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Journal of Applied Microbiology 2004, 96, 700–708
doi:10.1111/j.1365-2672.2004.02177.x
Adhesive properties of a LamB-like outer-membrane protein
and its contribution to Aeromonas veronii adhesion
R.C. Vàzquez-Juárez, M.J. Romero and F. Ascencio
Departamento de Patologı´a Marina, Centro de Investigaciones Biológicas del Noroeste (CIBNOR), La Paz, México
2003/0742: received 24 August 2003, revised 3 November 2003 and accepted 5 November 2003
ABSTRACT
R . C . V À Z Q U E Z - J U Á R E Z , M . J . R O M E R O A N D F . A S C E N C I O . 2004.
Aims: To identify and characterize nonfimbrial proteins from Aeromonas veronii involved in the attachment to
epithelial cells in vitro.
Methods and Results: Two Aer. veronii mucin- and lactoferrin-binding proteins with molecular masses of 37 and
48 kDa were identified by Western blot analysis. According to its N-terminal amino acid sequence, the 48-kDa
protein was identified as Omp48, an outer-membrane protein similar to LamB of Escherichia coli. LamB is a wellknown porin involved in maltose transport across the outer membrane in E. coli. In a microtitre plate assay, Omp48
bound to the immobilized extracellular matrix proteins collagen and fibronectin, and the mucin- and lactoferrinbinding activity was confirmed. Adhesion of Omp48 to mucin, lactoferrin and collagen was diminished by
preincubation with homologous glycoproteins or other carbohydrates, suggesting a putative Omp48 lectin-like
binding domain. Anti-Omp48 antiserum significantly inhibited the Aer. veronii adhesion to confluent HeLa cell
monolayers and pretreatment of cells with purified Omp48 elicited competitive inhibition of adhesion. Similarly,
cross-inhibition of Aer. hydrophila and Aer. caviae adhesion was achieved with the same treatments, indicating the
existence of a conserved surface protein among these species.
Conclusions: Taken together, these data indicate that Omp48 is involved in Aer. veronii adhesion to epithelial cells
and might be an alternative adhesion factor of this micro-organism.
Significance and Impact of the Study: The adhesive potential of Aeromonas spp. is correlated with
pathogenicity; however, the adhesion mechanism is complex and not well understood. This study provides evidence
of a putative adhesion factor that might be contributing to pathogenicity of Aer. veronii and could be used for
vaccine development.
Keywords: adhesion, Aeromonas veronii, collagen, lactoferrin, LamB, mucin, outer-membrane proteins.
INTRODUCTION
Aeromonas species are known to be of great importance
both economically and medically. They form a complex
group of ubiquitous Gram-negative bacteria that are widely
isolated from clinical, environmental and food samples and
are considered opportunistic human pathogens and important pathogens of fish and a variety of animals (Janda and
Correspondence to: F. Ascencio, Mar Bermejo 195. Col. Playa Palo Santa Rita.
La Paz, B.C.S. 23090, Me´xico (e-mail: [email protected]).
Abbott 1998; Austin and Austin 1999). Aeromonas veronii
has been reported as the aetiological agent of gastrointestinal infections (Hickman-Brenner et al. 1987; Stelma et al.
1988; Neves et al. 1990), and a wide spectrum of extraintestinal infections including wound infection, septicaemia, urinary tract and soft tissue infection and septic
arthritis in humans (Joseph et al. 1991; Abbott et al. 1994;
Hsueh et al. 1998; Steinfeld et al. 1998). Aeromonas veronii
is also the causative agent of the bacterial haemorrhagic
septicaemia (motile aeromonads septicaemia) of cultured
warm-water fish, and like Aer. salmonicida and Aer.
ª 2004 The Society for Applied Microbiology
LAMB-LIKE ADHESIN OF AER. VERONII
hydrophila, it is increasingly considered a major economic
problem for the aquaculture industry (Austin and Austin
1999).
A pivotal step in pathogenesis of virulent strains is the
adhesion to and colonization of host surfaces. The mechanisms underlying the adhesion of Aeromonas spp. to
epithelial cells are not well understood and seem to be a
complex process, apparently involving the occurrence of
sequential or simultaneous factors. Aeromonas spp. adhesion
factors that have been described include lipopolysaccharide
(LPS) O-antigen (Merino et al. 1996), polar and lateral
flagellum (Rabaan et al. 2001; Gavin et al. 2002) and a longwavy pili (type-IV pili) (Kirov et al. 1999). However,
phenotypic variation among adhesive clinical isolates (i.e.
strains lacking the LPS O-antigen, poorly or nonfimbriated
isolates) suggests the presence of alternative adhesion factors
(Sakazaki and Shimada 1984; Nishikawa et al. 1991, 1994;
Kirov et al. 1995). Adhesiveness to extracellular-matrix
proteins and mucus constituents promotes bacterial colonization (Westerlund and Korhonen 1993; Carnoy et al.
1994). We previously found that Aeromonas strains isolated
from diverse sources bind mucus glycoproteins (mucin and
lactoferrin) and extracelullar-matrix proteins (fibronectin,
laminin and collagen) (Ascencio et al. 1990, 1991, 1992).
Several proteins from Aeromonas spp. responsible for mucin
binding were identified (Ascencio et al. 1998).
In this study, we report on the interaction of the Aer.
veronii LamB-like protein Omp48 with extracellular-matrix
components and mucus glycoproteins, and its role in
adhesion to HeLa cells.
MATERIALS AND METHODS
Bacterial strains and growth conditions
The Aeromonas strains used in the study were obtained from
the Microbial Culture Collection of the Hospital of the
University of Lund, Sweden, and were kindly provided by
Prof. T. Wadström. Aeromonas veronii biotype veronii strain
A186 was isolated from a patient with a diarrhoea disease
and was identified by its fatty acids profile (Sherlock
System) at Auburn University, AL, USA (Bacterial Strain
Identification and Mutant Analysis Service). Aeromonas
hydrophila strain A205 and Aer. caviae strain 4019 were
isolated from diseased fish and human infection, respectively. Bacterial strains were grown in Luria-Bertani (LB)
liquid medium at 37C with vigorous shaking.
