Feb 9, 1995 - Proteus mirabilis strains were isolated from dogs with urinary tract infection. (UTI) and fimbriae were prepared from two strains. The N-terminal ...
Microbiology (1995), 141, 1349-1 357
Printed in Great Britain
Nucleotide sequences of two fimbrial major subunit genes, pmpA and ucaA, from canineuropathogenic Proteus mirabilis strains Isaac G. W. Bijlsma, Linda van Dijk, Johannes G. Kusters and Wim Gaastra Author for correspondence: Isaac G . W. Bijlsma. Tel:+31 30 534888. Fax: +31 30 540784. e-mail : bijlsma @ vetmic.dgk.ruu.nl
Department o f Bacteriology, Institute of Infectious Diseases and Immunology, Faculty o f Veterinary Medicine, University o f Utrecht, Yalelaan 1, PO Box 80.165, 3508 TD Utrecht, The Netherlands
Proteus mirabilis strains were isolated from dogs with urinary tract infection (UTI) and fimbriae were prepared from two strains. The N-terminal amino acid sequences of the major fimbrial subunits were determined and both sequences appeared identical to the N-terminal amino acid sequence of a urinary cell adhesin (UCA) (Wray, S. K., Hull, S. I.,Cook, R. G., Barrish, J. & Hull, R. A,, 1986, Infect lmmun 54,4349). The genes of two different major fimbrial subunits were cloned using oligonucleotide probes that were designed on the basis of the N-terminal UCA sequence. Nucleotide sequencing revealed the complete ucaA gene of 540 bp (from strain IVB247) encoding a polypeptide of 180 amino acids, including a 22 amino acid signal sequence peptide, and the pmpA (,.mirabilis p-like pili) gene of 549 bp (from strain IVB219) encoding a polypeptide of 183amino acids, including a 23 amino acid signal sequence. Hybridization experiments gave clear indications of the presence of both kinds of fimbriae in many UTI-related canine P. mirabilis isolates. However, the presence of these fimbriae could not be demonstrated in P. wlgaris or other Proteus-related species. Database analysis of amino acid sequences of major subunit proteins revealed that the UcaA protein shares about 56% amino acid identity with the F17A and F l l l A major fimbrial subunits from bovine enterotoxigenic Escherichia coli. In turn, the PmpA protein more closely resembled the pyelonephritis-associated pili (Pap)-like major subunit protein from UTI-related E. coli. The evolutionary relationship of UcaA, PmpA and various other fimbrial subunit proteins is presented in a phylogenetic tree. Keywords: Proteus mirabilis, fimbriae, major subunit gene, urinary tract infection
INTRODUCTION Protetls spp. can cause many kinds of extra-intestinal infections, such as otitis media and externa, meningitis, septicaemia and infections of wounds, and respiratory and urinary tract infections. The most frequent site of infection is the urinary tract and the majority of these infections is caused by Protetls mirabih, whereas Protetls vdgaris is of minor importance (Adler e t al., 1971). P. mirabilis is not only an important cause of urinary tract ............................... ..........,................................................................................................................ Abbreviations: Pap, pyelonephritis-associated pili; UCA, uroepithelial cell adhesin; UPEC, uropathogenic kherichia coli; UPPM, uropathogenic Proteus mirabilis; UTI, urinary tract infection. The EMBL accession numbers for the nucleotide sequences reported in this paper are X77611 (pmpA) and X77612 (ucaA). 0001-9484 0 1995 SGM
infection (UTI) in humans but also in dogs. In both hosts, the majority of UTI is caused by Escherichia coli and about 10% by P. mirabili~(Gaastra e t al., 1995). To exert their pathogenicity, P. mirabilis strains are equipped with several virulence factors, including the production of urease (Johnson e t al., 1993; Jones e t al., 1990), IgA proteases (Loomes e t al., 1992), haemolysin (Mobley e t al., 1991), flagella (Allison e t al., 1992) and fimbriae (Ahrens e t al., 1993; Bahrani e t al., 1991; Garcia e t al., 198813; Sareneva eta]., 1990; Wray et al., 1986). In most bacterial infections, adherence is accepted as an indispensable virulence factor, in this case to protect the Prateus bacteria from being washed out of the urinary tract by the urine flow. Adherence in UTI by means of fimbriae has been extensively studied in both human and canine uro1349
I. G. W. BI J L S M A and OTHERS
Table 1. Results of Southern blot analysis of Pstl-Hindlll-digested chromosomal DNA from a number of Proteus strains and Proteus-relatedspecies ~
Bacterial species
IVB strain no.*
Source
Size of hybridizing fragments (kb) UCA-band(s)t
Proteus mirabilis
Proteus vdgaris Morganella morganii
Providencia rettgeri
80 219 225 227 229 236 237 238 240 241 242 243 244 245 246 247 253 254 255 256 257 260 271 276 292 1297 900 1315 911 1316 1317 1318 1319
Dog urine Dog urine Dog urine Dog urine Dog urine Dog urine Dog urine Dog prepuce Dog faeces Dog urine Dog urine Dog urine Dog urine Dog faeces Dog uterus Dog urine Dog urine Dog vagina Dog faeces Dog urine Dog faeces Dog urine Dog vagina Dog faeces Dog urine Dog ear Cow milk Horse Hedgehog nose Horse Cow organs Dog urine Dog ear
1 and 2.8 5 2.8 2 and 2.8 6 2.8, 4 5 and 11-5 2.8 and 4 5 2 2 2 2.8 2.8 2.8 2.8 2 28 2.8 2.8 2 and 5 -
11.5 and 2.8 -
Pmp-band$ (Pap-like) 6 23 6 6 2.3 23 6 6 6 6 6 23 23 23 2.3 6 6 6 6 2.3 -
6
-
6.5 23 6 -
-
-, no bands observed.
