(~ INSTITUT P,~.S]EUR/ELSr~VlbR Paris 1992
Re$. MicrobioL
1992, 143., 449-4.5,7
Expression of M6 protein gene of Streptococcus pyogenes in Streptococcus gordonii after chromosomal integration and transcriptional fusion G, Pozzi 0, z)(*~, M.R. Oggioni (t~, R. Manganelli (Ij and V.A. Fis~.hetti (3~ o~ Dipartimento di Biologia Molecolare. Se~ione di Microbiologia, Universitit di Siena, Via Laterina, 53100 Siena (Italy), tej lstitulo di Mictobiologta, Universit~ di Vetona, Slruda Le Grazie, 37134 Verona (I'~aty) and t3/ The Rockefeller University. 1230 York Ave., New York, N Y 10021-6399
SUMMARY
The M 6 protein uf Streptococcus pyogenes was expressed on the cell surface and secreted in Streptococcus gordon# Challis (formerly Streptococcus sanguis) after chromosomal integration of a promoterless M 6 protein gene ~emm-6.11. The ermC gene, conferring resistance to erythromyc!.~, was cloned downstream of emm-6. 1, within the same Clal fragment. The initiation codon of emrn-6. 1 was 19 bp downslream of a Clal site, so that Clal cleavage would leave the gene promoterless. The Cfa! fragment containing the promoterless emm-6.1 and ermC was iigated in vitro with a Clal digest of S. gordonii chromosomal DNA. Random chromosomal integration of the haterologous DNA was obtained by using the ligation mixture to transform the naturally competent S. gordon# Challis. Twenty-eight percent of transforrnants selected for erythromycin resistance also expressed M6. ~,mong the best M6 producers, 10 clone~ were selected for the stability of their phenotype. Nine of the 10 c;ones were shown to harbour one intact copy ef the emm-6. T/ermC Clal fragment integrated into the chromosome, These strains both expressed M6 protein on the surface and secreted different amounts of the molecule, since in each case the protein was produced after a transcriptional fusion of emm-6.7 with a different chromosomal promoter. A S. gordonii strain expressing large amounts of surface iVI6 protein, as judged by immunofluorescsnce and Western blot, was compared to the M - parental stra,n in a standard opsonophagecytosis assay. Of the isogenic pair, M 6 ~ S. gordonfi survived better in human blood and was phagocytosed at a slower rate.
Key-words: M protein, Gene emm-6. 1, Streptococcus pyogenes, Streptococcus gordoniJ; Transcription, Chromosomic DNA, Genetic engineering.
INI'RODUCTION
et eL, 1989; Guss et aL, 1984; Haanes and
Sequcnce analyses of sur.L-.c~ proteins from Gram-positive cocci (Ferretti et al., 1989; Frithz
C!ear:e, 1989 ; Heath and Cleary, 1989; Hollingshead et al., 1986; Kok et al., 1988; Okahasifi el al., 1989; Olsson et al., 1987 ; Schneewirl,rl ef
Submitted J~lovember25, 1991, acccpterl February 25, ]992. (*] Currcspo~[mg ~uOl~}r.
,$50
G. P O Z Z i E T AL,
al., 1990; Simpson et ~L. 1990) have revealed a similar arrangement of hydrophobic amino acids and charged tails at the C terminus of these molecules. Furthermore, a hexapeptidc seouence, with the consensus sequence L P S T G E , which begins 9 residues N-terminal to the hydrophebie t ¢gion, is found in all these surface proteins (Fischetti et al., !990). This highly conserved motif suggests a common mode of attachment for these different surface molecules. For this reason, it should be possible, after appropriate genetic manipulations, to express these surface proteins in heterologous Gram-positive hosts. Unfortunately, however, the ability to express M protein in ether Gram-posit!re hosts has met wire limited success.
a promoterless emm-6,1 gene into the chromosome, By this method, transcriptional fusions of e m m - 6 , 1 are m a d e with c h r o m o s o m a l promoters, exploiting the natural competence for genetic transformation of S. gordonii (Frakula et al., 1958 ; Macrina et al., 1980 ; Pozzi el a t , 1990), Our approach to genetic manipuiatiun of :~treptococci is based on c h r o m o s o m a l integration o f re:ombinant D N A molecules rather than on self-replicating plasmid vectors (Pozzi and Guild, 1985 ; Pozzi et al., I988, 1991 ; Oggioni and Pozzi, 1990). The M6-prodncing recombinant strains obtained by this method proved to have a stabIe phenotype and a normal doubling time. Part o f this work was presented at the 3rd International ASM Conference on Streptococcal Genetics (Minneapolis, 1990).
M protein is a fibrillar surface molecule of Streptococcus pyogenes (Fischetti et al., i988; Fischetti, 1989), which is considered to play a major role in pathogenicity by virtue o f its an-. tiphagocitic activity (Lancefield, 1962). In previous studies, the cloned M6 protein gene (emm-6.1) (Scott and F!~chetti, 1983; Hollingshead et al., 1986), containing its putative promoter, was integrated into the conjugative transposon f/6001 (now calted Tn5253) and transferred to the c h r o m o s o m e o f different streptococci (Oggioni and Pozzi, 1990). In these experiments, no expression o f M6 protein was observed in either Streptococcus gordon# Challis (formerly Streptococcus sanguis, see Kilian el al., 1989), Streptococcus pneumoniae, or S. pyogenes. An Escherichia coil~Streptococcus shuttic plasmid (pVA838) was also used to introduce the e m m - 6 (Scott et al., 1896) and e m m - 5 (Poirier et al., 1989) genes ip,.o S. gordonii Challis. M6 was not expressed on the surface of S, gordonii (Fischetti, unpublished results), and M5 was expressed very poorly (Poirier et a t , t989).
MATERIALS AND MEi'HODS Plasmids, I~acteria and growth conditions
Plasmids and bacteria are descrihed in table I. Streptococcal strains were grown in brain-heart ~nr fusion (BH! ; Difeo Lab., Detroit, Mich.) or in ToddHewitt broth (TInIB; Difco Lab.). Agar (I..5 %) was added to BHI to obtain solid media. All cultures were incubated at 37°C. Recombinant D N A techniques
Standard procedures were used for subcloning and Southern blot analysis (Maniatis et al., 1982). Transforma!ion
Frozen cells of naturally competent S. gordonii Challis were prepared and transformed as already described (Pozzi e¢ al., 1990). Chromosomal DNA o! GP204, containing a streptomycin resistance marker, was used at a concentration of 0.5 ~g/ml of competent cells to control the level of competence
Here we describe the construction of strains of S. gordonff, lhat express M6 protein on the cell surface, obtained by random integration of
}~,HI CFU Em-T rDAb
=
bra itl l~zarI iltlu~doa b r o t h . co[ofl y-f of I p.i]lg ~.lllit. erylhronlyci3 ~esistam. fil 0 P.O[lO.rl~.J ~ n l i b o d ~ .
i i
OD PHS THB
=
optical density. phosphate buft~led saline. Tod, d~Hewht broth.