Protein fractionation
The proteins were precipitated from culture supernatant of
exponentially growing Aer. veronii with ammonium sulphate
at 80% (w/v) saturation, centrifuged at 18 000 g for 30 min
701
at 4C, and dissolved in distilled water. Proteins were
exhaustively dialysed against 0Æ01 mol l)1 ammonium bicarbonate and the protein concentration was determined using
Bradford solution (Bio-Rad, Hercules, CA, USA).
SDS-PAGE and blotting
Proteins from the 80% ammonium sulphate precipitation
fraction (F80) were heat-denatured and separated electrophoretically on 12% SDS-PAGE at 20 mA for ca 2 h in the
Mini-Protean II system (Bio-Rad), according to the method
of Laemmli (1970). Proteins were electro-transferred from
the gel to a PVDF membrane (Millipore, Billerica, MA,
USA) using a semi-dry Trans-Blot Cell system (Bio-Rad)
with transfer buffer solution [25 mmol l)1 Tris-20% (v/v)
methanol] for 2 h at 100 mA. The membranes were blocked
with phosphate-buffered saline (PBS, 137 mmol 1)1 NaCl,
2Æ7 mmol 1)1 KCl, 10 mmol 1)1 Na2HPO4, 2 mmol 1)1
KH2PO4, at pH 7Æ4) containing 3% (w/v) bovine serum
albumin (BSA) for 30 min at 22C and washed three times
with PBS containing 0Æ05% (v/v) Tween-20. Peroxidaselabelled porcine mucin (POD-mucin) or peroxidase-labelled
bovine lactoferrin (POD-lactoferrin) (30 lg ml)1 in PBS)
was added to the membrane and incubated at 22C for
90 min, and washed four times with PBS containing 0Æ05%
(v/v) Tween-20. The colour reaction was developed with
diaminobenzidine solution (0Æ25 mg ml)1 in 50 mmol l)1
sodium acetate buffer, at pH 5Æ0) containing 0Æ25 ll hydrogenperoxide per millilitre. The reaction was stopped with
0Æ1 mol l)1 sodium metabisulphite.
Isolation and N-terminal amino acid sequencing of
proteins with affinity for mucus constituents
Proteins from F80 were boiled for 5 min, electrophoresed by
preparative 12% SDS-PAGE and the bands corresponding
to the mucus constituents-binding proteins were purified by
electro-elution, using a Model 422 Electro-Eluter, following
the manufacturer’s instructions (Bio-Rad). The purified
proteins were subjected to N-terminal sequencing by
automated Edman degradation at the Instituto de Biotecnologı́a, UNAM, México.
Isolation of outer-membrane proteins (OMPs) and
Omp48 purification
The Aer. veronii OMPs were isolated as described previously
(Vazquez-Juarez et al. 2003). Bacteria were harvested in the
mid- to late-exponential growth phase by centrifugation at
8000 g for 20 min at 4C, and washed twice with PBS. The
cell pellet was suspended in 5 ml of sonication buffer
(20 mmol l)1 Tris, pH 7Æ4, containing 100 lg DNase ml)1
and 100 lg RNase ml)1), and the cell suspension was
ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 96, 700–708, doi:10.1111/j.1365-2672.2004.02177.x
702 R . C . V À Z Q U E Z - J U Á R E Z ET AL.
sonicated eight times for 30 s, each time. Unbroken cells
were removed by centrifugation at 3000 g for 20 min at 4C
and the supernatant was centrifuged at 20 000 g for 90 min
at 4 C to pellet the cell envelopes. The supernatant was
discarded and the pellet was dispersed in 5 ml of
20 mmol l)1 Tris (pH 7Æ4) containing 0Æ75% (w/v) SDS
at 37C for 20 min. The suspension was centrifuged at
20 000g for 90 min at 4C to pelletize the insoluble outer
membranes. Finally, the outer-membrane fraction was
suspended in deionized water and protein concentration
was determined using Bradford solution (Bio-Rad). OMPs
were electrophoresed as described above and stained with
Coomassie Blue R-250 (Laemmli 1970). For Omp48 purification, OMPs were fractionated in a preparative 15%
native-PAGE, stained with Coomassie Blue R-250, and
major bands were carefully excised out from the gel. The
proteins were separately electro-eluted from the excised gel
slices as described above. The eluate was lyophilized in a
Savant Speed-Vac concentrator (GMI, Albertville, MN,
USA) and Omp48 (48 kDa protein) was identified by SDSPAGE.
Preparation of polyclonal antiserum and Western
blot
Polyclonal anti-Omp48 antiserum was raised in rabbits by
immunization with Omp48 from Aer. veronii strain A186
according to Harlow and Lane (1988). Briefly, electro-eluted
Omp48 was electrophoresed by preparative 12% SDSPAGE and the 48-kDa band, stained with Coomassie Blue,
was excised out from gel. Gel slices were minced, homogenized with sterile PBS buffer, and injected subcutaneously
into the back of a New Zealand white rabbit. Two booster
doses were given at intervals of 2 weeks. Reactivity and
specificity of the anti-Omp48 antiserum were assessed by
Western blot as follows: OMPs were fractionated by SDSPAGE, and electro-transferred to a PVDF membrane as
described above. The membrane was blocked with PBS-3%
(w/v) BSA for 30 min at 22C, washed three times with
PBS-0Æ05% (v/v) Tween-20, and incubated for 90 min
at 22C with anti-Omp48 antiserum diluted in PBS-0Æ5%
(v/v) Tween 20 (1 : 1000). After three washes, the membrane
was incubated for 90 min at 22C with goat anti-rabbit IgG
peroxidase-conjugated diluted in PBS-0Æ5% (v/v) Tween 20
(1 : 2000), washed again, and the colour reaction was
developed as described above.