* Strain numbers from our collection are preceded by the abbreviation IVB for Institute for Veterinary Bacteriology. t UCA bands obtained by hybridization with the EcoRI-PstI probe (about 700 bp) from IVB1433 (Fig. 1). $ Pmp-bands obtained by hybridization with the ClaI probe (648 bp) from IVB1430 (Fig. 1).
pathogenic E. coli (UPEC) (Garcia e t al., 1988a, b; Marklund e t al., 1992; Kuehn e t al., 1994). In human UPEC, P-fimbriae, which bind to glycosphingolipids of uroepithelial cells, are the most prevalent type of fimbriae (Garcia e t al., 1988a; Johnson, 1991; Lund e t al., 1988). They are encoded by the pap (pyelonephritis-associated pili) operon of which the papA-gene encodes The major fimbrial subunit, whereas the papG gene encodes a receptor-binding protein, located at the tip of the fimbriae (Hultgren & Normark, 1991; Hal Jones e t al., 1992). 1350
Several serological variants of P-fimbriae exist (Denich e t al., 1991; Garcia e t al., 1992; Kusters & Gaastra, 1994). Human and canine UPEC isolates have different binding specificity, which can be attributed to differences in the PapG protein (Garcia e t al., 1988a, b; Marklund e t al., 1992).
A similar host-specific adhesion could not be demonstrated for uropathogenic P. mirabilis (UPPM) strains (Garcia e t al., 1988b). UPPM strains generally produce
Canine P. mirabilis fimbrial genes fimbriae, but these fimbriae have been studied far less than those of UPEC. Thus far, the existence of four fimbrial structures has been reported for human UPPM. Wray e t al. (1986) isolated a fimbrial uroepithelial cell adhesin (UCA) and determined the N-terminal amino acid sequence of the major fimbrial subunit. Subsequently, Bahrani and co-workers identified mannose-resistant/ Pratezls-like (MR/P) fimbriae (Bahrani & Mobley, 1993) and P. mirabilis fimbriae (Bahrani e t a/., 1993). The nucleotide sequences of the major fimbrial subunit genes, mrpA and p m f A , were determined. In addition, the total mrp operon was sequenced (Bahrani & Mobley, 1994). Finally, Massad e t al. (1994) identified a new ambienttemperature fimbria (ATF) in UPPM. The N-terminal amino acid sequence of ATF did not show significant similarity to that of any previously described fimbrial protein. Several authors (Bahrani & Mobley, 1993; Duguid & Old, 1980; Mobley & Chippendale, 1990; Old & Adegbola, 1982; Silverblatt, 1974) demonstrated that different kinds of fimbriae may be found in the same strain of P. mirabilis. Similarly, UTI-related E. coli strains have been shown to express as many as four types of fimbriae (Rhen, 1985). In this study, the isolation of two genes that encode different fimbrial subunits in P. mirabilis isolates from canine UTI is reported. The nucleotide sequences of the genes were determined and the deduced amino acid sequences have been compared with the major subunit sequences of other fimbriae from uropathogenic isolates.
METHODS Bacterial strains and plasmids. The strains of P. mirabilis, P. vukaris, Morganella morganii and Providencia rettgeri used in
this study were obtained from the Veterinary Microbiological Diagnostic Centre of Utrecht University, The Netherlands, and are listed in Table 1.
