May 6, 1985 - Four major classes of antigens cover the surface of enteric bacteria : kapsular (K) ..... S. (1984) J . B ~ c t e ~ i o l . 1-57. 330-. 333. (1955) .1.
Eur. J. Biochem. 151, 573-577 (1985) K' FEBS 1985
Comparison of the nucleotide sequences of the genes encoding the KS7lA and F7 fimbrial antigens of uropathogenic Escherichia coli Mikacl R H E N
I,
Irma van DIE', V u o k k o R H E N ' and Hans BERGMANS'
' Departincnt of General Microbiology, University of Helsinki
' Dcpartmcnt of Molecular Cell Biology, State University of Utrecht (Rcccived May 6, 1985) - EJB 85 0478
DNA fragments encompassing the genes for the KS71 A and F71 fimbrial subunits of Esc/lrrichiti c d i strains KS71 ( 0 4 : K 1 2 ) and AD1 10 ( 0 6 : K 2 ) , respectively, have been subjected to DNA sequencing. The nucleotide sequences of tlie two fimbrillin genes were identical and they encode a polypeptide of 187 amino acids of which 31 amino acids probably will constitute the signal sequence. The primary structure of these fimbrillitis showed significant homology with the primary structure of other E. coli fimbrillins.
Four major classes of antigens cover the surface of enteric bacteria : kapsular (K), lipopolysaccharide 0-side chain (O), flagellar (H) and fimbrial (F) antigens [ l , 21. E.schrric/iilr c d i fimbriae have been subjected to intense investigation a s the expression of fiinbriae has been correlated with extraintestinal infectious diseases. K88, K99, CFAjl and CFA/II fimbriac occur on enterotoxigenic strains [3], whereas P fimbriae occur on human pyelonephritogenic strains [4, 51. P-fimbriatcd bac1 -4)-P-~-Galportion of P-bloodteria recognize tlie cc-~-Gal-( group antigens [6, 71. Indeed, bacterial adherance to uroepithelial P-blood-group antigens has been considered as a prerequisite for ascending pyelonephrilis in children [8]. B. t ~ d fimbriae i are proteinacous filaments that protrude from the bacterial cell surface [9]. Each filament is built up of hundreds of identical subunits called fimbrillins [I 01. A number of antigenic types of P fimbriae occur on E. c d i [ I ] and one strain may possess more than one type of P fimbriae [ I l l . Our model strains, E. coli KS71 and AD110, each produce two types of P fimbriae, termed KS71A, KS71B and F71, F72, respectively [12, 131. In addition both strains produce type 1 fimbriae and non-haemagglutinating fimbriae called KS71C or type 1 C. Each of these fimbriae appears to be encoded by a separate, chromosomally inherited set of genes [ 12 171. The serological diversity of P fimbriae has hampered the construction of typing schemes and of fimbrial vaccines against pyelonephritis. One way to resolve the serological diversity is to map antigenic determinants within the primary structure of the fimbrillins. Two gcncs for P fimbriac (PAP and F72) have rcccntly been sequenced [ l X , 191. In this communication we present the primary structure, predicted from the nucleotide sequence of the fimbrial-subunit gene, the hydrophobicity profile and predicted secondary structure for a third antigenic type of P fimbriae. ~
CorrcJspondeence to M. Rhen, Yleisen Mikrobiologian Laitos. Hclsingin Yliopisto, Mannerheimintie 172. SF-00280 Helsinki 28, Finland Ahhwvintion. kb, 1000 base pairs.
