Download Cloning and nucleotide sequence of a gene upstream of the eaeA

EISEVIER
FEMS Microbiology
Letters 133 (1995) 35-39
Cloning and nucleotide sequence of a gene upstream of the eaeA
gene of enterohemorrhagic Escherichia coZi 0157:H7
Shaohua Zhao a, Sharon E. Mitchell b, Jianghong Meng a, Michael P. Doyle a,*,
Stephen Kresovich b
’ Centerfor
Enhancement and Department qf’ FoodScience and
Unilvrsity of Georgia, GrifJin, GA 30223, USA
Unit, USDA/ARS,
Unir ersity of Georgia, Griffin. GA 30223,
Food Safety and Quality
’ Genetic Resources
Technology.
USA
I August 1995; accepted 28 August 1995
Received
Abstract
A DNA segment located immediately
upstream
of the eaeA gene of enterohemorrhagic
Escherichia
coli 0157:H7 strain
HAI was cloned and sequenced. This segment contained an open reading frame encoding a predicted protein of 156 amino
acids. A database search identified similar open reading frames upstream of the eaeA gene in two other bacterial pathogens,
i.e. enteropathogenic
E. coli and Citrobacter freundii. The predicted amino acid sequence of the enterohemorrhagic
E. coli
protein shared 96.8% and 94.2% identity with the enteropathogenic E. coli and C. freundii sequences, respectively. Because
the open reading frame is located within the locus of enterocyte effacement region of the E. coli chromosome, a ‘hot spot’
for insertion of virulence factor genes, and shares high sequence homology with attaching and effacing EPEC and C.
freundii, this protein may be associated with pathogenicity of E. co/i 0157:H7.
Keywords;
Escherichia
co/i 0
1S7:H7; Hemorrhagic
colitis; Enterohemorrhagic
1. Introduction
Enterohemorrhagic
E~cherichia
colt’ (EHEC)
0157:H7 is the most common cause of hemorrhagic
colitis and hemolytic uremic syndrome [ 11. Although
the precise mechanism of pathogenicity has not been
fully elucidated, it is generally thought that E. coli
0157:H7 causes disease by adherence to the host
cell membrane followed by production of one or
more Shiga-like toxins (SLTs) [2,3]. The structure
* Corresponding
author. Tel.: + 1 (404) 228 7284; Fax:
(404) 229 3216; E-mail: [email protected].
037%1097/95/$09,50
0
SSDI 037X-1097(95)00330-4
1995 Federation
of European
+
I
Microbiological
Escherichia
co/i
and function of SLT genes and their products have
been extensively studied 141. The mechanism of bacterial adherence, however, is less clear, although
some adherence factors have been identified [2,3].
Like the enteropathogenic
E. coli (EPEC), E. coli
0157:H7 can colonize the intestine of humans and
experimental animals, producing a characteristic attaching and effacing (AE) lesion. Two genes involved in the generation of these lesions have been
characterized:
eaeA encodes intimin, a protein required for intimate attachment [5] and eaeB encodes
a secreted protein necessary for intimate attachment
and transduction
of signals to epithelial cells in
EPEC [6]. The eaeB gene is located approx. 4.5 kb
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downstream from the 3’ end of the eaeA gene. Both
eaeA and eaeB have been sequenced and characterized [6-81. Studies have shown that although eaeA
is important for the production of lesions, this gene
alone is insufficient to confer AE activity [9]. The
function of eaeB of EHEC has not been fully elucidated.
An approx. 35kb conserved region containing the
earA, eaeB, and sep (secretion of E. coli protein)
genes has been reported in both EHEC and EPEC
[3]. This region, the locus of enterocyte effacement
(LEE), is not present in normal flora E. coli, E. co/i
K12 or enterotoxigenic
E. cc&, but is present in
other AE bacteria, including
Citrobacter ,freundii
biotype 4280, Hafnia alcei, and E. coli strain
RDEC-I. In EPEC and EHEC, the LEE region is
inserted into the E. coli K12 chromosome at approx.
82 min which is also the location of a large (approx.
70 kb) insert in the uropathogenic E. coli. This large
insert or ‘pathogenicity
island’ contains virulence
factor genes in uropathogenic
E. coli. The fact that
the LEE of EHEC and EPEC is located at the same
chromosomal site suggests that this region is a ‘hot
spot’ for insertion of virulence factor genes. In the
present study, we report the DNA sequence of the
region immediately flanking the 5’ end of the EHEC
eaeA gene, and compare the predicted amino acid
sequence of an open reading frame (ORF) located
within this region to those from similarly located
ORFs in EPEC and C. ,freundii.