E X P R E S S I O N OF/146 P R O T E I N G E N E I N S T R E P T O C O C C U S G O R D O N I I o f S. gordonii in each transformation experiment. Plating and scoring of transformants on multilayered 01ates was also as previously described (Pozzi et al., 1987)." e r ~ h r o m y c i n was added at 5 I~g/ml and slreptomycin at I000 ~ g / m l in the overlay,
451
i~ranes as described (Fischetti et al., 1985), The presence ol" M protein was visualized using m A b 10B6 as described above. immunoflunrescence
"Streak blot" T r a n s f o r m a n t s were streaked on the surface of a blood agar plate by toothpick transfer o f colonies f r o m the selection plates. Each plate (9 cm o f diameter) contained streaks from 50 different transform a n t s , a n d M6 + (S, pyogenes D471) and M 6 (S. gordonii V2g8) controls. After 36 h of incubation at 37°C, a nitrocellulose m e m b r a n e was applied to the surface o f the plate a n d kept there for 20 mln at room temperature. T h e m e m b r a n e was then incubated 30 rain at 37"C and 15 rain at 80°C in a v a c u u m oven. The presence o f M6 protein b o u n d to the nitrocellulose was visualized by the m e t h o d o f Blake et aL (1984), using the monoclonal antibody (mAb) 10B6, raised against recombinant M6 protein purified from E. coil {ColiM6) (Jones et al., 1985).
Semiquanlilative dot b l o t assay A n exponentially growing liquid culture at an optical density (OD) of 0.6 was used for this assay. Turbidity was measured by determiuing the O D at 590 n m with a " B e c k m a n D U - T ' s p e c t r u p h o t o m e ter, using cuvettes with a 10-ram path. After a cent r i f u g a d o n step, cells were r e s u s p e n d e d in phosphate-bu_rfered saline (PBS). T h e equivalent of 0.5 ml o f culture, resuspended in 50 ~zl o f PBS, and 4-fold dilutions o f it were applied to a 96-well dotblot a p p a r a t u s (Bethesda Research Laboratories, Gaithersburg, Md), assembled with a nitrocellulose m e m b r a n e . T h e m e m b r a n e s were reacted with M6-specific m A b a n d processed as described for Western blots.
Bacteria were grown in ] l i B harvested in the late exponential phase, washed, and resuspended in PBS to 20 % o f the growth volume. Ten mierolitres were then applied on a clean glass slide, air-dried, a n d fixed for one rain in methanol. Rabbit anti-Co.qM6 s e r u m (lgG fraction) tagged with fiuoresccm isothiocyanate (van de Rijn et aL, 1977) was auded to the surface and the slides were incubated for 30 min at room temperature in a moist chamber. Slides were ~hen washed in PBS, air-dried, and a cover Jlip was placed over 10 pl o f quencher (pphenylenediamine, l m g / m l in 90 o70 glycerol). Bacteria were observed under a "Zeiss'" fluorescence microscope (Carl Zeiss, Inc., Yhornwood, NY) using an t 0 0 × oil objective. Opsonophagoeytosis ~ssay This is a modification o f a previously described assay (Fischetti, 1983). Briefly, bacteria grown lo ¢,~ponential phase were diluted in T H B and 0.1 m~ were added to sterile 9 - m m glass tubes containing 0.4 ml o f heparini,'ed (10 units/ml) h u m a n bloc, O. Two capped test tubes were prepared for each inoculum : one was rotated end over-end, and the second remained stationary. After 3 h o f ~ncubation o, 37~C, the content of the tubes was entirely plated. Plates were incubated 36 h at 37°C, and colonies were carefully counted..
Cldi
~0
Her ~,la t ~
A~tl A'~n
Extractian o[ M protein and Western blot Bacteria were grown in 5.0 ml o| T H B , harvested in the late exponential phase (0.50Ds~r,) a n d washed once in 0.02 M p h o s p h a t e buffer, pH 7.0. After sedimentation, cells were resuspended in 200 pl o f the s a m e buffer containing 30 % ruff lapse. Mutanolysin (Sigma C h e m . Co., St. Louis. Mo'~ was a d d e d to a final concentration o f 50 t~g/ml and the mixture was incubated at 37°C for 1 h. T h e protoplasts were sedimented by centrifugation and :l~e supernatant, containing the M protein, ',',as separated on 14 % sodium dodecyl sulphate/polyacrylamlde gels and electrotransferred onto nitrocellulose mem~
V.I,I]
[Cl,I]llI!,p])
[ ; I ~l I
Fi~. I. Ech~-:maKi¢ r~pr=.~,¢ntation of the 3.4 kb C'/a| f~-J.g ment Of p V M B3 containing ,.~mm-6_/ and ~rmC.
Oal cteava.ee oi' pVMB3 leave~ 'chc e m m 6.1 prnrno~crless, hul wi~h an int-ict ribosemal I~inding sin:, '.,Vhi'~e: 2.2-kb C'/al fragment of pVV3:M6A (HruOy el at.. 1"98g1: Erey: 1.2-kb ?.-ts/~[:Girl fra~mcnl nf pF i94 (Flnrinmtchi and w¢isbtum, 19~2!.
452
G, P O Z Z I E T AL, RESULTS
Construction of the restriction fragment to be
DHS~x, a clone was isolated with a plasmid, pVMB3, containing the Ctal fragment shown in figure 1.
integrated A construct was made where e r m C (Horinouchi and Weisblum, 1982), a gene conferring resistance to erythromycin, was cloned downst;eam of e m m - 6 , ! , so that both genes would be within the same Clal fragment. In this construct, the initiation codon of e m m - 6 . 1 was 19 bp downstream of one of the ClaI sites, so that Clal cleavage would leave e m m - 6 . l promoterless (fig. 1). Plasmid pVV3;M6A (Hruby et aL, 1988), comaining this ClaI site upstream of the ernm-6.l-coding sequence, was used in the experiments (table I). The 2.0-kb M s p l fragment of pE194, containing ermC, was ligated with a partial Clal digestion o f pVV3:M6A. After transformation of E. coli
Chromosomal integration The 3.4-kb Clai fragment of pVMB3, containing the promoterless e m m 6 . l and ermC, was figated with chromosomal DNA of S. g o r d o n i i also cut with Clal. The ligation mixture was used to tranzform the naturally transformable S. gord o n i i "Challis", strain V288. By this method, the e m m - 6 . 1 / e r m C Clal fragment (fig, I) was integrated at random into the chromosome. The chromosomal DNA figatcd to the Clal fragment provided the homology for integration during transformation. Erythromycin-resistant (Em-r) transformants were selected (table 11) and analysed for production of M6 protein by streak
Table I. Bacterial strains and plasmids. Strains/plasmids
Relevant properties
S. gordon# Challis V288 GP204 GP221-GP230 GP23 [ S. pyogenes D471 pVV3:M6A pE194 pVMB3
Reference
transformation recipient str-204 emm-6, l / e r m C emm-6, l / e r m C str-204
Macrina et aL, 1980 Pozzi et aL, 1988 This study Thi~ study Scott and Fisehetri, 1983 Hruby et aL, 1988 Hor~oouchi and Weisblum, 1982 This study
emm-6.1 emm-6.1 er~nC emm-6, l / e r m C
~ l r - 2 0 4 - chram~3somal point ro mali.On conferring resistance to slreptomycin; e m m - 6 , 1 - gone encoding for type 6 M protein ; e r m C = geae encoding for resistance to crythromycin; e m m - 6 , 1 / e r m C = 3.4-kb Clui fragmenl contaieing bolh genes (fig. 1).
