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Journal of Bacteriology, November 2001, p. 6717-6720, Vol. 183, No. 22
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.22.6717-6720.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Molecular Characterization of a Secreted Enzyme
with Phospholipase B Activity from Moraxella
bovis
Jacinta L.
Farn,1,2,*
Richard A.
Strugnell,2
Peter A.
Hoyne,3
Wojtek P.
Michalski,1 and
Jan M.
Tennent1,
CSIRO Livestock Industries, Geelong,
Victoria, Australia 3220,1 and
Department of Microbiology and Immunology, The University of
Melbourne,2 and CSIRO Health Sciences
and Nutrition,3 Parkville, Victoria, Australia
3052
Received 30 April 2001/Accepted 29 August 2001
 |
ABSTRACT |
A candidate for a vaccine against infectious bovine
keratoconjunctivitis (IBK) has been cloned and characterized from
Moraxella bovis. The plb gene
encodes a protein of 616 amino acids (molecular mass of ~65.8
kDa) that expresses phospholipase B activity. Amino acid sequence
analysis revealed that PLB is a new member of the GDSL
(Gly-Asp-Ser-Leu) family of lipolytic enzymes.
| |
TEXT |
Moraxella bovis is the
etiologic agent of infectious bovine keratoconjunctivitis (IBK), the
most common ocular disease that occurs in cattle (2, 13,
15). IBK is highly contagious and, if left untreated, can result
in corneal ulceration and temporary or permanent blindness. In vitro
studies have suggested that M. bovis produces a number of
virulence determinants, including type IV pili (3, 23), a
secreted hemolysin (28), proteases, fibrinolysins, and
phospholipases (9). To date, only type IV pili and the
hemolysin have been substantively linked to the virulence of M. bovis (4, 10, 16, 17, 20, 26). Here we describe the
molecular characterization of an M. bovis gene encoding a phospholipase B activity and discuss the role this enzyme may play in
the virulence of this veterinary pathogen.
The Australian M. bovis strains used in this study have been
defined according to their pilus serotype (25): S276R
(serotype A), 3WO7 (serotype B), Dalton 2d (serotype C), R593L
(serotype D), Tat849 (serotype E), 218R (serotype F), and Fleur462
(serotype G). Other M. bovis strains used, Q220 and Epp63,
were isolated in the United Kingdom and the United States,
respectively. All of these M. bovis strains were positive
for lipase activity when tested on Tween 80 medium (30),
as indicated by a zone of opalescence around areas of growth.
Cloning and sequence analysis of the M. bovis plb
gene.
Genomic DNA from M. bovis strain Dalton 2d
(serotype C) was purified with cetyltrimethylammonium bromide-NaCl and
phenol chloroform extractions followed by isopropanol precipitation.
The genomic DNA was partially digested with Sau3A under
conditions that maximized the amount of DNA in the size range of 1 to 2 kb. Fragments <200 bp were removed by passing the digested DNA through
a Microspin S-400 HR column (Pharmacia). The 1- to 2-kb fragments were
ligated with pBR322 (5), previously digested with
BamHI, and electroporated into Escherichia coli
strain MC1061 (31). The partial genome library was
screened on Tween 80 medium, and 28 out of 24,000 clones were found to
be positive for lipase expression.
All positive clones contained a 5.4-kb fragment in common. The size of
the insert DNA in one positive clone, pMB1, was reduced from 5.4 kb by
restriction endonuclease digestion to 2.2 kb (pMB4, positive for lipase
expression) and 2.0 kb (pMB5, negative for lipase expression). It is
interesting to note that neither MC1061/pMB1 nor MC1061/pMB4 was found
to be positive for protease or hemolytic activity when plated onto agar
containing skim milk or erythrocytes.
Recombinant pMB4 DNA was isolated by using the Wizard Plus SV Minipreps
DNA purification kit (Promega), and the nucleotide
sequence of the
2.2-kb insert was determined (GenBank accession
no.
AY032849). An open
reading frame of 1,851 bp with the potential
to encode a 616-amino-acid
protein with a predicted molecular
mass of 65.8 kDa was identified
together with a potential Shine-Dalgarno
site preceded by putative
promoter sequences. The sequence following
the open reading frame has
the potential to encode a transcriptional
terminator with a
G value of
15 kJ mol
1. Based on
subsequent protein and biochemical analyses described
below, this gene
was designated
plb.
Identification of the plb gene product.
