P fimbriae of Escherichia coli : Molecular cloning of DNA fragments containing the structural genes

June 24, 2017 | Autor: Timo Korhonen | Categoria: Biological Sciences, Escherichia coli, Molecular cloning, Sperm Dna Fragmentation
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FEMS MicrobiologyLetters 19 (1983) 119-123 Published by Elsevier

119

P fimbriae of Escherichia coli: Molecular cloning of D N A fragments containing the structural genes Mikael Rhen

,a,

J o n a t h a n K n o w l e s b, M e r j a E. Penttil~i Korhonen a

b

M a t t i Sarvas c a n d T i m o K.

a Department of General Microbiology, University ofHelsinki, b Technical Research Centre of Finland, Biotechnical Laboratory, Espoo, and c National Public Health Institute, SF-00280, Helsinki 28, Finland

Received 21 March 1983 Accepted 23 March 1983

1. I N T R O D U C T I O N The

adhesion properties of p a t h o g e n i c coli strains are often mediated by filamentous proteinaceous surface organelles called fimbriae [1]. Fimbriae specifically recognize structures on mammalian cell surfaces and are believed to anchor bacteria to their cognate hosts [2-5]. The ability to adhere to the site of infection is considered to be a prerequisite for enterotoxigenic dairrhoea in piglets, calves, lambs and humans and in human urinary tract infection caused by E. coli. Pyelonephritogenic human isolates of E. coli possess a special class of fimbriae termed the P fimbriae [3,6,7]. P fimbriae mediate adhesion of E. coli to human urinary tract epithelial cells by binding to the a-D-Gal-(1-4)-fl-D-Gal part of the P b l o o d group antigens [3-9]. Adhesion to uroepithelial cells is a major virulence factor in human childhood pyelonephritis [10]. The serological analysis of P fimbriae has been hampered by the multiplicity of fimbrial antigens in pyelonephritogenic E. coli strains [3]. The model strain used by us, E. coli KS71, displays two

Escherichia

* Correspondence to: M. Rhen, Department of General Microbiology Mannerheimintie 172, SF-00280, Helsinki 28, Finland.

different antigenic types of fimbriae when grown on agar plates [8,9]. The A-B fimbrial antigens are responsible for P blood group-specific haemagglutination. They are serologically related [9], whereas the the C fimbriae of KS71 lack demonstrable haemagglutination activity and do not cross-react immunologically with the A-B fimbriae [8,9]. In addition, KS71 express the common type 1 fimbriae in static broth cultures. These fimbriae do not cross-react immunologically with the A, B or C fimbriae [3,9]. This communication describes the molecular cloning of the structural genes for the A, B and C fimbriae.

2. M A T E R I A L S A N D M E T H O D S 2.1. Bacterial strains and cultivation

The pyelonephritogenic E. coli strain KS71 ( 0 4 : K12) was kindly donated by Dr. G. K~illenius (Statens Bakteriologiska Laboratorium, Stockholm, Sweden). The P. fimbriae isolated from this strain have recently been characterized [3,8,9]. As a recipient we used the E. coli K12 derivative HBI01 (leu, pro, lac, hsdS, gal, strA, thi, recA). Strains were cultivated on Luria broth or on Luria agar. Recombinant strains were cultured on media supplemented with ampicillin (100/~g/ml).

0378-1097/83/0000-0000/$03.00 © 1983 Federation of European Microbiological Societies

120

2.2. Enzymes The restriction endonucleases BamHI (Boehringer-Mannheim), EcoRI SalI and Sau3A (all three obtained from Amersham International) were used under conditions recommended by the manufacturers. T4 ligase was obtained from New England Biolabs.

2.3. Cloning procedure Whole-cell D N A was isolated from a 100 ml broth culture of KS71 grown to the exponential phase of growth essentially as described previously [11]. Long D N A (over 100 kilobases (kb)), most likely chromosomal, was partially digested with Sau3A restriction enzyme. Digests were electrophoresed in 0.5% L G T agarose gels. Fragments of 50-35 kb were cut out from EtBr-stained gels and D N A isolated by electroelution. Isolated KS71 D N A fragments (400 ng) were ligated with 200 ng of BamHI-treated pHC79 cosmid D N A [12] in 6 #1 of ligation buffer containing 200 U of T4 ligase. The suspension was maintained at + 16°C for 18 h and then packed in vitro into )~ phage particles [131. Plasmid D N A was isolated as described in reference [14] and HB101 cells were transformed according to [15].