Binding of Omp48 to extracellular matrix (ECM)
and mucus glycoproteins
Analysis of Omp48 binding to immobilized mucosal and
ECM glycoproteins was performed by ELISA, essentially as
described by Sperandio et al. (1995). Briefly, ELISA plates
were coated overnight at 4C with 100 ll (10 lg ml)1 in
carbonate buffer, at pH 9Æ5) of the following glycoproteins:
collagen types I, III, IV, VI, plasma fibronectin, mucin type
II (porcine, crude), mucin type III (porcine, partially
purified), bovine lactoferrin, and as negative control, BSA
(Sigma Chemical). Plates were blocked with PBS-0Æ5%
(v/v) Tween 20 containing 1% (w/v) BSA for 2 h at 22C.
OMP fraction (100 ll; 10 lg ml)1 in PBS-0Æ5% Tween 20)
was added to each well, incubated for 2 h at 22C, and
washed three times with PBS-0Æ5% (v/v) Tween 20. Wells
were incubated for 2 h at 22C with anti-Omp48 serum
diluted in PBS-0Æ5% (v/v) Tween 20 (1 : 100) and after
washing, were incubated with goat anti-rabbit IgG peroxidase-conjugated (1 : 1000). Colour reaction was developed
with 100 ll of peroxidase substrate for 30 min at 22C in the
dark. Reaction was stopped by adding 100 ll H2SO4
1 mol l)1, and the A490 was determined. Assays were
performed in triplicate.
Inhibition of Omp48 binding to immobilized mucin,
lactoferrin, and collagen
For binding inhibition assays, 100 ll of OMP fraction
(10 lg ml)1 in PBS-0Æ5% Tween 20) was incubated at 22C
for 1 h with 1 mg ml)1 of various carbohydrates and
glycoconjugates. Then, binding assays to mucin type-III,
bovine lactoferrin and collagen type-I were performed as
mentioned above.
Bacterial adhesion to epithelial cells
The adhesion assays were conducted as follows: HeLa cells
were grown to confluence at 37C (95% humidity and 5%
CO2) in 24-well culture plates in RPMI-1640 culture media
supplemented with 10% (v/v) foetal calf serum, 2 mmol l)1
)1
L-glutamine and gentamycin (40 lg ml ). Before the
adhesion assay, cell monolayers were washed with sterile
PBS (pH 7Æ4). Aeromonas strains were grown in LB liquid
medium with vigorous shaking and submitted to mechanical
shearing to remove filamentous structures, such as flagella,
that might contribute to bacterial adhesion (Thornley et al.
1996; Rabaan et al. 2001; Gavin et al. 2002). An inoculum of
107 bacteria per well was added to the cell monolayers in
1 ml RPMI-1640 culture media and incubated for 1Æ5 h at
37C. After incubation, the medium and nonadherent
bacteria were removed by washing three times with sterile
PBS. Then, 200 ll 0Æ1% (v/v) Triton X-100 in PBS buffer
were added to each well and the plates were rocked at 37C
for 30 min. Adhering bacteria were recovered by adding
800 ll RPMI-1640 culture media and vigorously pipetting
the suspension up and down. Bacterial suspensions were
diluted serially in sterile PBS, plated on LB agar, and after
24 h incubation, the CFU were determined. Adhesion is
ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 96, 700–708, doi:10.1111/j.1365-2672.2004.02177.x
LAMB-LIKE ADHESIN OF AER. VERONII
expressed as the amount of bacterial inoculum recovered
from the HeLa cells after incubation for 2 h. For inhibition
of bacterial adhesion assay, 107 bacteria were mixed with
anti-Omp48 serum (1 : 10 or 1 : 100) diluted in RPMI-1640
and pre-incubated for 30 min at 37C. Bacteria preincubated in the same conditions with rabbit serum (1 : 10) were
used as negative control. For competitive inhibition assay,
50 lg per well of purified Omp48 were added into the
respective wells and incubated for 30 min at 37C. For
controls of respective strains, no protein was added. After
incubation, quantitative adhesion assays were performed as
described above. Experiments were carried out in triplicate
wells and at least two separate assays were performed.
(a)
M
1
2
(b) 3
703
4
97.4
66.2
45.0
31.0
Statistical analysis
Data from Omp48 binding to mucus and ECM glycoproteins and from bacterial adhesion assays were expressed as
means ± S.D. The difference among mean values was
analysed by Student’s t-test. A value of P < 0Æ05 was
considered significant.
Fig. 1 (a) SDS-PAGE (12%) and (b) Western blot of precipitated
proteins from Aeromonas veronii culture supernatant. Lane M,
molecular mass markers expressed in kDa; lane 1, F80 proteins; lane 2,
purified 48 kDa protein (Coomassie stain); lanes 3 and 4, F80 proteins
blotted and revealed with peroxidase-labelled porcine mucin or
peroxidase-labelled bovine lactoferrin, respectively
RESULTS
and Yersinia enterocolitica. Furthermore, analysis revealed
100% homology to the N-terminal sequence of the mature
form of the OMP from Aer. veronii Omp48, whose gene was
recently cloned and characterized (Vazquez-Juarez et al.
2003). Proteins of the LamB porin family, including
Omp48, are involved in the permeation of maltose and
maltodextrins across the bacterial outer membrane (Jeanteur
et al. 1992; Boos and Shuman 1998).
Identification of proteins with affinity for mucus
constituents
SDS-PAGE of the precipitated protein fraction (F80)
stained with Coomassie blue revealed the presence of several
proteins with a wide range of molecular masses. Protein
bands with apparent molecular mass of 33, 37, 48 and
55 kDa were present (Fig. 1a). A preliminary survey
investigated whether Aer. veronii produces extracellular
proteins with affinity for mucus constituents, where the F80
was subjected to Western blot analysis, using POD-mucin or
POD-lactoferrin as a probe. As shown in Fig. 1b, two main
reactive proteins with apparent molecular mass of 37 and
48 kDa were detected, indicating the affinity of F80 proteins
for mucin and lactoferrin. Purification of the 48-kDa protein
by electro-elution rendered a single discrete band as
observed by SDS-PAGE (Fig. 1a). For reasons that are
not clear, this purification method yielded very small
amounts of the 37-kDa protein (data not shown).