MATERIALS A N D METHODS Rrit~tericilstrcritis. phriges, p1a.snzid.s aiid iiirtlitr
LY. c d i HBlOl (IistlS. r w A ) and JA221 ( r w A , IisdA) [20] wcre grown for 16- 18 h at 37 'C in Luria broth o r on Luria agar supplemented with ampicillin, 100 pg x 1111 (Wycth Laboratories, USA); tetracycline 15 pg x m - * (Sigma Chemical Co., USA) o r with chloramphenicol, 20 pg x m - ' (Sigma). 'The phage M I 3 derivatives mp8 and mp9 [23] were harboured in E. co/i JMlOl [23]. Recombinant plasmids used in this study are suinmarized in Table 1. Cloiiiiig procwliircs trml restriction intipping
Restriction endonucleases and T4 DNA ligase obtained from Boehringer Mannheim (FRG) and Amersham International ( U K ) were used under conditions recommended by Maniatis et al. [24]. Plasinid and phage DNA was isolated from cell lysates by ethidium broniide/caesium chloride equilibrium gradient centrifugation [24]. For screening
T;i
ble I . ~ W O H ~ J I ~ Mp/~.s~Iiids ~I~
Thc 8. c.o/i strains in which the recombinant plasmids were harboured are given i n purcnthcsis. A limbrial antigcn in parcnthcsis indicates t h a t fimbriae arc not cxprcssed on the cell surface in strains carrying thc plasmid Reconibi na n t plasmid
Fimbrial antigen encoded
pKTH3024 (HB101) pKTH3011 (JA221) pKTH3028 (H BIOI ) p KT CI 3003 ( H B 101) pI'lL110-75 (HB101) pPILl10-753 (JA221) pPIL110-35 (JA221) pPILl10-51 (HRIOI) pPAP5 (HB101)
KS71A KS71 B (KS71A) KS71C F7 1 (F~I) F72 typc 1c PAP
Reference
574
i
2
la -c--
4
3
5
----------J
6
F-
I \
\
- -m
Fig. 1. Sequencing strcrtc'gy of ihe KS71A ( a ) and t.'71 ( h ) ,firnhrinlsuhunit genes locared on plusmicts pKTI1.7028 und pPILI10-75,i . e . y ) i ~ c . t i i , c l ~ ~ . The thicker lines indicate D N A of the cloning vector pBR322 and the thin lines indicate insert D N A . The numbers under thesc lines indicate the size in bases x 10- '. The sequenced regions arc shown as enlarged sections. Arrows underneath thc cnlarged regions indicate the direction of the sequenced DNA fragments. Hatched boxes show the location of the fimbrial genes, the signal pcptide region being tnarkcd by diff'erent hatching
purposes the DNA was isolated by the alkaline lysis method of Birnboini and Doly [25]. Ligated D N A as well a s isolated plasmid and phage DNA were introduced into recipient strains by transformation of CaCI,-treated cells [26]. Restriction mapping was performed as described [17].
C o n p t c r tmalysis
The hydrophobicity profile was calculated according to [28] and the predicted secondary structure according to Chou and Fasman [29].
D N A scguencing
RESULTS A N D DISCUSSION
The chain termination method of Sanger et al. [27] was used employing recombinant M I 3 D N A as template. Sequencing primer and DNA polymerase I Klenow fragment were purchased from Boehringer Mannheim, whereas dATP[35S] was from Aiiiersham International.
Scq ticti cirig strcr trgy
Agglutination tcsts
The antiscra against KS71A, KS71B, KS71C or ER2B1 fimbriae were those described previously [I 1, 171. Bacterial agglutination with antiscra was performed as described [12].
The KS71A limbrillin gene has previously been localized within the 1.6-kb SmuI-CIuI fragment of pKTH3028 [21] (Fig. 1 a), whereas the F71 gene is located within the 3.1-kb CkcrI-BumH1 fragment o f pPILl10-753 [16] (Fig. 1b). Various restriction fragments from the regions proposed to contain the fimbrilliii germ were cloned in phage M I 3 vectors mp8 and mp9. Thcsc fragments were then sequenced as shown in Fig. 1. A fragment encompassing 1000 bp was sequenced in both directions from pKTH3028, similarly, a fragment of 645 bp was sequenced from pPILl10-753.