2.5. Isolation of fimbriae Fimbriae were purified by the immune precipitation method [8] employing only one precipitation with 5 ~1 of anti-KS71ABC serum per 100 #g of detached antigen. Purity of fimbriae preparations was assayed by sodium dodecyl sulphate gel electrophoresis (SDS-PAGE) [16] and by electron microscopy. Electron microscopy was performed with phosphotungstic acid-stained preparations in a JEM-100 transmission electron microscope operating at 80 kV. Electron micrographs were taken at the Department of Electron microscopy, University of Helsinki.

a

b

c

d -94 -67 -43 -30

2.4. Agglutination tests Bacterial agglutination was performed on glass slides using antisera against the total fimbrial fraction from agar grown KS71 cell (anti-KS71ABC) or against the purified KS71A, KS71C or E. coli 2131 type 1 fimbriae [3,8,9]. Screening for fimbriated recombinants was performed by mixing single colonies in 50 ~1 of an antiserum suspension (antiKS71 diluted 1/50 in 0.01 M Tris-HC1, 0.9% NaC1 buffer, pH--= 7.5). Titrations with anti fimbriae sera were performed by mixing 25/.tl of a bacterial suspension (approx 3 - 1 0 9 cells/ml in phosphate-buffered saline) with an equal volume of a two-fold dilution of antiserum. Haemagglutination of h u m a n Pj and erythrocytes in the presence of 5% ( w / v ) c~methyl-mannopyranoside was performed as previously described [9].

-20.1

S-~ I

-14.4

Fig. 1. SDS-polyacrylamidegel electrophoresis (155~ gel) of purified fimbriae from E. coli strains KS71, EH824, EH825 and EH826. Lane a shows the A, B and C fimbriae from agar grown KS71 cells. Fimbriae purified from recombinant strain EH824 consists of the A peptide only (lane b), whereas fimbriae from recombinant strains EH825 (lane c) EH826 (lane d) contain only the B and C peptides, respectively.The positions of Mr markers (kDa) are indicated at the right.

121 3. R E S U L T S

a

A total of 200 ng of insert D N A was packed in vitro into )~ phage particles and used to transfect the HB101 recipient. The HB101 cells were plated onto Luria plates containing ampicillin and about 6700 A m p r colonies were obtained. A total of 1500 A m p r colonies were tested for the presence of fimbriae by means of agglutination with antiserum against the K S 7 1 A B C fimbriae. Seven strongly agglutinating colonies were obtained. The fimbrial fraction was purified from the strains that were agglutinated by anti-KS71ABC serum. W h e n such fimbriae preparations were analyzed in S D S - P A G E , it was found that a given recombinant strain produced only one of the three KS71 fimbrial peptides. Three different strains, each representing one of the three fimbrial peptides, were selected for further investigation. The strain EH824 produced the KS71A fimbrial peptide, whereas the strains EH825 and EH826 produced the KS71B and KS71C peptides, respectively (Fig. 1). Fimbriae preparations obtained from the three strains had identical morphology, i.e. they were 7 n m in diameter and 0.3 to 2 /~m

C

Fig. 2. Electron micrographs of non-fimbriated and fimbriated strains. No fimbriae are seen on the recipient strain HB101 (a). Numerous fimbriae are seen on the recombinant strains EH824 (b), EH825 (c) and EH826 (d). The bar represents 200 nm.

E. coli

122

long. The strains EH824, EH825 and EH826 were clearly fimbriated when viewed in the electron microscope in contrast to the recipient strain HB101 (Fig. 2.). The agglutination properties of the recombinant strains are shown Table 1. All recombinant strains were agglutinated by the polyspecific antiKS71ABC serum, EH824 and EH825 were also agglutinated by the anti-KS71A serum but not by the anti-KS71C serum. EH826 was not agglutinated by anti-KS71A serum significantly but high agglutination titers were obtaind with the antiKS71C serum. Antiserum against type 1 fimbriae of E. coil 2131 did not react significantly with KS71, EH824, EH825, EH826 or HB101. Human P~, but not ~, erythrocytes were agglutinated by EH824 and EH825. The ~ erythrocytes are not agglutinated by P fimbriae since they lack the P-blood-group antigens [3,17]. EH826 failed to agglutinate either type of erythrocytes. Hybrid plasmids were isolated from EH824, EH825 and EH826 and the plasmids were named pKTH143, pKTH144 and pKTH145, respectively. The plasmids were then treated with E c o R I and S a i l restriction enzymes to measure the insertion size. The inserted DNA fragments were in the size range for cosmid inserts [13] (approx. 40 kb in pKTH144, approx 35 kb in pKTH143 and pKTH145). The presence of the fimbrial genes in the plasmids was checked by transforming HB101 cells with isolated hybrid cosmid DNA. Transformants produced the fimbrial type of the clone from which the plasmid was isolated.

4. DISCUSSION In this work the genes encoding the A, B and C fimbriae of E. coli KS71 were transferred to and cloned in the HB101-recipient strain. Although the parent strain KS71 possesses three different fimbriae when grown on agar plates [3,8,9] all the fimbriated recombinants obtained in this study produced only one fimbriae each. The simplest explanation for this phenomenon is that the genes encoding the three fimbriae are located on different parts of the chromosome, although other explanations cannot as yet be excluded. However, analogous findings have been reported by Hull et al. [11]. In their study, the genes encoding mannose-resistant and mannose-sensitive fimbriae were shown to be on different parts of the chromosome of E. coli. In previous works we have shown that the KS71A and KS71C peptides are the subunits of two different fimbrial species [8,9]. However, the fimbrial nature of the KS71B peptide has not been clearly demonstrated. The recombinant strain EH825 produced fimbriae, consisting of KS71B peptides only, in a same manner as EH824 and EH826 produced the KS71A and KS71C fimbriae, respectively. Thus, it is concluded that the KS71B peptides originate from yet another type of fimbriae. As suggested previously [8], the KS71B fimbriae were immunologically cross-reactive with KS71A fimbriae but not with KS71C fimbriae (Table 1). EH824 and EH825 both agglutinated human erythrocytes in a P-blood-group specific