N-terminal sequence analysis
The N-terminal amino acid sequence of the purified 48-kDa
protein was determined and the peptide corresponds to the
sequence VDFHGYMRSG. BLAST amino acid sequence
analysis revealed significant homology to LamB-like proteins
of various Gram-negative bacteria, including Aer. salmonicida, Vibrio cholerae, V. parahaemolyticus, Salmonella enterica
serovar. Typhimurium, Escherichia coli, Klebsiella pneumoniae
Purification of Omp48 and binding to ECM and
mucus glycoproteins
The OMP profile of Aer. veronii is shown in Fig. 2a. In
SDS-PAGE Coomassie blue stained, two major OMPs with
an estimated molecular mass of 38 and 48 kDa (Omp48)
were evident in the boiled OMPs. Omp48 was purified as
described in Materials and Methods section and is presented
in Fig. 2a. The reactivity and specificity of the polyclonal
antibodies raised against Omp48 was tested by SDS-PAGE
and Western blot analysis of the OMP fraction. Anti-Omp48
antiserum specifically recognized the 48-kDa protein
(Fig. 2b).
In a solid-phase binding assay, Omp48 showed high
binding to plasma fibronectin and collagens, including type I
and IV, which are the most abundant of the ECM
components. Additionally, the ability of Omp48 to act as a
potential ligand for mucin and lactoferrin was confirmed,
indicating a broad range to its binding ability. Omp48 bound
to crude and partially purified mucin and lactoferrin to a
similar extent (Fig. 3).
ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 96, 700–708, doi:10.1111/j.1365-2672.2004.02177.x
704 R . C . V À Z Q U E Z - J U Á R E Z ET AL.
M
(a)
97.4
66.2
45.0
31.0
1
2
(b)
M
3
97.4
66.2
45.0
carbohydrate moieties of mucin, lactoferrin and collagen
seem to be involved in Omp48 binding, as virtually all
carbohydrates tested were able to inhibit the binding
(Table 1). These results suggest that binding to immobilized mucin, lactoferrin and collagen involves different
regions of Omp48 and is likely mediated by a lectin-like
domain.
31.0
Role of Omp48 in Aer. veronii adhesion
to HeLa cells
21.5
14.4
21.5
14.4
Fig. 2 (a) SDS-PAGE and (b) Western blot analysis of outermembrane proteins (OMPs) extracted from Aeromonas veronii. Lanes
M, molecular mass markers expressed in kDa; lane 1, OMP fraction;
lane 2, purified Omp48 (Coomassie stain); lane 3, OMPs blotted and
probed with anti-Omp48 antiserum
2
Absorbance at 490 nm
1.8
1.6
1.4
The ability of Aer. veronii to adhere to HeLa cell
monolayers was evaluated. Aeromonas veronii showed a
high level of adhesion to HeLa cells after incubation for
1Æ5 h (Fig. 4). To investigate whether Omp48 is involved
in Aer. veronii adhesion, adhesion-inhibition assays were
performed with polyclonal antibodies raised against
Omp48. As shown in Fig. 4, preincubation of the bacteria
with the anti-Omp48 antiserum (diluted 1 : 10 or 1 : 100)
significantly inhibited Aer. veronii adhesion by more than
40%, from 4Æ2 · 107 to 2Æ4 · 107 CFU ml)1, (P < 0Æ05);
whereas rabbit serum did not show any inhibition
(P > 0Æ05). In addition, preincubation of HeLa cells with
purified Omp48 elicited a significant competitive inhibitory
effect (P < 0Æ01) on Aer. veronii adhesion. Almost 60%
inhibition, from 4Æ2 · 107 to 1Æ78 · 107 CFU ml)1, was
reached with when 50 lg of Omp48 was added to each
1.2
Table 1 Effect of carbohydrates and glycoconjugates on Omp48
binding to immobilized mucin, lactoferrin and collagen
1
0.8
Percentage of binding inhibition*
0.6
0.4
Inhibitor
0.2
0
BSA CnI CnIII CnIV CnVI Fn MuII MuIII Lf
Glycoprotein
Fig. 3 Binding of Omp48 to immobilized extracellular matrix and
mucus glycoproteins. Bovine serum albumin (BSA), wells coated with
BSA as negative control; CnI, CnIII, CnIV, and CnVI, collagen type I,
III, IV and VI, respectively; Fn, plasma fibronectin; MuII, mucin type
II (crude); MuIII, mucin type III (partially purified) and Lf,
lactoferrin. The error bars indicate S.D. (*P < 0Æ05)
Effect of carbohydrates and glycoconjugates on
Omp48 binding
The effect of carbohydrates and glycoconjugates on Omp48
binding to immobilized mucin, lactoferrin and collagen was
evaluated. Binding ability was specific, as it could be
significantly inhibited by homologous glycoproteins and to
a lesser extent by heterologous glycoproteins. Additionally,
Control (without inhibitor)
Carbohydrates
Galactose
Glucose
Fucose
Mannose
N-acetyl-neuraminic acid
Ramnose
Dextran
Dextran sulphate
Glycoconjugates
Collagen
Fetuin
Fibronectin
Heparin
Lactoferrin
Mucin
Orosomucoid
Mucin
Lactoferrin
Collagen
0
0
0
46
0
15
27
25
11
23
12
46
12
22
20
21
16
15
52
35
0
16
43
0
15
49
64
0
14
0
80
28
103
4
42
0
0
0
90
11
13
30
0
0
12
31
9
22
*Inhibition data represent the mean value of triplicate measurements.
The S.D. for each value is <10%.
ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 96, 700–708, doi:10.1111/j.1365-2672.2004.02177.x
LAMB-LIKE ADHESIN OF AER. VERONII
705
DISCUSSION
Fig. 4 Adhesion of Aeromonas veronii A186 to HeLa cells. Before
adhesion assays, bacteria and monolayers were pretreated as follows: (i)
untreated bacteria; (ii and iii) bacteria incubated with anti-Omp48
antiserum diluted 1 : 10 and 1 : 100, respectively; (iv) monolayers
treated with Omp48, 50 lg per well; (v) bacteria incubated with rabbit
serum diluted 1 : 10. The arrow indicates the amount of initial
inoculum. The error bars indicate S.D. (*P < 0Æ05)
Table 2 Adhesion of Aeromonas spp. to HeLa cells in the presence or
absence of anti-Omp48 antiserum or Omp48 protein
Bacteria recovered from HeLa cells
(·107 CFU ml)1) and percentage
of bacterial adhesion* after incubation with
Treatment
Aer. hydrophila
Aer. caviae
No antiserum
Anti-Omp48 (1 : 10)
Anti-Omp48 (1 : 100)
Omp48 (50 lg)
Rabbit serum (1 : 10)
4Æ49
2Æ43
2Æ57
2Æ22
4Æ3
4Æ36
2Æ47
2Æ66
2Æ27
4Æ03
±
±
±
±
±
0Æ33 (100%)
0Æ06 (54Æ1%)
0Æ17 (57Æ3%)
0Æ15 (49Æ5%)
0Æ1 (95Æ8%)
±
±
±
±
±
0Æ19
0Æ14
0Æ07
0Æ07
0Æ35
(100%)
(56Æ6%)
(61Æ0%)
(52Æ0%)
(92Æ4%)
*The amount of bacteria recovered from HeLa cells in the absence of
antiserum and Omp48 was taken to be 100% adhesion.
P < 0Æ05.
P > 0Æ05.
well (Fig. 4). Taken together, these data demonstrate that
Omp48 is involved in Aer. veronii adhesion to epithelial
cells and might be an alternative adhesion factor of this
micro-organism.
The ability of Omp48 to cross-inhibit the adhesion of
other Aeromonas spp. to epithelial cells was evaluated. As
shown in Table 2, Omp48 (50 lg per well) was able to
cross-inhibit adhesion of Aer. hydrophila A205 and Aer.
caviae 4019 to HeLa cells by around 50%. Similarly, antiOmp48 antiserum (diluted 1 : 10 and 1 : 100) significantly
diminished adhesion of both species (Table 2).
To initiate infection, bacterial pathogens must first be able
to attach to and eventually colonize an appropriate host
tissue or its associated components. The ability to bind
mucus glycoproteins (such as mucin and lactoferrin) and
ECM components (fibronectin, laminin and collagen) is a
common property of mesophilic aeromonads, which might
contribute to enhancing adhesiveness of pathogenic strains
(Ascencio et al. 1990, 1991, 1992, 1998).
An Aer. veronii OMP with affinity for mucin and
lactoferrin, namely Omp48, was identified. The omp48 gene
was recently cloned, and deduced amino acid sequence
analysis clearly indicated that this is a protein similar to
E. coli LamB porin, and together with the maltoporins from
Aer. salmonicida, V. cholerae and V. parahaemolyticus, they
form an independent group (Lang and Ferenci 1995; Boos
and Shuman 1998; Vazquez-Juarez et al. 2003). Although
Omp48 is proposed as a maltose-inducible protein, its
expression was substantial when Aer. veronii was grown in
LB medium in the absence of maltose. This unexpected
result is similar to that observed with the homologous
protein porin I from Aer. hydrophila where it was constitutively expressed in LB broth, regardless of the presence or
absence of maltose in the medium (Jeanteur et al. 1992). It is
likely that LB, as a complete rich medium, contains maltoselike molecules that are inducing Omp48 expression.
The ECM is a mixture of secreted proteins composed
primarily of collagens, fibronectin, laminin and proteoglycans located on epithelial and endothelial cell surfaces
(Aumailley and Gayraud 1998). A number of pathogens
adhere to ECM components, which contribute to virulence
by promoting bacterial colonization (Westerlund and Korhonen 1993). As an opportunistic pathogen, Aer. veronii
may take advantage of the ECM-binding property of
Omp48, as these molecules could become exposed after a
primary infection or after a tissue trauma following a
mechanical or chemical injury. The two major forms of
fibronectin are found in blood (plasma fibronectin) and on
the cell surface (cellular fibronectin) (Aumailley and Gayraud 1998). Doig et al. (1992) hypothesized that binding of
bacterial surface to soluble fibronectin, may serve to
camouflage bacteria from immunological recognition mechanisms by masking immunogenic epitopes. Mucin is the
main component of gastrointestinal mucous and the ability
of Aeromonas strains to bind to and use mucin from diverse
sources as the sole source of carbon and nitrogen has been
demonstrated (Ascencio et al. 1998). Thus, Omp48 mucin
binding might be useful to the bacterium to remain in
intestinal mucus, use mucin as a nutrient, replicate, and
eventually reach enterocytes, and colonize. Lactoferrin is an
iron-binding glycoprotein secreted by glandular epithelia
and is presented in mucosal secretions limiting the availab-
ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 96, 700–708, doi:10.1111/j.1365-2672.2004.02177.x
706 R . C . V À Z Q U E Z - J U Á R E Z ET AL.
ility of free iron in the extracellular body fluids. It is
essential for microbial growth. Bacteriostatic and bactericidal effects of lactoferrin against Gram-negative microorganisms have been demonstrated (Ellison et al. 1988). In
contrast to antibacterial activity, lactoferrin supports growth
of pathogenic species: Aer. salmonicida(Hirst and Ellis 1996),
Aer. hydrophila(Stintzi and Raymond 2000), Pseudomonas
fragi, V. parahemolyticus and Helicobacter pylori when
cultured under iron-limited conditions (ChampomierVerges et al. 1996; Wong et al. 1996; Dhaenens et al.