2. Materials and methods
2.1. Bacterial media and chemical reagents
Luria broth (LB) and Luria plates were used for
routine culturing of E. coli strains. All media were
obtained
from Difco, Detroit,
MI. Ampicillin,
kanamycin,
nalidixic acid, 5-bromo-4-chloro-3-indolyl-b-galactoside
(X-gal),
and isopropyl-P-Dthiogalacto-pyranoside
(IPTG) were purchased from
Sigma Chemical Co., St. Louis, MO.
2.2. Bacterial strains and cectors
our
E. coli 0157:H7 strain HAI was maintained in
laboratory.
HA1 was derived from E. coli
0157:H7 strain 932. The parent strain was previously used for developing the monoclonal antibody
(mAb) 4E8C12 that specifically reacts with a unique
outer membrane protein (OMP) of E. coli 0157:H7
[ 101. E. coli 0157:H7 strain A10 was created in our
laboratory by Tn phoA mutagenesis [I I] and did not
express this unique OMP. E. coli strain JM 109
(Promega, Madison, WI) was used as the host cells
for plasmid vector pUC 19 (Promega).
2.3. Construction
screening
of expression
library and imrnuno-
Genomic DNA of E. coli 0157:H7 strain HAI
was prepared and purified by cesium chloride gradient centrifugation
using the procedure described by
Sambrook et al. [ 121. The chromosomal
DNA of
HA1 and plasmid DNA of pUC 19 were digested to
completion with BamHI and EcoRI according to the
manufacturer’s
protocol (Gibco BRL, Gathersburg,
MD). 50 ng of pUC 19 and I50 ng of E. coli
0157:H7 digested DNAs were mixed together and
ligated by adding 5 units of T4 DNA ligase (Gibco
BRL). The reaction mixture was incubated at 4°C
overnight and recombinant plasmids were then transformed into E. coli JM 109. Cells were plated onto
LB plates containing 50 pg ampicillin ml-‘, 25 ,ug
IPTG ml ~~’ and 25 pg X-gal ml-‘.
The white,
ampicillin-resistant
E. coli colonies were selected
and immunoscreening
was performed by the method
described by Sambrook et al. [ 121 using mAb 4E8C I2
(detects an OMP-specific to E. coli 0157:H7) as the
first antibody, and a goat anti-mouse IgG conjugated
to alkaline phosphatase (Sigma, St. Louis, MO) as
the second antibody. Western blots of proteins from
one colony showing a strong positive result in the
immunoblot
assay (clone 6-F) were probed with
mAb 4E8C 12.
2.4. Southern blot analysis
Southern blot hybridization [ 121 was performed to
determine if clone 6-F (positive in the immunoblot
assay) contained the same DNA sequence as in the
region flanking the TnphoA insertion site in the
OMPmutant strain, AlO. Strain A10 does not
express the protein detected by mAb 4E8C 12. Genomic DNAs from E. coli 0157:H7 strains HA 1 and
S. Zhao rt 01. / FEMS Microbiology
A10 were digested with EcoRV and probed
purified insert DNA from clone 6-F.
2.5. DNA sequencing
with
und analysis
Plasmid clone 6-F was sequenced directly by Taq
cycle sequencing using fluorescence-based
chain termination chemistry (Perkin Elmer/Applied
Biosysterns, Foster City, CA) and an automatic DNA sequencer (Perkin Elmer/Applied
Biosystems, model
373 A). Electropherograms
were edited and DNA
contigs were assembled with the aid of the computer
program Sequencer 2.1 (Gene Codes Corp., Ann
Arbor, MI). Additional analyses were performed using programs included in the GCG (Genetics Computer Group, Madison, WI) package [ 131. The entire
798-bp DNA sequence from 6-F and the deduced
amino acid sequence from one complete open reading frame were used to search the combined data
bases of the National Center for Biotechnology Information (Washington,
D.C.) via the BLAST network service. The database searches employed the
alignment algorithm of Altschul et al. [ 141.
2.6. Nucleotide
sequence and accession
number
The DNA sequence data reported in this article
have been submitted to GenBank database and assigned the accession number U323 12.
1
Letters
133
ClYY5l 35-39
31
3. Results and discussion
Several thousand
colonies
from the E. coli
0157:H7 library were screened in the immunoblot
assay. One colony containing
a plasmid with an
approx. 800-bp insert, 6-F, consistently reacted with
mAb 4E8C12. This antibody detects an OMP unique
to serotype 0157:H7
and a few SLT-producing
serotypes of E. coli [lo]. Western blots, however,
revealed no reaction with mAb 4E8C12 despite the
fact that protein samples from clone 6-F were prepared using several different methods.
Southern blot analysis was performed to determine if clone 6-F contained the same gene interrupted by Tn phoA in the OMP- mutant strain, A 10.