E. coli PC2495 (Phabagen Collection, Department of Molecular Cell Biology, Utrecht University, The Netherlands),a hsdS recA
derivative of E. coli JMlOl was used as host for pBluescript (Stratagene) and its derivatives. Isolation and analysisof fimbriae. The purification of fimbriae, assay of the purity of these preparations and total cell protein profiles by SDS-PAGE and Western blotting were performed as described by Mooi et al. (1987). The N-terminal amino acid sequence of purified fimbrial protein was determined at the Gas Phase Sequenator Facility (Department of Medical Biochemistry, State University of Leiden, The Netherlands). The instrument used was an Applied Biosystems Model 470A Protein Sequencer, equipped with an on-line Model 120A PTH Analyser. Electron microscopy of purified fimbriae preparations was done as described by Garcia e t al. (1988a). Recombinant DNA techniques. Chromosomal DNA was isolated as described by Garcia e t a/. (1988a). DNA restriction fragments were prepared by digestion of chromosomal DNA with the appropriate restriction endonucleases and the desired fragment was isolated after separation by electrophoresis of the digestion mixture in 1% (w/v) agarose gel and extraction from the agarose using the Geneclean kit (BIO 101). Ligation of DNA fragments into pBluescript was performed as described by Gaastra & Hansen (1984). Transformation of CaC1,-treated PC2495 cells was carried out according to standard procedures (Sambrooketal., 1989). The strategy for cloning and subcloning of DNA fragments from P . mirabilis chromosomal DNA is shown in Fig. 1. pBluescript derivatives containing restriction fragments derived from P. mirabilis chromosomal DNA were isolated using the Qiagen plasmid kit as specified by the manufacturer. PCR. After transformation, colonies containing cloned chromosomal DNA fragments from P. mirabilis in pBluescript were screened by PCR. DNA amplification with Taq polymerase in Taq polymerase buffer (Promega) was performed with the primers T3 (5’ATTAACCCTCACTAAAG 3’) and T7 (3’ GATATCACTCAGCATAA 5’) flanking the insert in pBluescript. Colonies were aseptically transferred to a 100 p1 reaction mix for amplification. PCR reactions were performed in an Omnigene Temperature Cycler (Hybaid) with 35 cycles of 1 min at 95 OC, 1 min at 48 OC and 1 min at 72 OC. After the final cycle, the mixture was incubated for 10 min at 72 OC to complete the last polymerase reaction.
UCA major subunit gene from /? mirabilis (strain IVB247) ucaA o 540 bp HE
IVB1433
LI
D I
E I
E I
IVB1441
C I
D
P
12kb
I
E
P
1
I
+ P I
-
IVB1444
E I
2.7 kb
2.1 kb
Pap-like major subunit gene from /? mirabilis (strain lVB219) H
IVB1425
I
IVB1430 IVB1431 IVB1437
H 1
c I
c c I
ps
549 bp
CP I I c
c
2.3 kb 648bp
CP I I
+
-
P
1
H I
H
I
5kb 2.7 kb
.................................................................... ........................ Fig. 1. Physical maps of the cloned and subcloned chromosomal DNA fragments containing the indicated major subunit gene or parts thereof. The positions of both genes are indicated by open boxes, preceded by the signal sequences in black boxes. The arrows under each clone indicate the sequencing strategy. Restriction sites: C, Clal; D, Dral; E, EcoRl; H, Hindlll; P, Pstl.