Secreted protein samples were prepared from M. bovis Dalton
2d and E. coli strains MC1061/pMB1, MC1061/pMB4, and
MC1061/pMB5 by ammonium sulfate precipitation of cell supernatant
fluids from overnight cultures, separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (12.5%
polyacrylamide) (19), and visualized by staining
with Coomassie blue. A dominant band running at approximately 66 kDa
was identified in the extracts from MC1061/pMB1 and MC1061/pMB4, but
not in those from MC1061/pMB5 (Fig. 1A,
lanes a, b, and c, respectively). Due to the large number of proteins
present in the M. bovis culture supernatant (contributed by
the membrane blebs that slough off M. bovis during normal
growth), Western blot analysis with antiserum raised in rabbits
immunized with the recombinant lipase was required to confirm the
presence of the lipase in the concentrated extract of M. bovis Dalton 2d-secreted proteins (Fig. 1B, lane d).
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FIG. 1.
Identification of native (M. bovis) and
recombinant (E. coli) lipase. (A) SDS-PAGE gel stained
with Coomassie brilliant blue. (B) Western blot of the gel in panel A
probed with antisera raised in rabbits against heat-inactivated
recombinant lipase. Lanes: a, MC1061/pMB1; b, MC1061/pMB4; c,
MC1061/pMB5; d, M. bovis Dalton 2d. Molecular mass
markers are indicated in kilodaltons to the left.
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|
The dominant 66-kDa band identified in the MC1061/pMB1 preparation
(Fig.
1A, lane a) was electrotransferred to polyvinylidene
difluoride
membrane, excised, and subjected to automated (Edman
degradation)
amino-terminal sequence analysis (
7). The resultant
data
confirmed the identity of the 66-kDa protein as the product
of
plb and furthermore identified the presence of a signal
peptide
cleavage site between residues 25 and 26 of the predicted
translation
product, designated PLB. This putative signal peptide has
similar
characteristics to classical signal peptides described by
Pugsley
(
27), suggesting that PLB may be secreted across
the cytoplasmic
membrane via an
s-dependent
pathway.
Amino acid sequence comparisons.
Comparison of the PLB
sequence with the GenBank sequence databank by using the BLAST program
(1) revealed a number of similar proteins. PLB had its
closest identity (28% identity and 42% similarity) to a lipase or
esterase from the plant pathogen Xylella fastidiosa (accession no. D82761). Three other proteins, including a putative autotransporter protein (PapA; accession no. CAC14200) and an esterase
(EstA; accession no. G83006) from Pseudomonas aeruginosa and
an outer membrane esterase from Salmonella
enterica serovar Typhimurium (AAC38796),
displayed 23, 23, and 31% identity and 35, 35, and 43%
similarity, respectively, to PLB. M. bovis PLB was also
found to be related to a group of prokaryotic and eukaryotic proteins
by virtue of the presence of a highly conserved amino acid sequence
motif, Gly-Asp-Ser-Leu (GDSL) (29), located in the
N-terminal regions of each of these proteins (Fig. 2). While variable in size and appearing
to have different functions (29, 32), the proteins aligned
share five discrete blocks of relatively high sequence similarity that
occur in the same relative location in each protein. The serine residue
within the GDSL motif has been shown to be part of the catalytic triad
of the Aeromonas hydrophila glycerophospholipid-cholesterol
acyltransferase (14) and may therefore be essential for
enzymatic activity, and possibly the virulence, of members of this
family. We propose that the PLB from M. bovis is a member of
the GDSL family and as such may play a role in the pathogenesis of the
IBK infection. The precise nature of this role will be investigated by
constructing a plb mutant and testing the pathogenicity of
the mutant in the bovine IBK model system (20).
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FIG. 2.
Conserved blocks of amino acids in the GDSL family
(members b to m) of lipolytic enzymes. New family member M.
bovis PLB (member a) has been compared with proteins of those
organisms shown to express a protein belonging to the GDSL family. The
accession number for each protein sequence is indicated, as is the
function of each enzyme. Asterisks indicate residues that are conserved
in six or more of the proteins. Numbers in parentheses indicate the
number of amino acid residues between the conserved regions. ORF, open
reading frame. The figure was adapted from data provided in the study
of Upton and Buckley (29).
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|
It has been suggested that one member of the GDSL family, the EstA
esterase of
P. aeruginosa, may also be a member of the
autotransporter protein family (
32). Autotransporters are
secreted
across the cytoplasmic membrane via the
s-system
and then secreted
across the outer membrane by a unique process that
does not require
any energy or additional accessory factors (
12,
22). The C-terminal
region of PLB was found to display homology
to members of the
autotransporter family. Taken together with the
observation that
PLB probably has a classical signal peptide, we
propose that PLB
is a bona fide member of the autotransporter
family.