The insert DNA from clone 6-F was purified and
hybridized to EcoRV-digested genomic DNAs from
E. coli 0157:H7
strains HA1 (OMP+) and A10
(OMP-1. In strain AlO, the EcoRV fragment containing TnphoA
was approx. 9.4 kb [ 111. Since
EcoRV does not cut within TnphoA, this fragment
includes the complete transposon plus approx. 2 kb
of flanking sequence. Therefore, if clone 6-F contained DNA sequences homologous to those flanking
the transposon insertion site in strain AlO, a 9.4-kb
fragment should have been detected for EcoRV-digested A 10 DNA and an approx. 2-kb fragment
should have been detected for similarly digested
120
38
S. Zhao et al. / FEMS Microbiology Letters 133 f 19951 35-39
1
100
KPAEKIGnxISFNmRLCSFAIDEIWIS IsD?4iDEYmIYGVCGKFPT
DNSNPALBIL
NANLWFAENG
GPYLCYEAGA
QSLLLALRPP
EH!x MSSRSELLLE
______*--- --_____s__ __________
EPGC _____----fJ R_-----V-S __________ -A--______ _--_A---- __________ --p------__~~______ __________ ~-p------- ----__R--- ----_-_s__ __________
CBFR ________-E ~------~-~ ___^_.____ -v-------156
101
NEmwvKSMBNLYLVLHNQ
GITLENmmK
IEBISSSDNK
HYmGR’
BHBC LmJATPBKLE
___A_-____
__________
___.---___
__________
-_____--_______
EPF.C
----______ ______N_____-.___
CBQR ---v-_____ _--.______ ___.---.--
Fig. 2. Alignment of the predicted amino acid sequences encoded by ORFs located upstream of the eaeA gene in: E.wherichia coli
0157:H7 strain HAI (EHEC), EPEC E2348/69
([17]; EMBL/GeneBank/DDBJ
Nucleotide Sequence Data Libraries accession no.
M3405 I) and Cirrobacterfreundii strain 4280 (CBFR) ([15]; accession no. Ll 1691). Dashes represent sequence identity.
wild-type DNA (strain HAl).
Results from the
Southern analysis revealed, however, that insert DNA
from clone 6-F hybridized to a 3.5-kb EcoRV fragment in both HAI and A10 (data not shown). We,
therefore, concluded that clone 6-F did not contain
the gene encoding the unique OMP and that the
positive reaction with mAb 4E8C12 was due to
non-specific binding.
The complete DNA sequence of the clone 6-F
insert (798 bp) is shown in Fig. 1. This clone
contained 110 bp of the 5’ region of the EHEC eaeA
gene (encoding 36 N-terminal amino acids of the
intimin protein), and 688 bp of upstream DNA sequence. The sequence upstream of the eaeA gene
included an open reading frame potentially encoding
a 156-amino acid protein (Fig. 1). Results from the
BLAST search indicated that this putative protein
has significant homology to predicted proteins, also
located upstream of eueA, in two pathogenic bacteria, EPEC and C. ,freundii. EPEC produce diarrhea in
humans, whereas C. freundii is the causative agent
of transmissible murine colonic hyperplasia [ 151.
The amino acid sequence alignment of the predicted protein in EHEC, EPEC and C. freundii is
shown in Fig. 2. The protein sequence of EHEC
shared 96.8% and 94.2% identity with the sequences
from EPEC and C. freundii, respectively. Three out
of a total of five amino acid substitutions between
EHEC and EPEC occurred within the first 18 Nterminal residues of the predicted protein, whereas
the pattern of amino acid substitution between EHEC
and C. freundii was more uniform.
Recent studies have revealed that the eaeA, eaeB,
and spa (cfm) genes are all located within an approx. 35-kb region that is present in EPEC and E.
coli 0157:H7
[3]. This so-called LEE region is not
present in normal flora E. coli, E. coli K- 12, or
enterotoxigenic
E. coli but is present in other AE
bacteria, including
C. freundii biotype 4280, H.
alvei, and E. coli strain RDEC-1 that causes diarrhea in rabbits, and is thought to be a hot spot for
insertion of virulence factor genes in the E. co/i
chromosome. Lai and Donnenberg [ 161 reported that
genes located between eueA and eaeB as well as
downstream of eaeB are required for attaching and
effacing activity. Characterization
of genes in the
LEE region should help clarify the biochemical
mechanisms involved in the pathogenicity of E. coli
0157:H7. We propose that the open reading frame
immediately upstream of the eueA gene might encode a virulence factor and participate in the formation of AE lesions. Expression and mutation analyses
of this gene are presently underway, and these studies may determine if genes located upstream of eueA
are important in E. coli 0157:H7 pathogenesis.
Acknowledgements
This study was supported in part by USDA Special Grant 93-COOP-2-959 I.