Table 11, Transformation of S. gordonii Challis. Donor DNA
Ligase treatment
Transformants
Genetic analysis
emm-6, l / ermC + chrom. DNA
Yes
1.8 × l03
emm-6.1/ermC+ chrom. DNA
28 % Em-r, M6* 72 % Era-r, M6-
No
: 104 7.1 × 103 8.5× 103
900 32 32 3
1.7X ]05 5.8x 104 8.i × 10~ 4.0× 103
'Two test 'tube.~ ',,.ere prepared for ea,.:h inoeulum : one ",,,'aslefl on the rack (sto.tJ.,..t~ary) und one was rotated, for 3 h at 37°C. Results V;ere expressed a~ mtai C [ U present in the ~esl tube (0.6 ml volume).
EXPRESSION OF M6 PROTEIN GENE IN STREPTOCOCCUS GORDONIi
455
promoterless M6 protein gene was linked in vitro to an e r y t h r o m y e i n resistance m a r k e r , a n d the restriction f r a g m e n t c o n t a i n i n g b o t h genes was ligated to r a n d o m fragments o f S. gordonff chrom o s o m a l DNA. [[sing this ligation mixture to t r a n s f o r m the naturally c o m p e t e n t S. go~'donii Challis, r a n d o m integration o f the heterologous D N A (emm-6. l/erraC) was o b t a i n e d . In naturally c o m p e t e n t streptococci, heterologous D N A ligated to c h r o m o s o m a l sequences can be integrated i n t o the c h r o m o s o m e d u r i n g t r a n s f o r m a t i o n (Vasseghi et al., 1981; Vasseghi a n d Claverys, 1983; Mannarelli a n d Lacks, 1984; M o r r i s o n et al., 1984; Pozzi a n d Guild, 1985). As previously discussed (Pozzi and Guild, 1985), t h e usual product o f this process is the integration o f o n e copy o f the heterologous D N A between t w o direct repeats o f the h o m o l o g o u s c h r o m o s o m a l segment. M o r e t h a n o n e q u a r t e r (28 % ) o f t h e t r a n ~ f o r m a n t s , in which the heterologous D N A was integrated, expressed the M6 protein, In these t r a n s f o r m a n t s , a functional t r a n s c r i p t i o n a l fusion o f emm-6.1 with chrom o s o m a l promoters h a d occurred. As expected, the t r a n s f o r m a n t s expressed different a m o u n t s o f M6. Each was p r e s u m a b l y the p r o d u c t o f a difl'erent intcgrational event, which h a d led to a fusion with a different p r o m o t e r . Screening several t r a n s f o r m a n t s allowed t~or the selection o f the best producers. A m o n g t h e m , it was possible to select strains with a stable phenotype and with a single copy o f emm-6.1 integrated into the chromosome.
sion is complex and not completely u n d e r s t o o d ( C a p a r o a a n d Scott, 1987 ; Robbins et al., 1987; S i m p s o n et al., 1990). Previous a t t e m p t s to express cloned e m m genes from their own p r o m o t e r s in various streptococcal hosts always failed to produce stable a n d " h e a l t h y " reeomb i n a n t s (Scott et aL, 1986; Poirier et aL, 1989; Oggioni a n d Pozzi, 1990'~ This was due, in our opinion, to two types of problems : (1) those connected with ~he regulation o f expression of the e m m gene away from its c h r o m o s o m a l locus, a n d (2) those connected with plasmid instability. In the present study, these problems were overcome by integrating the coding sequence of emm-5, l into the c h r o m o s o m e o f S. gordonii, downstream o f strong c h r o m o s o m a l promoters.
I m m u n o f l u o r e s c e n t staining of the bacterial surface a n d Western blot analysis s h o w e d that M 6 was expressed o n the surface o f S. gordonii a n d also secreted in the m e d i u m . The presence o f M6 o n the surface o f S. gordonii e n a b l e d the o r g a n i s m s to survive f r o m ~0 to 30 times better in h u m a n blood when c o m p a r e d to the M ~ o r ganisms. This antiphagocytic activity is ]~ot as p r o n o u n c e d as w h e n the M molecule is o n the native S. pyogenes. However, S. gordonii is p r o b a b l y ustfited to test the antiphagocytic activity o f the M molecule in the classic bactericidal assay, since, unlike S. pyogenes, it dt>es not grow weI[ in h u m a n blood.
Expression du gbne de la proleine M6 de Strepzococcus pyogenes ehez Streptoeoccu~ gordon# apr~s integration chromosomique et fusion lranscriptionnelle
Regulation o f M protein gene (emrn) expres-
This new method allows (1)expression of heterologous genes in S. gordonii, (2) selection of strong p r o m o t e r s , a n d (3) selection of stable ]'econqDincqi'tis. ,.-r : . . .tylJt~ . . . o f ap~J~ tL JlJ~, . . . . . . Ua~jLJ . C,~ii ~0~ Of great help in the c o n s t r u c t i o n o f streptococcal strains to be used in further genetic engineering experiments utilizing heterologous genes.
Acknowledgements This work was s~lpported in part by grants from CNR {Progetto Finaliz;,ato ingegneria Generica. n. 91.000!6.90) and ISS (IV progetto AIDS, n. 6207-011)to GP. NIH (Gran~ A111822) to VAF. and NATO (Collaborative Research Grant n. 0840/88).