575 1
51
I
A
T
102
C
A
T
K
A
103
T
15M
C
X 203 ATG A T t M G TCG GlT
159
T
A'A
T
52
C
I
T
C
M
X
M
W
A
m
net -21
I l e L y s Ser V a l
204
AU A-U CCGGU UAUUU
243
rn Gcc ma ocz ATG GCA GTG GTG m m GGT I l e A l a G l y A l a V a l A l a Met A l a V a l V a l Ser Phe C l y
A l l GCC
-16
-4
244
282
Fig. 3. Possible
stem arid loop structure fi)rrned by tlw r q i o n of 789 mu' 803) nt tke t?iR N A level
ujvad
.>j~ttitiictr.r(hcJtwcwpositions
GCA M T 6CA CCT CCA TCT ATC CCT CM GGA C M GGT G M A l a Asn A l a A l a A l a Ser I l e pro G l n G l y G l n ~ l ~y l u
-3 283
-'A'
10
321
GTA AGT m AM u;C ACT CTT CTT GAC GCT CCG TGT GGT V a l Ser Phe L y s G l y Thr V a l V a l A s p A l a Pro C y s G1 t1 25
322 360 AR GAA ACT CAO m OCA AM CM; GM A n GAT m GGT 11e G l U Thr G h Ser A h L y s C l n G l u I l e A s p Phe G 1
24 361
38 399
CAC ATC rcT AM rrC rrC CFG CM GAG
GGC GGA GAG ACT
G l n 110 Ser L y m S e r P h e Leu G l n G l u G l y G l y G l u Thr
31
48
400
438 GAC As
CM CCG U A 6hT Zn; M T ATT Mc CIT GTG M T TGT G l n Pro L y s Asp Leu Asn I l e L y s L e u V a l A s n C y s
so
6E
439 477 A T T ACT M T Tni AM CM; CIT CM GGC CGG GCA GCT AM I l e Thr A s n Leu t y s C l n Leu C l n G l y G l y A l a A l a L y s
75
63
478 516 A M GGT ACA OPG %A rrG kca m %A G m on; CLT GCA L y s C l y Thr V a l Ser L e u Thr P h e Ser G l y V a l P r o A l a
88
76
517
555 G M M T Cca GAT =C ATG CFC CM ACA CTP GGA GAT ACT G l u A m A l a A s p Amp net Leu G l n Thr V a l G l y A s p Thr
89
101
556 MT ACT GCG A n GIT
594
m ACT
GAT TCG ACT GGA AM
coc
A s n Thr A l a l l e V a l V a l Thr A s p Ser Scr G l y L y s A r
112
102
633 GTG AM m GAT GGA GCC A C T GAG ACC CGG CCT TCA M T V a l L y s Phe Asp G l y A l a Thr G l u Thr G l y A l n scr A n n 115 I?? 595
672
634 ESG A m M T
Gcc GAT MT ACA A m
712
150
CCC M T Tn: AAC C T G A t T TAT CM; A l a &'he Ser A l a V a l A l a Aan P I I ~ . A '; lt Leu 'Thr Tyr ( ; I t \ 154 166 CIY: l'CA GCA
151 HOl TAACrCTMTACCATMrCnoCTCCCCTMACA(;('(;OI~
UUI
The aniino acid sequtwce encoded by the.fimhrilliiz genes A region corresponding to the NH,-terminus of the KS71A and F71 fimbrillins [30, 311 starts at position 253 (Fig. 2). The region between this position and the initiation triplet at position 188 corresponds to the P-fimbrial signal sequence of PAP and F72 [IS, 191 suggesting that KS71A and F7, are also synthesized as precursor proteins. The deduced signal sequence consists of 21 amino acids and contain a hydrophobic center Val-Val-Ser-Phe encoded by nucleotides 228 to 240 (Fig. 2). The Ala-Asn-Ala triplet preceding the cleavage site is in agreement with known signal sequences [32]. The hydrophobicity profile and predicted secondary structure of the KS71 A and F71 Gmbrillins is shown in Fig. 4. The signal sequence is within a hydrophobic region (residues - 21 to - 1). Other hydrophobic centers are around residues 20, 60, 85, 110, 140 and 160. There are six strong predicted reverse turns, five of which are located at hydrophilic peaks (at residues 5 , 104, 114, 135 and 148) and may be involved in the formation of antigenic sites.
CAT TIT ACT GCA
L e u I l e Asn G l y Asp Ann Thr I l e IIis I'hc Thr A l a Phe 120 140 673 1 11 GTT MG AM GAT MT AGT ccc MC AAT t m W P C:AA CGT V a l L y s L y a h a p Amn P a r G l y L y s A s n Val n l a Glo ti1 141 15s CCC
rcading frame of 582 base pairs, starting at an ATG triplet at position 168 and ending at a TAA triplet at position 751. A region of dyad symmetry is located between nucleotides 789 and 803 (Figs 2 and 3). Possibly this region determines a transcriptional termination loop.