References
[I] Doyle. M.P. (199 1) Escherichia coli 0 157:H7 and its significance in foods. Int. J. Food Microbial. 12, 289-301.
[2] Tesh, V.L. and O’Brien, A.D. (1992) Adherence and colonization mechanisms of enteropathogenic
and enterohemorrhagic Escherichia cd. Microb. Pathog. 12, 245-254.
[3] Kaper,
J.B. (1994)
Molecular
pathogenesis
of enteropathogenic
Escherichia co/i. In: Molecular Genetics of
Bacterial Pathogenesis (Miller, V.L., Kaper, J.B., Portnoy,
D.A. and Isberg, R.R., Ed%), pp. 173- 195. American Society
for Microbiology, Washington, D.C.
S. Zhao et nl. / FEMS Microbiology
[4] O’Brien,
A., Tesh, V., Donohue-Rolfe,
A., Jackson, M.,
Olsnes, S., Sandvig, K., Lindberg, A. and Keusch, G.T.
(1992) Shiga toxin: biochemistry,
genetics mode of action
and role in pathogenesis.
Curr. Top. Microbial. Immunol.
180, 65-94.
[5] Donnenberg. MS, Tzipori, S., McKee, M.L.. O’Brien, A.D.,
Ahoy, J. and Kaper, J.B. (1993) The role of the eae gene of
enterohemorrhagic
Escherichia
coli in intimate attachment
in vitro and in a porcine model. J. Clin. Invest. 92, 14181224.
[6] Donnenberg, MS, Yu, J. and Kaper, J.B. (1993) A second
chromosomai gene necessary for intimate attachment of enteropathogenic
Escherichicl cob to epithelial cells. J. Bacteriol. 175, 4670-4680.
[7] Beebakhee. G., Louie, M., De Azavedo, J. and Brunton. J.
(1992) Cloning and nucleotide sequence of the eae gene
homologue from enterohemorrhagic
Escherichia co/i. FEMS
Microbial. Lett. 91, 63-68.
[8] Yu. J. and Kaper, J.B. (1992) Cloning and characterization
of
the eae gene of enterohemorrhagic
Escherichia
co/i
0157:H7.
Mol. Microbial. 6, 41 I-417.
[9] Lottie, M.. De Azavedo, J.C.S., Handelsman, M.Y.C., Clark,
C.G., Ally, B., Dytoc, M., Sherman, P. and Brunton, J.
(1993) Expression and characterization
of the eaeA gene
product of Escherichia cdi serotype 0157:H7. Infect, Immun. 6 I, 4085-4092.
[ 101 Padhye. N.V. and Doyle, M.P. (1991) Production and characterization of a monoclonal antibody specific for enterohemorrhagic Eschrrichia co/i of serotypes 0157:H7 and 026:HI I.
J. Clin. Microbial. 29. 99-103.
Letters 133 (/YY5135-39
39
[I I] Zhao, S., Meng. J., Doyle. M.P. and Wang, G. (1995) A
unique outer membrane protein associated with colonization
of Eschrrichia co/i 0157:H7 on human intestinal epithelial
cells, Abstr. B9, p. 167. Abstr. 95th Gen. Meet. Am. Sot.
Microbial. 1995. American Society for Microbiology, Washington, D.C.
[ 121 Sambrook. J., Fritsch, E.F. and Maniatis. T. (1989) Molecu-
[ 131
1141
[I51
[I61
[I71
lar Cloning: A Laboratory Manual, 2nd edn. Cold Spring
Harbor Laboratory, Cold Spring Harbor, NY.
Devereaux, J., Haeberli, P. and Smithies, 0. (1984) A comprehensive set of sequence analysis programs for the VAX.
Nucleic Acids Res. 12. 387-395.
Altschul, S.F., Gish, W., Miller. W., Myers, E.W. and Lipman, D.J. (1990) Basic local alignment search toll. J. Mol.
Biol. 215, 403-310.
Schauer. D.B. and Falkow, S. (1993) The etie gene of
Citrohrrcter ,freundii biotype 4280 is necessary for colonization in transmissible murine colonic hyperplasia. Infect. Immun. 61. 2486-2492.
Lai. L.-C. and Donnenberg, M.S. (1995) Novel loci within
the eae gene cluster are necessary for attaching and effacing
activity of enteropathogenic
Escherichirr coli (EPECJ. Abstr.
84, p. 167. Abstr. 95th Gen. Meet. Am. Sot. Microbial.
1995. American Society for Microbiology, Washington, D.C.
Jerae, A.E., Yu. J., Tall, B.D. and Kapper, J.B. (1990) A
genetic locus of enteropathogenic
Escherichia co/i necessary
for the production of attaching and effacing lesions on tissue
culture cells. Proc. Natl. Acad. Sci. USA 87. 7839-7843.