1351
I. G. W. B I J L S M A a n d O T H E R S
Southern blotting and DNA hybridization. Chromosomal DNA digests and the PCR products were run in a 1 % agarose gel and transferred to nitrocellulose filters (BA/85, Schleicher & Schuell) by Southern blotting using standard procedures (Sambrook e t al., 1989). Screening for cloned DNA fragments larger than 3 kb was done by colony blotting. For that purpose, colonies were transferred in duplicate to LB agar plates supplemented with 100 pg ampicillin ml-'. After growth, one of the plates was used for blotting colonies onto nitrocellulose filters (BA/85). Colonies were lysed and DNA was immobilized on the filters (Sambrook e t al., 1989). Oligonucleotide probes ( < 30 bp) were end-labelled with [y-32P]ATP,whereas larger fragments ( > 200 bp) were labelled with [a-32P]ATPusing a random primer labelling kit (Promega). Before prehybridization (2 h), filters were wetted in 5 x SSPE (Sambrook e t a/., 1989). Prehybridization and hybridization with larger probes were carried out at 42 OC, subsequent washing being at 50 OC. Hybridization and washings with oligonucleotide probes were done at 37 OC. After hybridization (16 h) and washing, filters were dried and mounted for autoradiography. Nucleotide sequence analysis. The sequence of the cloned DNA fragments was determined with an Automated Laser Fluorescent DNA sequencer (Pharmacia). In addition to the fluorescein isothiocyanate (F1TC)-labelled SK, KS and T7 Bluescript primers, FITC-labelled internal primers (Pharmacia) were used to determine ucaA and p m p A sequences. Computer analysis. Nucleotide sequences were analysed using the PC/GENE program (release 6.70, Genofit). Similar amino acid sequences were located by searching all available databases with the BLAST programs (Altschul e t al., 1990) as provided by the National Center for Biotechnology Information (NCBI) BLAST E-mail Server. Sequences were retrieved from the NCBI databases and aligned with MULTALIN (Corpet, 1988). A phylogenetic tree (Fitch & Margoliash, 1967) was derived from the amino acid difference matrix calculated from these alignments with PHYLIP (Phylogeny Inference Package) version 3 . 5 ~ (Felsenstein, 1989).
RESULTS UcaA fimbrial subunit Fimbriae from the dog UTI-associated P. mirabilis strains IVB219 and IVB292 were isolated and the sequence of the first 25 amino acid residues of the major subunit protein was determined (Fig. 2). The sequence appeared identical to the N-terminal amino acid sequence of the UCA fimbriae major subunit protein from human P. mirabilir isolates, reported by Wray e t al. (1986). Upon SDSPAGE, the purified fimbrial protein revealed only one
band corresponding to a mass of approximately 17-5kDa. In electron microscopy, we only observed thin fimbriae with a diameter of 4 nm. These data further confirmed the resemblance to the results of Wray et al. (1986). We designed an oligonucleotide probe, B92-96, based on the sequence of amino acids 12-20 of the N-terminal amino acid sequence of the UCA major subunit protein. The neutral base inosine (I) was used in third positions of three- or fourfold ambiguity (Fig. 2). This probe hybridized with a 2 kb fragment in a PstI-Hind111 digest of chromosomal DNA from strain IVB247 which eventually resulted in strain IVB1433 (Fig. 1). Subsequently, we isolated an EcoRI fragment of 2.7 kb from the chromosomal DNA of strain IVB247 by means of a DraI-PstI fragment from the 2 kb fragment as a probe. Following the strategy as indicated in Fig. 1, we sequenced the gene for the UCA major subunit. Analysis of the derived nucleotide sequence yielded a 540 bp ORF encoding a polypeptide of 180 amino acids, including a signal peptide of 22 amino acids (Fig. 3). The molecular mass of the processed polypeptide of 158 amino acids was calculated to be 16724 Da. The first 25 amino acids of this mature subunit protein were identical to the N-terminal amino acid sequence of the UCA fimbrial subunit as determined by Wray e t al. (1986) for fimbriae from human P. mirabilis isolates.
Pap-like fimbrial subunit PmpA A second oligonucleotide probe was designed on the basis of amino acids 2-11 of the N-terminal amino acid sequence of the UCA major subunit protein. T o minimize the degeneracy of this probe, we took into account the homology with the K99 major subunit that is found particularly in this region (Roosendaal et al. , 1984; Wray e t al., 1986). This probe 220 (Fig. 2) was used to screen PstI-HindIII-digested chromosomal DNA from P. mirabilis strains IVB219 and IVB292 in Southern hybridization. A hybridizing fragment of approximately 2.3 kb from strain IVB219 was cloned into pBluescript and yielded strain IVB1425 (Fig. 1).In the strategy for cloning and sequencing (Fig. l), the subcloned ClaI fragment in pIVB1430 was used as a probe to isolate the 5 kb Hind111 fragment for subsequent cloning into pIVB1431. DNA sequence analysis revealed a 549 bp ORF. Downstream of the ORF, at positions 1058-1080, a potential inverted repeat was found (Fig. 4). Computer
1 5 10 15 20 25 Tyr A s p Gly Thr Ile Thr Phe Thr Gly Lys Val Val A l a Gln Thr Cys Ser Val A s n Thr A s n A s p Lys A s n Leu
G A GGT ~ ACT ATT ACC TT: Y
"
probe 220
ACT GGC A A ~ GTA GTI GCI CAA ACI TGC TCI GTI AAT AC w
probe 892-96
Fig- 2. Alignment of the UCA N-terminal amino acid sequence and the deduced oligonucleotide probes 220 and B92-96 that were used for the isolation of the pmpA and ucaA, respectively.