La prot~ine M6 de Streptococotspyogenes a ~t~ exprim6e fi la surface c~llulaire et s~cr&oe chez Strep(ococcus gordonii ChaI[is (ant,2ricurcment Streptococcus sanguis), ~pr~s inz6gration du gene (emm-6. l) de la prot~ine Me, sa:~s promoteur. Le g~ne ermC coat, rant la r~sistance 5. l'&ythromycine a et~ clone- en aval de emm-6.1, a l'int~rieur du m~mc fl-agmt nt CtaI. Le codon d'initiation du gene emm-6. Icst un fragment de 19 pb en aval du site Clal, de sorre que la coupure par Clal laisse le g~ne san~ promotet:r. Le fragment C/u[ contenant Ic gbne ermC e; te g ~ e emm-6. ! sans promoteur, a
456
C. P O Z Z I E T A L .
dt~ lid in vitro fi un fragment C/at d ' A D N c h r o m o somiclue de S. gordonii. L'int~gration c h r n m o s o m i que au hasard de I ' A D N h~t~rologue a ~t~ obtenue par l'utilisation du m d a n g e 1i$ pour t r a n s f o r m e r la s o o t h e de S. gordolffi Challis naturellement compStente. Vingt-huit pour cant des t r a n s f o r m a n t s s~lecAioun~s pour leur rds]stance ,h 1'Srythromyeine exp,iment aussi Ia prc, t~ine MS. Dix clone~ parmi los meilleurs producteurs de M6 a n t 6t~ sSlectionn~s sur la base de leur stabilit~ phSnotypique. Nenf des dix clones sont porteurs d ' u n e topic intacte du fragment Clal, emm-6.1/ermC, int~gr~ dans le c h r o m o s o m e . Ces souches expriment la protiine M6 A la surface et en secretcnt d¢~ qu,ttltitcs vari~es: dons c h a q u e cas la prot~ine a ~t~ produite aprgs fusion transcriptionnelle du g~ue ernra-6.1 avec uu p r o m o t e u r c h r o m o somique. Unc souchc dc S. gordpnff e x p r i m a n t de grandes quantitSs de protSine de surface M6, Svalu~es pat immunofluorescence et W e s t e r n blot, a ~t$ c o m p a rSe ~, la souche parentale M - par dosage standardis6 de l ' o p s o n o p h a g o c y t o s e . De la paire de s o o t h e s isog~niques, S. gordonii M6 ÷ ~urvit mieux dans 1¢ sang h u m a i n et elle est phagoeytSe plus lentement. Mots-clds : Prol~ine M, Streptococcus pyogenes, Streptococcus gordonii, G~ne e m m - 6 . 1 ; Transcription, A D N c h r o m o s o m i q u e , GSnlc gSn~tique.
Reterences
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region of surface proteins from 8ram-positive coccl.
MoL Microbial., 4, i(,03-1605. Fiscbelli, V.A., Parry, D,A,D., Trus, B.L., Scott, J.R. & Manjula, B.N. (1988), Conformational characteristic of the complete sequence of group A streptococcal M6 protein. Prot. $truct. FuncL Genetics, 3, 60-69. Frithz, E., Heden, L.-O. & Lindahl, G. {1989), Extensive sequence homology between IgA receptor and M protein in Streptococcus pyogenes. MoL Microbial., 3, 1Ill-1119. Guss, B., Uhlen, M., Nilsson, B., Lindherg, M., Sjoquist, J. & Sjodahl. J. (19841, Reaion X, the cell-wallattachment part of staphylococcal protein A. Europ. J. Biocl~em,, 138, 413-420. Haanes, E.J. & Cleary, P.P. (1989), Identification of a divergent M protein gone and an M protein related gone family in serotype 49 Streptococcus pyogenes, J. Boot., 171, 6397-6408. Heath, D.G. & Cleary, P.P. (1989), Fe-receptor and M protein genes of group A streptococci are products of gone duplication. Pro¢. hal Acad. SoL (Wash.), 86, 4741-4745. Hollingshead, S.K., Fischetti, V.A. & Scott, J.R. (1986), Complete nucleotide sequence of type 6 M protein of the group A streptococcus, d. bioL Chem., 216, 1677-1686. Ho, iaouchi, S. & Weisblam, B. (1982), Nucleotide sequence and functional map of pE 194, a plasmid that specifies resistance to macrolide, lincosamide, and streptogramin type B antibiotics. J. Boot., 150, 804-814. Hruby, D.E,, Hodge, W.M., Wilson, E.M., Franke, C.A. & Fischetti, V.A. (1988), Expression of streptococcal M protein in mammalian cells. Proc. nat. Acad. SoL (Wash.), 85, 5714-5717. Jonas, K,F., Manjula, B.N., Johnston, K,H., Hollingshead, S.K+, Scott, J.R. & Fischetti, V.A. (1985), Loeolian of variable and conserved epiropes among the multiple ~erotypcs of streptococcal M protein. J. exp. Med., 16t, 623-628. Kilian, M., Mikkelsen, L. & Henrichscn, J. (1989), Taxonomic study of viridans streptococci: description of Streptococcus gordonii sp. nov. and emended descriptions of Streptococcus sanguis (White and Niven, 1946), Streptococcus oralis (Bridge and Sheath, 1982), and Streplococcns mills (Andrewes and Harder, 1906). Int. J. sysl. Boot., 39, 471-484. Kok, J+, Leenhouts, K.J., Haandrikman, A.J., Ledeboer, A.M. & Vcnema, G. (1988), Nucleoiide sequence of the cell wall protcinam gone of Streptococcus eremori$ Wg2. AppL Environ. Microbial., 54, 231-238. Lancefield, R.C. (19fi21, Current knowledge of the type specific M antigens of group A streptococci. J. Immunol., 89, 307-313. Macrina~ F.L., Wood. P.H. & Jones, K.R. (1980), Genetic transformation of Streptococcus sanguis (Challis) with cryptic pin,raids from Streptococcusferua. Infect. lmmun., 28, 692-699. Maniatis, T., Fritsch, E.F. & Sambrook, J. (1982), Molecular cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, New York. Mannarell[, B.M. & Lacks, S.A, (1984), Ectopic integration of chromosomal genes in Screptococcua pnevmostiae. J. Boot., 160, 867-873. Morrison, D.A., Trombe, M.-C., Hayden, M.K., Waszak, G.A, &Chen, J.-D. (1984), Isolation of transfurma-