Table 1 Agglutination properties of parent, recipient and recombinant strains Bacterial strain

Agglutination titer with anti-KS71ABC

anti- KS71A

anti-KS71C

anti-type 1

H u m a n P~ erythrocytes

Human erythrocytes

KS71 HB101 EH824 EH825 EH826

5210 0 5210 5210 5 210

1 280 0 5210 2561 10

640 0 0 0 1280

10 0 10 10 20

32 0 64 32 0

0 0 0 0 0

Haemagglutination titer with

123

manner indicating that both KS71A and KS71B are P fimbriae. EH826 did not cause haemagglutination. These results confirm our earlier studies [9] which indicated that the P blood-group binding activity resides in the KS71A-B fimbriae but not in the C fimbriae. 13rskov et al. [18] have studied the fimbrial antigens from an E. coli O 6 : K 2 strain in crossed immunoelectrophoresis. The fimbrial antigens responsible for mannose-resistant haemagglutination in this strain was termed the F7 antigen. The Mr-value and N-terminal amino acid sequence of the F7 antigen are remarkably similar to those of KS71A, whereas another fimbrial antigen isolated from the O 6 : K 2 strain resembles the KS71C fimbriae [9,19]. Furthermore, the F7 antigen is reported to consist of two structures termed F7~ and F72, as two immune precipitation spurs were seen in crossed immunoelectrophoresis [18]. Thus, the fimbrial constitution of KS71 might be analogous to that of the E. coli 0 6 : K2 strain. This is the first communication that describes the cloning of fimbriae genes from an uropathogenic E. coil strain with well characterized fimbrial constitution. The availability of strains that differ in only one property, i.e. the type of fimbria expressed, will enable a detailed serological and functional analysis of P-fimbriae.

REFERENCES [1] Duguid, J.P., Smith, I.W., Dempster, G. and Edmunds, P.N. (1955) J. Pathol. Bacteriol. 70, 335-348. [2] Gaastra, W. and De Graaf, F.K. (1982) Microbiol. Rev. 46, 129-161. [3] Korhonen, T.K., V[iisAnen, V., SaxOn, H., Hultberg, H. and Svenson, S.B. (1982) Infect. Immun. 37, 286-291. [4] K/illenius, G., Svenson, S.B., M611by, R., Cedergren, B., Hultberg, H. and Winberg, J. (1981) Lancet ii, 604-606. [5] Korhonen, T.K. Eden, S. and Svanborg Ed6n, C. (1980) FEMS Microbiol. Lett. 7, 237-240. [6] K~illenius, G. M611by, R., Svenson, S.B., Helin, I., Hultberg, H. Cedergren, B. and Winberg, J. (1981) Lancet ii, 1369-1372. [7] Vais~nen, V., Elo, J., Tallgren, L.C., Siitonen, A., M~ikel/i, P.H., Svanborg Ed6n, C., K/illenius, G., Svenson, S.B., Hultberg, H. and Korhonen, T.K. (1981) Lancet ii, 1366-1369. [8] Rhen, M., Wahlstr6m, E. and Korhonen, T.K. (1983) FEMS Microbiol. Lett. 18, 227. [9] Rhen, M., Klemm, P., Wahlstrbm, E., Svenson, S.B., Kallenius, G. and Korhonen, T.K. (1983) FEMS Microbiol. Lett. 18, 233. [10] Svanborg Ed6n, C., Hans~a, L..~., Jodal, U., Lindberg, U. and Sohl Akerlund, A. (1976) Lancet ii, 490-492. [11] Hull, R.A., Gill, R.E., Hsu, P., Minshew, B.H. and Falkow, S. (1981) Infect. Immun. 33, 933-938. [12] Hohn, B. and Collins, J. (1980) Gene 11,291-298. [13] Collins, J. and Hohn, B. (1978) Proc. Natl. Acad. Sci. USA 75, 4242-4246. [14] Clewell, D.B. (1972) J. Bacteriol. 110, 667-678. [15] Mandel, M. and Higa, A. (1970) J. Mol. Biol. 53, 159-162. [16] Laemmli, U.K. (1970) Nature 227, 680-685. [17] Naiki, M. and Kato, M. (1979) Vox Sang. 37, 30-38. [18] Orskov, I., Orskov, F., Birch-Andersen, A., Klemm, P. and Svanborg Eden, C. (1982) in Seminars in Infectious Disease, Vol. IV. Bacterial Vaccines (Robbins, J.C., Hill, J.C., Sadoff, J., Eds.), pp. 97-1103. Thieme-Stratton, New York. [19] Klemm, P., 121rskov, I. and Orskov, F. (1982) Infect Immun. 36, 462-468.

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