1352
Canine P. mirabilis fimbrial genes CGTAACTATCAAAATCAACGCCATCGATCGAhAAAACCATCTTTACT
47
AAGTGATATTGAAATAATAATTCAATTATTATTTTATATTGCTACATCACCATTATTAATATCTTTATTATTT
126
ATATAATCTACAAATATAAACCTCACTATAMCTCTATTTAATAGGATTATATATATTTTATTATTCAATAATACTTTT SD C A T T T T T T T G A A A M C A T A A C T T A A C T T T A A T T T ~ T ~ A A T C A T A M T AATG T AAA AGA AAA GTT H K B K V
205
ATA GCA CTA GCT ACT ATT CTT TCT GCT GCA TTT GCT GGC TCA TCT ATG GCG TAT GAG GGA I A L A T I L S A A P A G S S H A Y D G
339
279
-22
-1
mob.
. .
B92-96
....
+1
..: ::
GTI GCI CAA ACI TGC TCI GTI AAT AC *
ACC 'k%'kT 6°C AAT ACA AAT GAT AAG T C S V N T N D K
399
AAT TTA GCG GTA ACA TTA CCT ACA GTA TCC ACC ACT ACA TTA AAT G M AAT GCG GCT ACT
459
GCA GGT CTT ACT CCA TTT ACT ATT CAT TTA ACT GGT TGC GCT ATT GGT ATG GAT GGT,GCA A G L T P P T I H L T G C A I G M D G A
519
ACA ATT ACA TTT ACA GGT A M GTT GTT GCG 6 T I T P T G K V V A Q N
CAA
Q
L
A
V
T
L
P
T
V
S
T
M
T
T
L
N
E
N
A
A
T
AGT GTC AAA ACA TAT TTT GAA CCT TCA AGT GAG ATT GAT GTA ACC ACA CAC AAC TTA S V K T Y P E P S S D I D V T T H N L
A M AAT ACT GCA CAA ACT AAA GCT GAT AAT GTT CAA GTT CAA TTA CTT AAC TCA GAT GCA K N T A Q T K A D N V Q V Q L L N S D A
579 639
GCA ACA ACA ATC CAG TTA GGT ACT GAT TCT GCA ACA CAA GAT GTC CAT CCA GTA CAA ATC A T T I Q L G T D S A T Q D V H P V Q I
699
GAG AAT GCT AAT GTA AAC CTC CCA TAT TTT GCT CAA TAT TAT GCA ACC GGA CAA TCT ACC D N A N V N L P Y P A Q Y Y A T G Q S T
759
GCT GGG GAT GTA AAA GCA ACC GTT CAT TAC ACC ATT GCC TAT GAG TAA GGTTTATTGGATTGC A G D V K A T V H Y T I A Y E t
822
TTTTATTTTACAGGGCAGACAGCCCCATTTCCTTCTAGAGGGATTAT
874
............................................................................................................
Fig. 3. Nucleotide sequence of the major subunit gene, ucaA, from P. mirabilis and the alignment of probe B92-96 with the hybridizing part of the sequence. Two points (:) indicate that the respective probe nucleotide is identical to the corresponding nucleotide in the pmpA gene; one point (J indicates that there is only identity to the ucaA gene (compare with Figs 1 and 4). The deduced amino acid sequence of the ORF is given underneath in single-letter code. The first amino acid of the mature subunit protein is numbered + 1. A Shine-Dalgarno (SD) sequence is indicated.
analysis calculated a AG (25 "C) of - 12.2 kcal mol-' (- 51 k J mol-l) for this palindromic structure. The ORF encodes a polypeptide of 183 amino acids, including a putative signal sequence of 23 amino acids. The molecular mass of the processed polypeptide of 160 amino acids was calculated to be 16399 Da. Because of its P-fimbrial nature and with reference to p m f A (Bahrani e t al., 1993) and smfA (Mizunoe e t al., 1988), the gene encoding the predicted amino acid sequence was designated p m p A (P. mirabilis P-like pili).
structural fimbrial proteins (Fig. 5). The unprocessed UcaA protein appeared to share 56.1 % amino acid identity with the F17 major fimbrial subunit as well as 58.3% identity with the F l l l major subunit. However, comparison of the mature UcaA amino acid sequence with that of the PmpA major fimbrial subunit revealed only 18.4 YOidentity. The identical amino acid residues were mainly found in the N- and C-terminal regions. It was especially remarkable that the successive PmpA Nterminal residues 4-13 were identical to residues 2-1 1 of UcaA.