1997). In addition to the siderophore iron-uptake mechanism of Aer. salmonicida, a second mechanism that requires
direct cell contact with lactoferrin has been reported (Chart
and Trust 1983). Whether the lactoferrin-binding ability of
Omp48 represents another mechanism for iron acquisition
by Aer. veronii requires further studies.
As binding to mucin, lactoferrin and collagen was inhibited
by sugars, Omp48 on the bacterial surface probably interacts
with the carbohydrate domains of the glycoproteins in a lectinlike fashion. Interestingly, Omp48 contains the sequence
YYQRHD, which is one of four conserved sequences that
form the maltodextrin-binding sites in amylases (Vihinen and
Mantsala 1989; Schneider et al. 1992; Vazquez-Juarez et al.
2003). This motif might be involved in the Omp48 lectin-like
interaction with carbohydrates, although nonspecific charge
interaction cannot be dismissed.
Glycoproteins such as mucin, lactoferrin and collagen
contain several N- and/or O-linked oligosaccharides that
may vary with the source. Such heterogeneity makes
glycoproteins difficult to define exactly in terms of what a
particular carbohydrate contributes as a potential receptor
for bacterial binding. However, we emphasize that the main
monosaccharide precursors of the oligosaccharide moieties
were able to inhibit binding.
Adhesion of Aeromonas spp. to epithelial cells seems to be
a multi-factorial process involving participation of sequential
or simultaneous factors, such as LPS O-antigen (Merino
et al. 1996), polar and lateral flagellum (Rabaan et al. 2001;
Gavin et al. 2002), and long-wavy pili (type IV pili) (Kirov
et al. 1999).
Some clinical isolates of Aeromonas spp. lacking the LPS
O-antigen or nonfimbriated strains are still adhesive
(Sakazaki and Shimada 1984; Nishikawa et al. 1991, 1994;
Kirov et al. 1995). Taking into consideration the ability of
Omp48 to bind host glycoproteins, the role of this LamBlike protein as a potential alternative adherence factor was
undertaken. To get an insight into this, epithelial HeLa cells
were used as a first approach in this study because they have
been used as a common model to study patterns of adhesion
in other enterobacteria (Albert et al. 2000) The use of more
suitable epithelial cells that better resembles in vivo
situation, such as primary fish epithelial cell lines, is planned
in new studies.
Anti-Omp48 antibodies in the rabbit serum significantly
inhibited bacterial adhesion to HeLa cells by blocking
Omp48 adhesive epitopes, while purified Omp48 showed a
similar effect by competitive inhibition. In addition,
Omp48 was able to cross-inhibit the adhesion of Aer.
hydrophila and Aer. caviae. As the Aer. veronii strain A186
used in this study lacks an S-layer, we suggest that Omp48
is directly interacting with the surrounding milieu in vitro,
allowing the bacteria to adhere to host surfaces by means
of this protein. Taken together, the data demonstrate that
Omp48 might be an alternative adhesion factor of Aer.
veronii and suggest that Aeromonas spp. expresses similar
surface proteins with conserved structural or lineal antigenic epitopes. Other LamB-like proteins, including
maltoporin from the psychrophilic Aer. salmonicida and
porin I from the mesophilic Aer. hydrophila, have been
described (Jeanteur et al. 1992; Dodsworth et al. 1993),
indicating their widespread occurrence in this genus. A
43-kDa OMP from Aer. caviae was also reported as a
potential adhesin; however, no additional data indicating
that this is the homologous protein of Omp48 was
provided (Rocha-De-Souza et al. 2001). Nishikawa et al.
(1991, 1994 reported that the adhesion and haemagglutination of highly adhesive nonfimbriated Aeromonas strains
was inhibited by fucose. Carbohydrate reactive OMPs
(CROMPs) from Aer. hydrophila, including a 43-kDa
LamB-like maltoporin, were proposed as adhesins (Quinn
et al. 1994). In agreement with our results, other authors
(Paerregaard et al. 1991; McKee et al. 1995; Sperandio
et al. 1995) have suggested that OMPs from other important enteric pathogens, such as OmpU from V. cholerae,
intimin from enterohaemorrhagic E. coli and YadA from
Yersinia pseudotuberculosis and Y. enterocolitica, are colonization factors.
A lectin-like interaction of Omp48 with ECM components and mucosal proteins may help Aer. veronii remain
in the extracellular milieu, and eventually reach host
epithelial cells. Aeromonas spp. adhesion via OMPs could
be mediated by a combination of (i) stereospecific lectinlike interaction with carbohydrate-rich cell surfaces of the
host and (ii) interaction with a specific receptor on
enterocytes.
It is not known whether Aeromonas spp. qualitatively and
quantitatively express the same OMP profile during in vivo
growth as they do in vitro. However, it is likely that during
the Aeromonas spp. colonization process, availability of
maltose and maltodextrins (from nutrient digestion), induce
the expression of LamB-like proteins such as Omp48.
Therefore, Omp48 is a potential target antigen for vaccine
development and induction of protective immunity through
inhibition of host colonization. This is one of the few studies
demonstrating the role of nonfimbrial proteins in Aeromonas
spp. adhesion.
ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 96, 700–708, doi:10.1111/j.1365-2672.2004.02177.x
LAMB-LIKE ADHESIN OF AER. VERONII
ACKNOWLEDGEMENTS
This work was supported by the Mexican Council of
Science and Technology, (CONACyT grant 39510A/1) and
the CIBNOR fiscal project PAC-11. R. C. Vazquez-Juarez is
the recipient of a doctoral fellowship (CONACyT grant
121100). We thank L. D. Possani (Instituto de Biotecnologı́a, UNAM, México) for N-terminal peptide sequencing and A. Sierra for assistance with HeLa cell cultures.