Characterization of PLB enzymatic activity.
To determine the
enzymatic specificity of PLB, thin-layer chromatography was used to
analyze the end products of phosphatidylcholine and
lysophosphatidylcholine hydrolysis (8). Ammonium
sulfate-precipitated cell supernatant fluids from E. coli
MC1061/pMB4 and M. bovis Dalton 2d hydrolyzed both
substrates to produce free fatty acids and glycerophosphorylcholine in
a manner similar to that observed for the commercial phospholipase B
(Fig. 3). Based on these data, we
concluded that the activity displayed by PLB was characteristic of a
phospholipase B.
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FIG. 3.
Thin-layer chromatographic analysis of the lipid
products generated by the action of the M. bovis lipase
or commercial phospholipases on two different substrates:
phosphatidylcholine (PC) (A) and lysophosphatidylcholine (LPC) (B).
Lanes: a, reference standards; b, phospholipase A2; c,
phospholipase B; d, phospholipase C; e, phospholipase D; f,
MC1061/pMB4; g, M. bovis Dalton 2d. Included as controls
in panel A are unprocessed phosphatidylcholine and
lysophosphatidylcholine. The other reference standards used were
monoacyl glycerol (MG), diacyl glycerol (DG), and free fatty acid
(FA).
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|
Expression of PLB by M. bovis.
To investigate
the frequency of expression of PLB among isolates of M. bovis, a Western blot using antisera raised in rabbits immunized
with the recombinant lipase was performed with protein extracts from
nine representative strains. Protein samples were made from overnight
cultures, separated by SDS-PAGE (12.5% polyacrylamide), transferred to
nitrocellulose, and incubated with recombinant lipase antisera. A
protein band equivalent in size (~66 kDa) to the lipase produced by
E. coli MC1061/pMB4 (Fig. 4,
lane j) was detected in each of the M. bovis strains (Fig.
4, lanes a to i), thus establishing the global nature of this enzyme
among the species.
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FIG. 4.
Distribution of PLB among M. bovis
strains. Secreted proteins present in cell supernatants prepared from
overnight cultures of M. bovis strains were precipitated
with ammonium sulfate at a final concentration of 60%. Proteins were
visualized by Western blot analysis with recombinant PLB antisera.
Lanes: a, S276R; b, 3WO7; c, Dalton 2d; d, R593L; e, Tat849; f, 218R;
g, Fleur462; h, Q220; i, Epp63; j, MC1061/pMB4; k, MC1061/pMB5.
Molecular mass markers are indicated in kilodaltons to the left.
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Previous studies have shown that the type IV pili produced by
M. bovis play a fundamental role in bacterial colonization of
the
bovine eye and mediate the essential first step in the pathogenic
process that leads to disease (
20). Following adhesion,
secondary
virulence factors are presumed to cause the clinical damage
observed
as pitting on the corneal surface (
18). Enzymatic
activity of
the
M. bovis phospholipase B on membrane
phospholipids could result
in cell lysis and lead to such clinical
symptoms. This damage
could be directly caused by the presence of
lysophosphatidylcholine,
one of the intermediate products of PLB
activity on phospholipids
(
6). Phospholipases are
recognized as major virulence determinants
in a number of bacterial
species, including
Listeria (
11) and
Corynebacterium pseudotuberculosis (
24).
Until now, development of a pilus-based vaccine against IBK has been
hampered by the need to construct multivalent vaccines,
since the pili
of specific serotypes are not cross protective
against disease caused
by heterologous challenge (
21). The failure
of such
vaccines supports the need for the identification of immunogens
that
are antigenically conserved across
M. bovis. The
identification
of PLB as one such conserved immunogen will likely prove
important
in the future development of an efficacious vaccine against
IBK.
| |
ACKNOWLEDGMENTS |
J.L.F. was a recipient of an Australian Postgraduate Award. R.A.S.
and J.M.T. are members of the Cooperative Research Centre for Vaccine Technology.
| |
FOOTNOTES |
*
Corresponding author. Present address: Department of
Veterinary Pathology, The University of Glasgow, Bearsden Rd., Glasgow, Scotland, United Kingdom G61 1QH. Phone: 0141 339 8855, ext. 0683. Fax:
0141 330 5602. E-mail: J.Farn{at}vet.gla.ac.uk.
Present address: CSL Animal Health, Parkville, Victoria, Australia 3052.
| |
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Journal of Bacteriology, November 2001, p. 6717-6720, Vol. 183, No. 22
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.22.6717-6720.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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