Occurrence of ucaA and pmpA in Proteus strains
Alignment of all the known sequences of the major fimbrial subunit proteins from UTI-related isolates of P. mirabili" and E. coli, as well as of homologous fimbrial sequences from other sources yielded a phylogenetic tree (Fig. 6) in which all the different Pap fimbrins of UPEC fell within one branch. The aligned amino acid sequence of PmpA showed an identity ranging from 40.8 to 43.7 % to the various Pap fimbrins (F7,-F16), including the F165,A sequence (Hare1 e t al., 1992; Maiti et al., 1994).
A total of 33 Protem strains and Proteas-related strains, listed in Table 1,were screened for the occurrence of both the acaA gene and the pmpA gene. Of 26 P. mirabiliJ strains, 22 were positive for the zlcaA probe, whereas 24 strains were positive for thepmpA probe. Sixteen of these P. mirabilis strains had been isolated from canine urine and all 16 strains were positive for both probes, except strain IVB227 which hybridized only with the p m p A probe. P. vzQaris or other Protetls-related species are isolated at a much lower frequency from patient materials (e.g. urine, faeces, organs). None of the seven isolates, other than P. mirabilis, reacted with the McaA probe or with the pmpA probe. Comparison of UcaA and PmpA amino acid sequences with other fimbrin sequences The deduced amino acid sequence of the UcaA major fimbrial subunit closely resembled both the F17 (Lintermans et a/., 1988) and the F111 (Lintermans, 1990)
DISCUSSION In this study, UCA fimbriae have been demonstrated in and isolated from a number of canine P. mirabilis strains. Since the N-terminal amino acid sequence of these fimbriae appeared identical to the N-terminal amino acid sequence of UCA fimbriae from human P. mirabilisisolates (Wray e t al., 1986), it was our prime goal to identify, isolate and sequence the major fimbrial subunit gene ~ l c a A from a P. mirabili~strain originating from a dog with UTI. We inadvertently first isolated a P-fimbriae-like major subunit gene, pmpA, merely because the amino acid 1353
I. G. W. B I J L S M A a n d O T H E R S
ATCGATMGMTAGAGGATCTGATTCTTTATTATTMGGMGMTATTGGAAATTTTATTAGAGMTCTCGTATACGTM
00
ATCTCTMCAGGAGCCCMTTAGGCGMTTACTTGATGTGAGTCAGCMCMATATCMGATATGAAAATGGMTMCM
160
GTATCAATATTGAAACATTAGATATGATATTAAMTTATTGGATGCTGACTGCGTCTGAGTTTTATCGAAAAGTTCTTGT
240
CGTAGATATTATAAAACATAAATT~TAGTGATTCGTTTCCATTCTATTTGCGTGTTTAGATGATTTATATCATCT
320
GTATAAATACAGTTTAAATTTTATATAAAAATTT~TAGTCATMGTTTTTAAAAGTMTGGMGTMCMTATCATC
399
TCTGGATGGGATGAGGATMTGTGTGTMCTATATATCATAGTTMTTTTTTMCGCTTTMTTCCTACGTTAAATATA
470
MGGTAAAAAT ATG A M TTG AAT A M TTA GCG ATG TTC GCT ATT GCA TCA ATG GCT TTT TCT H K L N K L A H F A I A S H A P S
540
-23
vroba 220
GA; GGT ACT ATT ACC TT; ACT GGC A A ~GTA :::
. . ..... . . ..... .
:::
::.
::
::: ::
GCG ACT GTT GCT C M GCG GCT TCA GGT GAT GGT ACT ATT ACT TTC ACT GGT A M GTT ATT T V A Q f , t , S G D G T I T P T G K V I A
600
GCT ATC GAT TTT GGT CAA ATC A I D P G Q I
660
GAT GCA CCT TGT GGT ATT GCT ACC G M AGT GCT AAC D A P C G I A T E S A N
CAA
Q
ATC CCA ATT AAA TTG I P I K L
720
GTT AAT TGT GAT TTA ACT A M GCC GGT TCT GAT ACT GGT GCT GCA GGT TCT TAT AAA GGC V N C D L T K A G S D T G A A G S Y K G
700
GTA AAA GTA ACC TTT AAT GGA M T ACT ATT ACT GGT GCA ACA GAA GAG TTA GCA ACA ACT V K V T P N G N T I T G A T E E L A T T
040
GGT AAT ACA GGG ACT GCT ATT GTT ATT TCA GGA ACA ACA ACT GGT TCA ATG GTT AAA TTC G N T G T A I V I S G T T T G S H V K P
900
AAT GAA GCA GGT G M TTA C M GCA CTG GGT M T M T GAT AAT ACG CTG ATG TAT ACA GCT N E A G E L Q A L G N N D N T L H Y T A
960
TGG GCT AAG AAA GCA ACA AAC GGA ACA ATT GCT GAA GGT GAG TTT AAC GCT ACA ACA AAC W A K K A T N G T I A E G E P N A T T N
1020
TTC ACA TTA GCC TAT GAA TAA AAMTATTAAATTTMCGAGAGCAGTTMTCTGCTCTCGATTATTTMGGC P T L A Y E t I \
1092
CAAAATCTATGTTTAAAAAAATC
1115
AGC S
AAA
K
AGC CTA TTA GAG M A GAT GGT ATT TCT S L L E K D G I S
C M
Q
GTT A M V K
C M
Q
............................................................................................................