E X P R E S S I O N O F M6 P R O T E I N GENI~" I N S T R E P T O C O C C U S G O R D O N I I finn deficient Streptococcus pneumoni~.c mutants defecting in controt of competence, using insertion duplication mulagenesis with the erythromycin resistance determinant of pAM[3I. J. Bact., 159, 870-876. Oggioni, M.R. & Pozzi, G. (1990), Conjugativ~ mobilization of the cloned M6 protein gene from Streptocm~ cus pneumoniae to Streptococcus pyosenes. Microbiologrctr, 13, 273-281. Okahashi, N., Sasakaw~, C., ¥oshikawa, S., Hamada, S. & Koga, T. (t989), Molecular characterization of a surface protein antigen gene from serotype C Streptococcus mutuns implicated in dental caries. MIoL MierobioL, 3, 673,678. Olsson, A,, Eliasson, M., Guss, B., Nilsson, B., Hellman, U., Lindberg, M. & Uhlen, M. (1987), 5*.rueture and evolution of the repelitive gene encoding, streptococcal protein G. Europ. J'. Biochem., t68, 319-324. Pakula, R., Fluder, Z., Hulanicka, E. & Walczak, W. (1958), Studies on transformation of streptococci. Bull. Acad. poL ScL Ser. Sci. Biol., 6, 319-323. Poirier, T.P., Kehee, M.A., Whitnack, E., Doekter, M.E. & Beachey, E.H, (1989), Fibrinogen binding and resistance to phagocytosis of Streptococcus songuis expressing cloned M protein of Streptococcus pyogenes. Infect. hnmun., 57, 29-35. Pozzl, G. & Guild, W.R. (1985), Modes of integration of heterotogous plasmid DNA into the chromosonae of Streptococcus pneumottiae. J. Buct., 161, 90%912. Pozzi, G., Musmauno, R,A., Lievens, P.M.-J., Oggioni, M.R., Plevani, P. & Manganelli, R. (1990L Me'~hod and parameters for genetic transformation of Streptococcus sanguis Challis. Res. MicrobioL, 141, 659-670. Pozzi, G., Musmanno. R.A., Renzoni, E.A.. Oggloni. M.R, &Cusi, M.G. (1988), Host-vector system for integration of recombinant DNA into chromosomes of transformable and non~trans formabte streptococci. J. Boot., 170, 1969-1972. Pozzi, G., Musmanno, R.A., Stellini, M. & Molina, A.M. (1987), Transformation of Streptococcus satrguis Challis with a plasm~.dof Streptococcus pneurnoniae. FEMS Mtcrobiol. Letters, 48, 189-194. Pozzi, G., Ogginni, M.R., Manganelli, R. & Plevani, P. (1991), Genetic manipulatior~ of streptococci by chro mosomal integration of recombiu~,u~ DNA, in
457
"Genetics and Molecular Biology of Streptococci, Lactococci, and Enteroeocci" (Dunny, G., Cleary, P. & MeKay. L) (pp. 59-61L American Society for Microbiology, Washington, DC, Kobbins, J .C., Spanier, J.G., Jones, S,J., Simpson, W.J. & Cleary, P.P. 11987). Streptococeuspyogenes type-t2 M protein gene regulatiorl by upstream sequences. J. Roct., I69, 5633-5640. Scimcewind, O,, Jones, K.F. & Fischetll. V A. (1990), Sequence and structural characterization of the trypsinresistant T6 surface protein or group A streptococci. J. Boer., 172, 3310-3317. Scott, J.R. & Fischetti, V.A. (1993), Expression of streptococcal M protein in Esct~eriehia coil Science, 221, 758-760. Scott, J.R., Guenthner, P.C., Malone, L.M. & Fischerti, V.A. (1986h Conversion of an M- group A streptococcus to M ' by transfer of a plasmid containing an M6 gene. J. exp. A'Ied., 164, 1641-1651. Signas, C., Raueci, G., Jonsson, K., Lindgren, P., Anantharamaiah, G.M., Hook, M. & Lindberg, M. (1989), Nucl~tide sequence of the gene [or fibronectinbinding protein from Staphylococcus aureus: use of this pcptide sequence in the synthesis of biologically active peptide:. Proc. nat. Acad. Set. (Wash.), 86, 699-703, Simpson, w . j . . LaPenta. D., Chen, C. & Cleary~ P.P. (1990), Coregulatior~ of type-12 M protein and streptococcal CSa pcplidase genes in grou~ A streptococci : evidence for a virulence regulon eonlroiled by the vb'R locus. ,L Bact.. 172, 696-700. Van de Rijn, I., Zabriskie, J.B. & McCarty, M. (1977}, Group A streptococcal antigens cross-reactive with myocardium. Purification of heart-reactive antibody and isolation and characterization of the streptococcal antigen. J. exp~ Med., 146, 579. Vasseghi, H. & Claverys, J.-P. (1983), Amplification of a eh[merlc plasmid carrying an ery~hromycln resistance determinant introduced taro the genome o[ Slreptococcus pneumoniae. Gene, 21, 285-292. Vasseghi, I--L, Claverys, J'. P. & Sicard. A.M. (19glL Mechanisms of integrating roreign DNA during transformation of &rept,~coccuspneumoniae, in "Transformation 1980" (Polsinelli, M. & Mazza, G.) (pp. 137-153). Cos~wo]d Press, Oxford.
Re$. MicrobioL
1992, 143., 449-4.5,7
Expression of M6 protein gene of Streptococcus pyogenes in Streptococcus gordonii after chromosomal integration and transcriptional fusion G, Pozzi 0, z)(*~, M.R. Oggioni (t~, R. Manganelli (Ij and V.A. Fis~.hetti (3~ o~ Dipartimento di Biologia Molecolare. Se~ione di Microbiologia, Universitit di Siena, Via Laterina, 53100 Siena (Italy), tej lstitulo di Mictobiologta, Universit~ di Vetona, Slruda Le Grazie, 37134 Verona (I'~aty) and t3/ The Rockefeller University. 1230 York Ave., New York, N Y 10021-6399
SUMMARY
The M 6 protein uf Streptococcus pyogenes was expressed on the cell surface and secreted in Streptococcus gordon# Challis (formerly Streptococcus sanguis) after chromosomal integration of a promoterless M 6 protein gene ~emm-6.11. The ermC gene, conferring resistance to erythromyc!.~, was cloned downstream of emm-6. 1, within the same Clal fragment. The initiation codon of emrn-6. 1 was 19 bp downslream of a Clal site, so that Clal cleavage would leave the gene promoterless. The Cfa! fragment containing the promoterless emm-6.1 and ermC was iigated in vitro with a Clal digest of S. gordonii chromosomal DNA. Random chromosomal integration of the haterologous DNA was obtained by using the ligation mixture to transform the naturally competent S. gordon# Challis. Twenty-eight percent of transforrnants selected for erythromycin resistance also expressed M6. ~,mong the best M6 producers, 10 clone~ were selected for the stability of their phenotype. Nine of the 10 c;ones were shown to harbour one intact copy ef the emm-6. T/ermC Clal fragment integrated into the chromosome, These strains both expressed M6 protein on the surface and secreted different amounts of the molecule, since in each case the protein was produced after a transcriptional fusion of emm-6.7 with a different chromosomal promoter. A S. gordonii strain expressing large amounts of surface iVI6 protein, as judged by immunofluorescsnce and Western blot, was compared to the M - parental stra,n in a standard opsonophagecytosis assay. Of the isogenic pair, M 6 ~ S. gordonfi survived better in human blood and was phagocytosed at a slower rate.
