~ hemolytique apr&s 25 min d'incubation b. 50~C.

498

G,M. M A T A R E T A L ,

Mots.cl~s: Listt~rloly~iue O, Virulence, Listeria monocytogenes; S6rotypes l / 2 a et 4b, Activit~ hemolytique.

References

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