inverted repedt
P17A
UcaA FlllA
Fig. 5. The amino acid sequence of the UcaA major subunit from the dog-uropathogenic P. mirabilis strain IVB247 compared to the amino acid sequences of the fimbrial subunits F17A (Lintermans et a/., 1988) and F l l l A (Lintermans, 1990) from bovine enterotoxigenicE. coli.
sequence chosen for the design of an oligonucleotide probe was identical in both the UcaA and PmpA polypeptides. Wray e t al. (1986) had already demonstrated significant homology of UcaA to the K99 N-terminal sequence, particularly among the first 10 residues (60 YO identity). Therefore, the K99 nucleotide sequence (Roosendaal e t al., 1984) was used to design probe 220, especially in third positions of three- or fourfold am1354
Fig. 4. Nucleotide sequence of the Pap-like pmpA gene from P. mirabilis and the alignment of probe 220 with the hybridizing part of the sequence. Two points (:) indicate that the respective probe nucleotide is identical to the corresponding nucleotide in the ucaA gene; one point (.) indicates that there is only identity to the pmpA gene (compare with Figs 1 and 3). The deduced amino acid sequence of the ORF is given underneath in single-letter code. The first amino acid of the mature subunit protein is numbered + l . An inverted repeat, following the stop codon (*), which might serve as transcription terminator, is underlined.
biguity. Yet, probe 220 appeared to match the p m p A nucleotide sequence much better than the m a A sequence (Fig. 4). Bahrani e t al. (1993) had a comparable situation when they identifiedpmfA using a probe also designed from amino acid residues (19-24) of the same UcaA Nterminal sequence. We designed a new probe on the basis of the subsequent amino acids (12-20) of the UCA Nterminal sequence and eventually succeeded in the isolation of the HcaA gene. The occurrence of UCA fimbriae has now been demonstrated both in canine isolates of P. mirabilis (this paper) and in human isolates (Wray e t al., 1986). The N-terminal amino acid sequences of the major subunits from the human and canine isolates appeared identical. Moreover, the amino acid composition derived from the translated ucaA gene is an exact match for the 25 amino acid residues of the N-terminal part of the UCA fimbrial subunit described by Wray e t al. (1986). Though we did not isolate Pmp fimbriae, there is also strong evidence for the occurrence of Pmp fimbriae on canine UPPM isolates. Bahrani e t al. (1991) isolated a mannose-resistant/Protetls-like (MR/P) type of fimbriae from a human UPPM strain and determined the Nterminal amino acid sequence of the major subunit which was designated MrpA. The N-terminal amino acid sequence of this MrpA subunit appeared identical to the first 20 amino acids of the processed polypeptide that we obtained after translation of the pmpA nucleotide se-
Canine P. mirabilis fimbrial genes F17
E. wli
F111
E. twli
UCA
P. mirabilis
PIL1
H. influemae
PIL3
K pneumoniae
SMF
S. marcBscens
MRP
P. mirabilis
F15
E. wfi
F12
E. coli
F16
E. wli
F7-2
E. cdl
F7- 1
E. wll
F14
E. wli
F9
E. wli
F165
E. cdi
F11
E. d l
F13
E. wll
F10
E. coli
F8
E. wli
PMP
P. mlrabills
PMF
P. mirabUis E. coli
K99
Fig. 6. Phylogenetic tree of the f . mirabilis PmpA and UcaA major fimbrial subunits and homologous fimbrial subunits. The tree was calculated with the assumption that there is an evolutionary clock and that all tip species are contemporaneous. This resulted in a rooted tree where the branches of the tree are constrained so that the total length from the root of the tree to any species is the same. References of the sequences: F17, Lintermans et a/. (1988); F111, Lintermans (1990); PlLl (Haemophilus influenzae), Whitney & Farley (1993); PIL3 (Klebsiella pneumoniae), Allen et a/. (1991); SMF, Mizunoe et a/. (1988); MRP, Bahrani & Mobley (1993, 1994); F9 and F12, Garcia et a/. (1992); F7-1, Van Die et a/. (1985); F7-2, Van Die et a/. (1984); F165, Harel e t a / . (1992) and Maiti et a/. (1994); F13, Bdga e t a / . (1983); F11, Van den Bosch e t a / . (1993); f . mirabilis fimbriae, Bahrani et a/. (1993); K99, Roosendaal et a/. (1984); the sequences of the F8, F10, F14, F15 and F16 serotypes of Ecoli P-fimbriae are unpublished results from W. Gaastra and L. van Dijk.