Key-words: M protein, Gene emm-6. 1, Streptococcus pyogenes, Streptococcus gordoniJ; Transcription, Chromosomic DNA, Genetic engineering.
INI'RODUCTION
et eL, 1989; Guss et aL, 1984; Haanes and
Sequcnce analyses of sur.L-.c~ proteins from Gram-positive cocci (Ferretti et al., 1989; Frithz
C!ear:e, 1989 ; Heath and Cleary, 1989; Hollingshead et al., 1986; Kok et al., 1988; Okahasifi el al., 1989; Olsson et al., 1987 ; Schneewirl,rl ef
Submitted J~lovember25, 1991, acccpterl February 25, ]992. (*] Currcspo~[mg ~uOl~}r.
,$50
G. P O Z Z i E T AL,
al., 1990; Simpson et ~L. 1990) have revealed a similar arrangement of hydrophobic amino acids and charged tails at the C terminus of these molecules. Furthermore, a hexapeptidc seouence, with the consensus sequence L P S T G E , which begins 9 residues N-terminal to the hydrophebie t ¢gion, is found in all these surface proteins (Fischetti et al., !990). This highly conserved motif suggests a common mode of attachment for these different surface molecules. For this reason, it should be possible, after appropriate genetic manipulations, to express these surface proteins in heterologous Gram-positive hosts. Unfortunately, however, the ability to express M protein in ether Gram-posit!re hosts has met wire limited success.
a promoterless emm-6,1 gene into the chromosome, By this method, transcriptional fusions of e m m - 6 , 1 are m a d e with c h r o m o s o m a l promoters, exploiting the natural competence for genetic transformation of S. gordonii (Frakula et al., 1958 ; Macrina et al., 1980 ; Pozzi el a t , 1990), Our approach to genetic manipuiatiun of :~treptococci is based on c h r o m o s o m a l integration o f re:ombinant D N A molecules rather than on self-replicating plasmid vectors (Pozzi and Guild, 1985 ; Pozzi et al., I988, 1991 ; Oggioni and Pozzi, 1990). The M6-prodncing recombinant strains obtained by this method proved to have a stabIe phenotype and a normal doubling time. Part o f this work was presented at the 3rd International ASM Conference on Streptococcal Genetics (Minneapolis, 1990).
M protein is a fibrillar surface molecule of Streptococcus pyogenes (Fischetti et al., i988; Fischetti, 1989), which is considered to play a major role in pathogenicity by virtue o f its an-. tiphagocitic activity (Lancefield, 1962). In previous studies, the cloned M6 protein gene (emm-6.1) (Scott and F!~chetti, 1983; Hollingshead et al., 1986), containing its putative promoter, was integrated into the conjugative transposon f/6001 (now calted Tn5253) and transferred to the c h r o m o s o m e o f different streptococci (Oggioni and Pozzi, 1990). In these experiments, no expression o f M6 protein was observed in either Streptococcus gordon# Challis (formerly Streptococcus sanguis, see Kilian el al., 1989), Streptococcus pneumoniae, or S. pyogenes. An Escherichia coil~Streptococcus shuttic plasmid (pVA838) was also used to introduce the e m m - 6 (Scott et al., 1896) and e m m - 5 (Poirier et al., 1989) genes ip,.o S. gordonii Challis. M6 was not expressed on the surface of S, gordonii (Fischetti, unpublished results), and M5 was expressed very poorly (Poirier et a t , t989).
MATERIALS AND MEi'HODS Plasmids, I~acteria and growth conditions
Plasmids and bacteria are descrihed in table I. Streptococcal strains were grown in brain-heart ~nr fusion (BH! ; Difeo Lab., Detroit, Mich.) or in ToddHewitt broth (TInIB; Difco Lab.). Agar (I..5 %) was added to BHI to obtain solid media. All cultures were incubated at 37°C. Recombinant D N A techniques
Standard procedures were used for subcloning and Southern blot analysis (Maniatis et al., 1982). Transforma!ion
Frozen cells of naturally competent S. gordonii Challis were prepared and transformed as already described (Pozzi e¢ al., 1990). Chromosomal DNA o! GP204, containing a streptomycin resistance marker, was used at a concentration of 0.5 ~g/ml of competent cells to control the level of competence
Here we describe the construction of strains of S. gordonff, lhat express M6 protein on the cell surface, obtained by random integration of
}~,HI CFU Em-T rDAb
=
bra itl l~zarI iltlu~doa b r o t h . co[ofl y-f of I p.i]lg ~.lllit. erylhronlyci3 ~esistam. fil 0 P.O[lO.rl~.J ~ n l i b o d ~ .
i i
OD PHS THB
=
optical density. phosphate buft~led saline. Tod, d~Hewht broth.
E X P R E S S I O N OF/146 P R O T E I N G E N E I N S T R E P T O C O C C U S G O R D O N I I o f S. gordonii in each transformation experiment. Plating and scoring of transformants on multilayered 01ates was also as previously described (Pozzi et al., 1987)." e r ~ h r o m y c i n was added at 5 I~g/ml and slreptomycin at I000 ~ g / m l in the overlay,
451
i~ranes as described (Fischetti et al., 1985), The presence ol" M protein was visualized using m A b 10B6 as described above. immunoflunrescence
"Streak blot" T r a n s f o r m a n t s were streaked on the surface of a blood agar plate by toothpick transfer o f colonies f r o m the selection plates. Each plate (9 cm o f diameter) contained streaks from 50 different transform a n t s , a n d M6 + (S, pyogenes D471) and M 6 (S. gordonii V2g8) controls. After 36 h of incubation at 37°C, a nitrocellulose m e m b r a n e was applied to the surface o f the plate a n d kept there for 20 mln at room temperature. T h e m e m b r a n e was then incubated 30 rain at 37"C and 15 rain at 80°C in a v a c u u m oven. The presence o f M6 protein b o u n d to the nitrocellulose was visualized by the m e t h o d o f Blake et aL (1984), using the monoclonal antibody (mAb) 10B6, raised against recombinant M6 protein purified from E. coil {ColiM6) (Jones et al., 1985).
Semiquanlilative dot b l o t assay A n exponentially growing liquid culture at an optical density (OD) of 0.6 was used for this assay. Turbidity was measured by determiuing the O D at 590 n m with a " B e c k m a n D U - T ' s p e c t r u p h o t o m e ter, using cuvettes with a 10-ram path. After a cent r i f u g a d o n step, cells were r e s u s p e n d e d in phosphate-bu_rfered saline (PBS). T h e equivalent of 0.5 ml o f culture, resuspended in 50 ~zl o f PBS, and 4-fold dilutions o f it were applied to a 96-well dotblot a p p a r a t u s (Bethesda Research Laboratories, Gaithersburg, Md), assembled with a nitrocellulose m e m b r a n e . T h e m e m b r a n e s were reacted with M6-specific m A b a n d processed as described for Western blots.