Special thanks to A. G. Torres (M & I, University of Texas
Medical Branch, Galveston) for reviewing the manuscript
and providing helpful ideas.
REFERENCES
Abbott, S.L., Serve, H. and Janda, J.M. (1994) Case of Aeromonas
veronii (DNA group 10) bacteremia. Journal of Clinical Microbiology
32, 3091–3093.
Albert, M.J., Grant, T. and Robins-Browne, R. (2000) Studying
bacterial adhesion to cultured cells. In Handbook of Bacterial
Adhesion. Principles, Methods and Applications ed. An, Y.H. and
Friedman, R.J. pp. 541–552. Totowa, New Jersey: Human Press.
Ascencio, F., Aleljung, P. and Wadstrom, T. (1990) Particle agglutination assays to identify fibronectin and collagen cell surface receptors
and lectins in Aeromonas and Vibrio species. Applied and Environmental Microbiology 56, 1926–1931.
Ascencio, F., Ljungh, A. and Wadstrom, T. (1991) Comparative study of
extracellular matrix protein binding to Aeromonas hydrophila isolated
from diseased fish and human infection. Microbios 65, 135–146.
Ascencio, F., Ljungh, A. and Wadstrom, T. (1992) Characterization of
lactoferrin binding by Aeromonas hydrophila. Applied and Environmental Microbiology 58, 42–47.
Ascencio, F., Martinez-Arias, W., Romero, M.J. and Wadstrom, T.
(1998) Analysis of the interaction of Aeromonas caviae, A. hydrophila
and A. sobria with mucins. FEMS Immunology and Medical Microbiology 20, 219–229.
Aumailley, M. and Gayraud, B. (1998) Structure and biological activity
of the extracellular matrix. Journal of Molecular Medicine 76, 253–
265.
Austin, B. and Austin, D.A. (1999) Bacterial Fish Pathogens: Disease in
Farmed and Wild Fish. New York: Springer Verlag.
Boos, W. and Shuman, H. (1998) Maltose/maltodextrin system of
Escherichia coli: transport, metabolism, and regulation. Microbiology
and Molecular Biology Reviews 62, 204–229.
Carnoy, C., Scharfman, A., Van Brussel, E., Lamblin, G., Ramphal, R.
and Roussel, P. (1994) Pseudomonas aeruginosa outer membrane
adhesins for human respiratory mucus glycoproteins. Infection and
Immunity 62, 1896–1900.
Champomier-Verges, M.C., Stintzi, A. and Meyer, J.M. (1996)
Acquisition of iron by the non-siderophore-producing Pseudomonas
fragi. Microbiology 142, 1191–1199.
Chart, H. and Trust, T.J. (1983) Acquisition of iron by Aeromonas
salmonicida. Journal of Bacteriology 156, 758–764.
Dhaenens, L., Szczebara, F. and Husson, M.O. (1997) Identification,
characterization, and immunogenicity of the lactoferrin-binding
protein from Helicobacter pylori. Infection and Immunity 65, 514–518.
707
Dodsworth, S.J., Bennett, A.J. and Coleman, G. (1993) Molecular
cloning and nucleotide sequence analysis of the maltose-inducible
porin gene of Aeromonas salmonicida. FEMS Microbiology Letters
112, 191–197.
Doig, P., Emody, L. and Trust, T.J. (1992) Binding of laminin and
fibronectin by the trypsin-resistant major structural domain of the
crystalline virulence surface array protein of Aeromonas salmonicida.
Journal of Biological Chemistry 267, 43–49.
Ellison, R.T. III, Giehl, T.J. and LaForce, F.M. (1988) Damage of the
outer membrane of enteric gram-negative bacteria by lactoferrin and
transferrin. Infection and Immunity 56, 2774–2781.
Gavin, R., Rabaan, A.A., Merino, S., Tomas, J.M., Gryllos, I. and
Shaw, J.G. (2002) Lateral flagella of Aeromonas species are essential
for epithelial cell adherence and biofilm formation. Molecular
Microbiology 43, 383–397.
Harlow, E. and Lane, D. (1988) Antibodies: A Laboratory Manual. Cold
Spring Harbor, NY: Cold Spring Harbor Laboratory.
Hickman-Brenner, F.W., MacDonald, K.L., Steigerwalt, A.G., Fanning, G.R., Brenner, D.J. and Farmer, J.J. (1987) Aeromonas veronii,
a new ornithine decarboxylase-positive species that may cause
diarrhea. Journal of Clinical Microbiology 25, 900–906.
Hirst, I.D. and Ellis, A.E. (1996) Utilization of transferrin and salmon
serum as sources of iron by typical and atypical strains of Aeromonas
salmonicida. Microbiology 142, 1543–1550.
Hsueh, P.R., Teng, L.J., Lee, L.N., Yang, P.C., Chen, Y.C., Ho, S.W.
and Luh, K.T. (1998) Indwelling device-related and recurrent
infections due to Aeromonas species. Clinical Infectious Diseases 26,
651–658.
Janda, J.M. and Abbott, S.L. (1998) Evolving concepts regarding the
genus Aeromonas: an expanding panorama of species, disease
presentations, and unanswered questions. Clinical Infectious Diseases
27, 332–344.
Jeanteur, D., Gletsu, N., Pattus, F. and Buckley, J.T. (1992)
Purification of Aeromonas hydrophila major outer-membrane proteins: N-terminal sequence analysis and channel-forming properties.
Molecular Microbiology 6, 3355–3363.
Joseph, S.W., Carnahan, A.M., Brayton, P.R., Fanning, G.R.,
Almazan, R., Drabick, C., Trudo, E.W. and Colwell, R.R. (1991)
Aeromonas jandaei and Aeromonas veronii dual infection of a human
wound following aquatic exposure. Journal of Clinical Microbiology
29, 565–569.
Kirov, S.M., Jacobs, I., Hayward, L.J. and Hapin, R.H. (1995)
Electron microscopic examination of factors influencing the expression of filamentous surface structures on clinical and environmental
isolates of Aeromonas veronii biotype sobria. Microbiology and
Immunology 39, 329–338.