a15
' rl-TA'rT'ri"T'~A C C C T A T T T A T C A T G A G A ~ ~ ' ~ ; A -
Fig. 2. Tlw iiiicleotiiie .wqutw'c of' tht, K S 7 l A ,fbnhrillin gcwc. The amino acid sequence of the predicted polypeptide is shown under the nucleotide scqucncc. Numbcrs above the line refer to the nucleotide position, whereas numbers below the line refer to the amino acid residue. The open arrowhead points to the signal peptide cleavage sitc. The region of dyad symmetry in the possible transcriptional termination site is underlined
Fig. 2 shows the nucleotide sequence determined for the KS71A fimbrillin gene. The nucleotide sequence of thc F7, fimbrillin gene appeared to be identical over the region whcre sequence data of F71 were available, i.e. from nucleotide 165 to nucleotide 760 in Fig. 2. Both sequences contain an open
Cot?ijJuri.sonof KS71A atid F71 wiih olher E. coli,fitnhrillins The serological relationship between type 1 C, PAP, F71, KS71A, F72 and KS71B fimbriae is shown in Table 2. Type 1 C fimbriae of AD110 are nonhaemagglutinating and reacted with anti-KS71C serum only. PAP, KS71A, F71, F72 and KS71B are P fimbriae but these could be grouped serologically. As expected, anti-KS71A serum reacted strongly with the homologous antigen and with F7, ; weaker reactions were observed with PAP, F72 and KS71B. Anti-KS71B serum reacted strongly with KS71A and F72 and weakly with KS71 A and F71, but not with PAP. A strong cross-reaction was observed with PAP and antiserum against E. coli ER2B1 fimbriac. The primary structure of these four fimbrillin antigens showed significant homology (Fig. 5), the non-cross-reactive Lype 1 C fimbria being less homologous. Yet, no less than 39 amino acids appeared to be conserved when compared with the P fimbriae. The conserved residues need not be involved in the formation of strong antigenic sites but may rather reflect structural requirements. When only the P fimbriae were compared, the number of conserved amino acids was 88. The two cysteine residues (at positions 22 and 63 in Fig. 5 ) could form a disulfide bridge and this may reflect the clustering of conserved amino acids around these residues.
576
-I0
1
10
50
100
150
AMINO ACID RES
Fig. 4. Hydropltilicifyp t ~ $ i / eand pletlicfrd stconcktry sfructwe (f’thc KS71A and F7, fimhriul s t h m i t s . Loops indicate x-helix. rigzag indicates /?-sheet structure, U-bands indicate turns and a horimntal line indicates random coil
PJ 1
25
55
56
80
110
111
135
165
type 1C
PAP
F72 KS71A/ P71
KS71A/
F71 type 1C
3 KV
Q
NLTYQ
p72 PAP KS71A/
’”
EiLTYQ
NLTYQ 166
Fig. 5 . Cornpurisori of /lie amino cicid sc~ciuenc~e.~ q f ’ f l wtypc I C , P A P , F72. KS7IA am/ F7, .fimhriaiJ.The amino acids arc given i n one letter codes. Homologous areas are framed. Deletions introduced in ordcr to present closer homology arc indicated by dashes Tablc 2. Agglufincition qf:fin?hriatedrecornhi~inntE. coli strains by anti-fimhria sera E. c,n/i HBlOl or JA221 not harbouring recombinant plasmids were not agglutinated Fimbrial antigen exprcsscd by rccombinant E. culi
KS71A KS71 B KS71C F7 1
F72 type 1C PAP
Agglutination titre with antiserum against ~~~
~
KS71A fimbria
KS71 R fimbria
KS72C fimbria
ER2BI fim bri a
5120 1280 < 20 5120 1280 < 20 640
40 51 20 < 20 20 5120 < 20 < 20
< 20 < 20 51 20 < 20 < 20 5120 < 20
20 1280 < 20 40-20 1280 < 20 1280
577 The genes encoding the KS71A and F71 fimbrillins appeared identical (Fig. I), also the nucleotide sequence of the KS71B and the F72 genes appears to be very similar, if not identical [I81 (and Tenhunen & Rhen, unpublished). Imniunologically the fimbrial constitutions of E. coli KS71 and AD1 10 also appeared similar (Table 2). These results suggest that the two strains possess a set of two or three fimbriae that are nearly or competely identical and will have the same F (fimbrial) serotype. This is surprising as the two strains have a different 0 and K serotype, suggesting that they have had a long separate history. I t is not very likely that this phenomenon has evolved just by chance. The possibility that this is due to transposability of virulence gene clusters within E. c d i [33] will be the subject of future research. The assistance of Hans de Cock and Caroline le Poole in part of the experimental work is gratefully acknowledged. We thank Wiel Hoekstra for critical reading of the manuscript and Matti Saraslc for help with the computer analyses. MR and VV-R were supported by thc Academy of Finland and by the Magnus Brnroth Foundation. We a l s o Licknowledge Dr M. BAga and Prof. S. Normark for providing us with the pPAPS clone.
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