Bacteria were grown in ] l i B harvested in the late exponential phase, washed, and resuspended in PBS to 20 % o f the growth volume. Ten mierolitres were then applied on a clean glass slide, air-dried, a n d fixed for one rain in methanol. Rabbit anti-Co.qM6 s e r u m (lgG fraction) tagged with fiuoresccm isothiocyanate (van de Rijn et aL, 1977) was auded to the surface and the slides were incubated for 30 min at room temperature in a moist chamber. Slides were ~hen washed in PBS, air-dried, and a cover Jlip was placed over 10 pl o f quencher (pphenylenediamine, l m g / m l in 90 o70 glycerol). Bacteria were observed under a "Zeiss'" fluorescence microscope (Carl Zeiss, Inc., Yhornwood, NY) using an t 0 0 × oil objective. Opsonophagoeytosis ~ssay This is a modification o f a previously described assay (Fischetti, 1983). Briefly, bacteria grown lo ¢,~ponential phase were diluted in T H B and 0.1 m~ were added to sterile 9 - m m glass tubes containing 0.4 ml o f heparini,'ed (10 units/ml) h u m a n bloc, O. Two capped test tubes were prepared for each inoculum : one was rotated end over-end, and the second remained stationary. After 3 h o f ~ncubation o, 37~C, the content of the tubes was entirely plated. Plates were incubated 36 h at 37°C, and colonies were carefully counted..
Cldi
~0
Her ~,la t ~
A~tl A'~n
Extractian o[ M protein and Western blot Bacteria were grown in 5.0 ml o| T H B , harvested in the late exponential phase (0.50Ds~r,) a n d washed once in 0.02 M p h o s p h a t e buffer, pH 7.0. After sedimentation, cells were resuspended in 200 pl o f the s a m e buffer containing 30 % ruff lapse. Mutanolysin (Sigma C h e m . Co., St. Louis. Mo'~ was a d d e d to a final concentration o f 50 t~g/ml and the mixture was incubated at 37°C for 1 h. T h e protoplasts were sedimented by centrifugation and :l~e supernatant, containing the M protein, ',',as separated on 14 % sodium dodecyl sulphate/polyacrylamlde gels and electrotransferred onto nitrocellulose mem~
V.I,I]
[Cl,I]llI!,p])
[ ; I ~l I
Fi~. I. Ech~-:maKi¢ r~pr=.~,¢ntation of the 3.4 kb C'/a| f~-J.g ment Of p V M B3 containing ,.~mm-6_/ and ~rmC.
Oal cteava.ee oi' pVMB3 leave~ 'chc e m m 6.1 prnrno~crless, hul wi~h an int-ict ribosemal I~inding sin:, '.,Vhi'~e: 2.2-kb C'/al fragment of pVV3:M6A (HruOy el at.. 1"98g1: Erey: 1.2-kb ?.-ts/~[:Girl fra~mcnl nf pF i94 (Flnrinmtchi and w¢isbtum, 19~2!.
452
G, P O Z Z I E T AL, RESULTS
Construction of the restriction fragment to be
DHS~x, a clone was isolated with a plasmid, pVMB3, containing the Ctal fragment shown in figure 1.
integrated A construct was made where e r m C (Horinouchi and Weisblum, 1982), a gene conferring resistance to erythromycin, was cloned downst;eam of e m m - 6 , ! , so that both genes would be within the same Clal fragment. In this construct, the initiation codon of e m m - 6 . 1 was 19 bp downstream of one of the ClaI sites, so that Clal cleavage would leave e m m - 6 . l promoterless (fig. 1). Plasmid pVV3;M6A (Hruby et aL, 1988), comaining this ClaI site upstream of the ernm-6.l-coding sequence, was used in the experiments (table I). The 2.0-kb M s p l fragment of pE194, containing ermC, was ligated with a partial Clal digestion o f pVV3:M6A. After transformation of E. coli
Chromosomal integration The 3.4-kb Clai fragment of pVMB3, containing the promoterless e m m 6 . l and ermC, was figated with chromosomal DNA of S. g o r d o n i i also cut with Clal. The ligation mixture was used to tranzform the naturally transformable S. gord o n i i "Challis", strain V288. By this method, the e m m - 6 . 1 / e r m C Clal fragment (fig, I) was integrated at random into the chromosome. The chromosomal DNA figatcd to the Clal fragment provided the homology for integration during transformation. Erythromycin-resistant (Em-r) transformants were selected (table 11) and analysed for production of M6 protein by streak
Table I. Bacterial strains and plasmids. Strains/plasmids
Relevant properties
S. gordon# Challis V288 GP204 GP221-GP230 GP23 [ S. pyogenes D471 pVV3:M6A pE194 pVMB3
Reference
transformation recipient str-204 emm-6, l / e r m C emm-6, l / e r m C str-204
Macrina et aL, 1980 Pozzi et aL, 1988 This study Thi~ study Scott and Fisehetri, 1983 Hruby et aL, 1988 Hor~oouchi and Weisblum, 1982 This study
emm-6.1 emm-6.1 er~nC emm-6, l / e r m C
~ l r - 2 0 4 - chram~3somal point ro mali.On conferring resistance to slreptomycin; e m m - 6 , 1 - gone encoding for type 6 M protein ; e r m C = geae encoding for resistance to crythromycin; e m m - 6 , 1 / e r m C = 3.4-kb Clui fragmenl contaieing bolh genes (fig. 1).
Table 11, Transformation of S. gordonii Challis. Donor DNA
Ligase treatment
Transformants
Genetic analysis
emm-6, l / ermC + chrom. DNA
Yes
1.8 × l03
emm-6.1/ermC+ chrom. DNA
28 % Em-r, M6* 72 % Era-r, M6-
No
: 104 7.1 × 103 8.5× 103
900 32 32 3
1.7X ]05 5.8x 104 8.i × 10~ 4.0× 103
'Two test 'tube.~ ',,.ere prepared for ea,.:h inoeulum : one ",,,'aslefl on the rack (sto.tJ.,..t~ary) und one was rotated, for 3 h at 37°C. Results V;ere expressed a~ mtai C [ U present in the ~esl tube (0.6 ml volume).