Kirov, S.M., O’Donovan, L.A. and Sanderson, K. (1999) Functional
characterization of type IV pili expressed on diarrhea-associated
isolates of Aeromonas species. Infection and Immunity 67, 5447–
5454.
Laemmli, U.K. (1970) Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 227, 680–685.
Lang, H. and Ferenci, T. (1995) Sequence alignment and structural
modelling of the LamB glycoporin family. Biochemical and Biophysical Research Communications 208, 927–934.
McKee, M.L., Melton-Celsa, A.R., Moxley, R.A., Francis, D.H. and
O’Brien, A.D. (1995) Enterohemorrhagic Escherichia coli O157:H7
ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 96, 700–708, doi:10.1111/j.1365-2672.2004.02177.x
708 R . C . V À Z Q U E Z - J U Á R E Z ET AL.
requires intimin to colonize the gnotobiotic pig intestine and to
adhere to HEp-2 cells. Infection and Immunity 63, 3739–3744.
Merino, S., Rubires, X., Aguilar, A. and Tomas, J.M. (1996) The
O:34-antigen lipopolysaccharide as an adhesin in Aeromonas hydrophila. FEMS Microbiology Letters 139, 97–101.
Neves, M.S., Nunes, M.P., Milhomem, A.M. and Ricciardi, I.D.
(1990) Production of enterotoxin and cytotoxin by Aeromonas veronii.
Brazilian Journal of Medical and Biological Research 23, 437–440.
Nishikawa, Y., Kimura, T. and Kishi, T. (1991) Mannose-resistant
adhesion of motile Aeromonas to INT407 cells and the differences
among isolates from humans, food and water. Epidemiology and
Infection 107, 171–179.
Nishikawa, Y., Hase, A., Ogawasara, J., Scotland, S.M., Smith, H.R.
and Kimura, T. (1994) Adhesion to and invasion of human colon
carcinoma Caco-2 cells by Aeromonas strains. Journal of Medical
Microbiology 40, 55–61.
Paerregaard, A., Espersen, F. and Skurnik, M. (1991) Role of the
Yersinia outer membrane protein YadA in adhesion to rabbit
intestinal tissue and rabbit intestinal brush border membrane
vesicles. APMIS 99, 226–232.
Quinn, D.M., Atkinson, H.M., Bretag, A.H., Tester, M., Trust, T.J.,
Wong, C.Y. and Flower, R.L. (1994) Carbohydrate-reactive, poreforming outer membrane proteins of Aeromonas hydrophila. Infection
and Immunity 62, 4054–4058.
Rabaan, A.A., Gryllos, I., Tomas, J.M. and Shaw, J.G. (2001) Motility
and the polar flagellum are required for Aeromonas caviae adherence
to HEp-2 cells. Infection and Immunity 69, 4257–4267.
Rocha-De-Souza, C.M., Colombo, A.V., Hirata, R., Mattos-Guaraldi,
A.L., Monteiro-Leal, L.H., Previato, J.O., Freitas, A.C. and
Andrade, A.F. (2001) Identification of a 43-kDa outer-membrane
protein as an adhesin in Aeromonas caviae. Journal of Medical
Microbiology 50, 313–319.
Sakazaki, R. and Shimada, T. (1984) O-serogrouping scheme for
mesophilic Aeromonas strains. Japanese Journal of Medical Sciences
and Biology 37, 247–255.
Schneider, E., Freundlieb, S., Tapio, S. and Boos, W. (1992)
Molecular characterization of the MalT-dependent periplasmic
alpha-amylase of Escherichia coli encoded by malS. Journal of
Biological Chemistry 267, 5148–5154.
Sperandio, V., Giron, J.A., Silveira, W.D. and Kaper, J.B. (1995)
The OmpU outer membrane protein, a potential adherence factor
of Vibrio cholerae. Infection and Immunity 63, 4433–4438.
Steinfeld, S., Rossi, C., Bourgeois, N., Mansoor, I., Thys, J.P. and
Appelboom, T. (1998) Septic arthritis due to Aeromonas veronii
biotype sobria. Clinical Infectious Diseases 27, 402–403.
Stelma, G.N., Johnson, C.H. and Spaulding, P.L. (1988) Experimental
evidence for enteropathogenicity in Aeromonas veronii. Canadian
Journal of Microbiology 34, 877–880.
Stintzi, A. and Raymond, K.N. (2000) Amonabactin-mediated iron
acquisition from transferrin and lactoferrin by Aeromonas hydrophila:
direct measurement of individual microscopic rate constants. Journal
of Biological Inorganic Chemistry 5, 57–66.
Thornley, J.P., Shaw, J.G., Gryllos, I.A. and Eley, A. (1996)
Adherence of Aeromonas caviae to human cell lines Hep-2 and
Caco-2. Journal of Medical Microbiology 45, 445–451.
Vazquez-Juarez, R.C., Barrera-Saldana, H.A., Hernandez-Saavedra,
N.Y., Gomez-Chiarri, M. and Ascencio, F. (2003) Molecular
cloning, sequencing and characterization of omp48, the gene
encoding for an antigenic outer membrane protein from Aeromonas
veronii. Journal of Applied Microbiology 94, 908–918.
Vihinen, M. and Mantsala, P. (1989) Microbial amylolytic enzymes.
Critical Reviews in Biochemistry and Molecular Biology 24,
329–418.
Westerlund, B. and Korhonen, T.K. (1993) Bacterial proteins binding
to the mammalian extracellular matrix. Molecular Microbiology 9,
687–694.
Wong, H.C., Liu, C.C., Yu, C.M. and Lee, Y.S. (1996) Utilization of
iron sources and its possible roles in the pathogenesis of
Vibrio parahaemolyticus. Microbiology and Immunology 40, 791–
798.
ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 96, 700–708, doi:10.1111/j.1365-2672.2004.02177.x