quence. However, in a subsequent paper (Bahrani & Mobley, 1993), the nucleotide sequence for a different major fimbrial subunit was designated as mrpA and the authors did not mention the previous MrpA sequence. For this reason and because of its P-fimbrial nature, the nucleotide sequence that we determined was designated PmPA.
It was surprising to find such high percentages in identity between fimbrial proteins not only produced by different bacterial species, but also by bacteria from different infection sites (the urinary tract versus the intestinal tract). The UcaA protein of P. mirabilis fimbriae appeared closely related to the F17 (Lintermans e t al., 1988) as well as the F111 (Lintermans, 1990) structural fimbrial proteins from bovine enterotoxigenic E. coli. The PmpA protein in turn showed a close relationship with the major subunits of the P-Yimbriae from UPEC. The close relationship of the F11
and F165 major fimbrial subunits (96% identity) is also interesting in this context, since F165 is associated with E. coli septicaemia in piglets (Harel e t al., 1992). It might therefore be speculated that the UCA type or the Pmp type of fimbriae on P. mirabilis isolates can be used for adhesion in the intestinal tract rather than in the urinary tract, in spite of the fact that it has been demonstrated that UCA fimbriae can mediate adhesion to uroepithelial cells (Wray e t al., 1986). Specific adhesion to uroepithelial cells should then be mediated by one of the other types of fimbriae produced by P. mirabilisisolates. This speculation is supported by the fact that most UTI-related P. mirabilis strains appeared to have genetic information for at least two different major subunits, UcaA and PmpA (Table l), and by the observation that faecal samples of dogs with recurrent UTI due to P. mirabilis contained lo4 Proteus cells per gram, whereas faecal samples of control dogs were negative (Gaastra e t al., 1995). A role for P-fimbriae of UPEC in the colonization of the large intestine has likewise been suggested (Wold e t al., 1988, 1992; Svanborg et a!., 1994). P-fimbriae-like sequences have been found in E. coli, P. mirabilis and Serratia marcescens strains isolated from UTI (Krogfelt, 1991;Kusters & Gaastra, 1994; Mizunoe etal., 1988). The MrpA protein appeared to share a high amino acid sequence identity with the SmfA subunit from a uropathogenic S. marcescens isolate and with the PapA subunit in fimbriae from UPEC (Bahrani & Mobley, 1993). So far, P. mirabilis is the only Proteus species that has been shown to have the genetic information for the production of this type of fimbria. The genetic information for these fimbriae could not be demonstrated in other Proteus-related species (Table 1). Bahrani e t al. (1993) obtained similar results for human P. mirabilis isolates. Therefore, we think that P. mirabilis has acquired the genes for these fimbriae recently in evolution by horizontal gene transfer, most probably from E. coli. Also, the fact that UPPM strains in contrast to UPEC strains do not exhibit host specificity in adhesion (Garcia et al., 1988b), is in favour of this hypothesis. Of the four major fimbrial subunit proteins of P. mirabilis for which the amino acid sequence is known, i.e. UcaA, MrpA, PmfA and PmpA, PmpA shows the closest relationship to the E. coli P-fimbrial major subunits. The evolutionary distance between the PmfA, MrpA and PmpA proteins is much larger. The similarity between PmpA and one of the P-fimbrial F serotypes is not higher than with any of the others, which indicates that P. mirabilis must have obtained the gene encoding this protein before the divergence of the various F serotypes in E. cob took place. On the other hand, K99 must have branched off earlier from the other P. mirabilis fimbrins on the phylogenetic tree (Fig. 6).
ACKNOWLEDGEMENTS We wish to thank Dr A. Agnes H. M. ter Huurne for her interest in our work and the lively discussions of this manuscript.
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