EXPRESSION OF M6 PROTEIN GENE IN STREPTOCOCCUS GORDONIi
455
promoterless M6 protein gene was linked in vitro to an e r y t h r o m y e i n resistance m a r k e r , a n d the restriction f r a g m e n t c o n t a i n i n g b o t h genes was ligated to r a n d o m fragments o f S. gordonff chrom o s o m a l DNA. [[sing this ligation mixture to t r a n s f o r m the naturally c o m p e t e n t S. go~'donii Challis, r a n d o m integration o f the heterologous D N A (emm-6. l/erraC) was o b t a i n e d . In naturally c o m p e t e n t streptococci, heterologous D N A ligated to c h r o m o s o m a l sequences can be integrated i n t o the c h r o m o s o m e d u r i n g t r a n s f o r m a t i o n (Vasseghi et al., 1981; Vasseghi a n d Claverys, 1983; Mannarelli a n d Lacks, 1984; M o r r i s o n et al., 1984; Pozzi a n d Guild, 1985). As previously discussed (Pozzi and Guild, 1985), t h e usual product o f this process is the integration o f o n e copy o f the heterologous D N A between t w o direct repeats o f the h o m o l o g o u s c h r o m o s o m a l segment. M o r e t h a n o n e q u a r t e r (28 % ) o f t h e t r a n ~ f o r m a n t s , in which the heterologous D N A was integrated, expressed the M6 protein, In these t r a n s f o r m a n t s , a functional t r a n s c r i p t i o n a l fusion o f emm-6.1 with chrom o s o m a l promoters h a d occurred. As expected, the t r a n s f o r m a n t s expressed different a m o u n t s o f M6. Each was p r e s u m a b l y the p r o d u c t o f a difl'erent intcgrational event, which h a d led to a fusion with a different p r o m o t e r . Screening several t r a n s f o r m a n t s allowed t~or the selection o f the best producers. A m o n g t h e m , it was possible to select strains with a stable phenotype and with a single copy o f emm-6.1 integrated into the chromosome.
sion is complex and not completely u n d e r s t o o d ( C a p a r o a a n d Scott, 1987 ; Robbins et al., 1987; S i m p s o n et al., 1990). Previous a t t e m p t s to express cloned e m m genes from their own p r o m o t e r s in various streptococcal hosts always failed to produce stable a n d " h e a l t h y " reeomb i n a n t s (Scott et aL, 1986; Poirier et aL, 1989; Oggioni a n d Pozzi, 1990'~ This was due, in our opinion, to two types of problems : (1) those connected with ~he regulation o f expression of the e m m gene away from its c h r o m o s o m a l locus, a n d (2) those connected with plasmid instability. In the present study, these problems were overcome by integrating the coding sequence of emm-5, l into the c h r o m o s o m e o f S. gordonii, downstream o f strong c h r o m o s o m a l promoters.
I m m u n o f l u o r e s c e n t staining of the bacterial surface a n d Western blot analysis s h o w e d that M 6 was expressed o n the surface o f S. gordonii a n d also secreted in the m e d i u m . The presence o f M6 o n the surface o f S. gordonii e n a b l e d the o r g a n i s m s to survive f r o m ~0 to 30 times better in h u m a n blood when c o m p a r e d to the M ~ o r ganisms. This antiphagocytic activity is ]~ot as p r o n o u n c e d as w h e n the M molecule is o n the native S. pyogenes. However, S. gordonii is p r o b a b l y ustfited to test the antiphagocytic activity o f the M molecule in the classic bactericidal assay, since, unlike S. pyogenes, it dt>es not grow weI[ in h u m a n blood.
Expression du gbne de la proleine M6 de Strepzococcus pyogenes ehez Streptoeoccu~ gordon# apr~s integration chromosomique et fusion lranscriptionnelle
Regulation o f M protein gene (emrn) expres-
This new method allows (1)expression of heterologous genes in S. gordonii, (2) selection of strong p r o m o t e r s , a n d (3) selection of stable ]'econqDincqi'tis. ,.-r : . . .tylJt~ . . . o f ap~J~ tL JlJ~, . . . . . . Ua~jLJ . C,~ii ~0~ Of great help in the c o n s t r u c t i o n o f streptococcal strains to be used in further genetic engineering experiments utilizing heterologous genes.
Acknowledgements This work was s~lpported in part by grants from CNR {Progetto Finaliz;,ato ingegneria Generica. n. 91.000!6.90) and ISS (IV progetto AIDS, n. 6207-011)to GP. NIH (Gran~ A111822) to VAF. and NATO (Collaborative Research Grant n. 0840/88).
La prot~ine M6 de Streptococotspyogenes a ~t~ exprim6e fi la surface c~llulaire et s~cr&oe chez Strep(ococcus gordonii ChaI[is (ant,2ricurcment Streptococcus sanguis), ~pr~s inz6gration du gene (emm-6. l) de la prot~ine Me, sa:~s promoteur. Le g~ne ermC coat, rant la r~sistance 5. l'&ythromycine a et~ clone- en aval de emm-6.1, a l'int~rieur du m~mc fl-agmt nt CtaI. Le codon d'initiation du gene emm-6. Icst un fragment de 19 pb en aval du site Clal, de sorre que la coupure par Clal laisse le g~ne san~ promotet:r. Le fragment C/u[ contenant Ic gbne ermC e; te g ~ e emm-6. ! sans promoteur, a
456
C. P O Z Z I E T A L .
dt~ lid in vitro fi un fragment C/at d ' A D N c h r o m o somiclue de S. gordonii. L'int~gration c h r n m o s o m i que au hasard de I ' A D N h~t~rologue a ~t~ obtenue par l'utilisation du m d a n g e 1i$ pour t r a n s f o r m e r la s o o t h e de S. gordolffi Challis naturellement compStente. Vingt-huit pour cant des t r a n s f o r m a n t s s~lecAioun~s pour leur rds]stance ,h 1'Srythromyeine exp,iment aussi Ia prc, t~ine MS. Dix clone~ parmi los meilleurs producteurs de M6 a n t 6t~ sSlectionn~s sur la base de leur stabilit~ phSnotypique. Nenf des dix clones sont porteurs d ' u n e topic intacte du fragment Clal, emm-6.1/ermC, int~gr~ dans le c h r o m o s o m e . Ces souches expriment la protiine M6 A la surface et en secretcnt d¢~ qu,ttltitcs vari~es: dons c h a q u e cas la prot~ine a ~t~ produite aprgs fusion transcriptionnelle du g~ue ernra-6.1 avec uu p r o m o t e u r c h r o m o somique. Unc souchc dc S. gordpnff e x p r i m a n t de grandes quantitSs de protSine de surface M6, Svalu~es pat immunofluorescence et W e s t e r n blot, a ~t$ c o m p a rSe ~, la souche parentale M - par dosage standardis6 de l ' o p s o n o p h a g o c y t o s e . De la paire de s o o t h e s isog~niques, S. gordonii M6 ÷ ~urvit mieux dans 1¢ sang h u m a i n et elle est phagoeytSe plus lentement. Mots-clds : Prol~ine M, Streptococcus pyogenes, Streptococcus gordonii, G~ne e m m - 6 . 1 ; Transcription, A D N c h r o m o s o m i q u e , GSnlc gSn~tique.
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