FEMS Microbiology Letters 139 (1996) 97-101

The 0:34-antigen lipopolysaccharide as an adhesin in Aeromonas hydrophila

Susana Merino, Xavier Rubires, Alicia Aguilar, Juan M. Ton-h *

Departamento de Microbiologia, Facultad de Biologia, Uniuersidad de Barcelona, Diagonal 645, 08071 Barcelona, Spain

Received 29 November 1995; revised 15 January 1996; accepted 29 January 1996

Abstract

We compared the ability of different Aeromonas hydrophila strains from serogroup 0:34 grown at different temperatures to adhere to Hep-2 cells. We found a high level of adhesion when the strains were grown at 20°C but not when they were

grown at 37°C. We previously described that these strains were able to form the O-antigen lipopolysaccharide when they grow at low temperature but not at high temperature. We also obtained by transposon mutagenesis mutants only devoid of the O-antigen lipopolysaccharide (rfb mutants), and they showed significantly lower levels of adhesion to Hep-2 cells than the smooth strains. All these results prompted us to conclude that the O-antigen LPS, in these strains, is an important adhesin.

Keywords: Aeromonas hydrophila serogroup 0:34; Adhesion to Hep-2 cells; O-antigen LPS

1. Introduction

Motile Aeromonas species are ubiquitous inhabi- tants of the aquatic environment and are also consid- ered to be part of the normal microflora of the intestinal tract of different animals [l]. Aeromonas

hydrophila is an opportunistic as well as primary pathogen of a variety of aquatic and terrestrial ani- mals, including humans; the clinical manifestations

range from gastroenteritis to soft-tissue infections, including septicemia, and meningitis [2]. Aeromonas

strains have been serotyped on the basis of the O-antigen lipopolysaccharide (LPS) 131, the polysac-

* Corresponding author. Tel.: + 34 (3) 402 1486; Fax: + 34 (3) 411 0592; E-mail: juant@porthos.bio.ub.es

charide chains in the smooth LPS, also known as the somatic antigen.

Serogroup 0:34 strains of A. hydrophila have been recovered from moribund fish [4,5] or from clinical specimens [6], being the single most com- mon Aeromonas serogroup accounting for 26.4% of all infections. Previous investigations have docu- mented 0:34 strains as an important cause of infec- tions in humans [7,8]. The results reported in adhe- sion to Hep-2 cells by some variants of A. sobria [9]

prompted us to study the role of the 0:3Cantigen

LPS as an adhesin to these cells, as a preliminary step in gastroenteritic infection.

We describe here the isolation of LPS mutants by transposon mutagenesis, and show that they were only devoid of the 0:34-antigen LPS without changes on the LPS core region. With these isogenic mutants

0378- 1097/96/$12tXl 0 1996 Federation of European Microbiological Societies. All rights reserved PII SO378-1097(96)00068-7

we studied the role of O-antigen LPS on the adhe- sion of A. hydrophilu serogroup 0:34 to Hep-2

cells.

2. Materials and methods

2.1. Bacteria, hucteriophuges and meditr

The A. hydtwphilu strains from serogroup 0:34 used are listed in Table I. Bacteriophages PM1 [lo] and PM2 [ 1 I] have been characterized previously by

us; they were propagated on A. h_vdrophila AH-3. The basal medium used for bacterial growth and phage propagation was tryptone soya broth (TSB) or TSB with 1.5% agar (TSA). To prepare soft agar we added 0.6% agar to TSB. Bacteriophage sensitivity or resistance was assayed by spot test.

Spontaneous rifampicin-resistant mutants were se- lected by plating IO” cells on TSA plus 100 Fg/ml of rifampicin. They were re-isolated by restreaking on the same plates.

Transposon Tn5 mutants were obtained after tri- parental mating using as donors E. coli S I7- 1 carry- ing the plasmid pSUP202 (a derivative of plasmid pBR322 unable to replicate on Aeromonus carrying the rnoh genes and transposon Tn5) [ 121 and a helper conjugative plasmid pRK2073 (spectino- mycin-resistant) in E. coli HB 101 [ 131, and using as

Table I

Strains and plasmida used, their relevant characterihtlca and origin

a recipient strain the rifampicin-resistant mutants from the A. hydrophilu wild-type strains. The tri-

parental matings were performed at 30°C and the selection was in TSA plates with 100 pug/ml of rifampicin and 25 pg/ml of kanamycin. The trans-

poson Tn5 mutants obtained were finally selected by immunoscreening after restreaking on the same se- lective plates.

2.2. Immunoscreening of‘ 0:34-antigen LPS murunts

Mutants grown on selective agar plates (plus 100 Fg/ml of rifampicin and 25 pg/ml of kanamycin) were overlaid with precut nitrocellulose filters (Mil- lipore Corp., Bedford, MA). Dried filters were then blocked, immunostained with specific 0:34-antigen rabbit antiserum (I: 1000) and stained with goat anti-rabbit immunoglobulin G alkaline phosphatase (Boehringer). The colonies that gave a negative sig- nal were purified and rescreened again by the same

procedure.

2.3. LPS isolution und anulvses

LPS was purified by the method of Westphal and Jann [ 141, as modified by Osborn [ 1.51 for mutants devoid of the O-antigen LPS. Purified LPS was

analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver stained

Strain Relevant properties Origin

A. hydrophih strains from serogroup 0:34 AH-3 Smooth when prowr. at 20°C. hut rough at 37’C

BaS Smooth when grown at 2o”C, but rough at 37°C.

AH-405 Rifampicin-resistant mutant from AH-3

AH-406 Rifampicin-resistant mutant from BaS

AH-22 Isogenic rough mutant with complete LPS core from AH-3

AH-35 Isogenic rough mutant with incomplete LPS core from AH-3

AH-457 0:34 with complete LPS core: Tn5 mutant derived from AH-405

AH-459 0:31 with complete LPS core: Tn5 mutant derived from AH-406

E. coli strains HBIOI

Sl7-I

Plasmids pRK2073

pSUP202 I

pro. /err. thi. I~icY. sir’. mdol , reul

,wY,. thi. HsdR-. HsdM+, rrc.4

Self-transmissible plasmid with resistance to bpectinomycin. streptomycin

Mobilizable transposon carrier plasmId TnS inserted with the mob genes from RP4

[I81 I181 this study

this study

[IO1

[I II this study

thib study

[I31 iI21

[l.il [I21

S. Merino et al. / FEMS Microbiology Letters 139 (1996) 97-101 99

by the method of Tsai and Frasch [16]. LPS core oligosaccharides, proteinase K digests of outer mem- brane fractions, were separated on 18% polyacryl- amide gels using a SDS-tricine buffer and visualized by silver staining as described [17]. 2-Keto-3-de-

oxyoctulosonic acid (Kdo) was measured by the thiobarbituric acid method as previously described by us [ 181. Monosaccharides were also analyzed as their alditol acetate derivatives by gas-liquid chro- matography on a 3% SP-3840 column (Supelco) as

previously described by us [lo].

min at 37°C with 5% CO, in air, coverslips were washed extensively with PBS, fixed in 70% methanol and stained with Giemsa. Coverslips were then mounted on glass microscope slides and adhesion was assessed by bright-field microscopy. For each

assay bacteria adhering to 30 randomly selected cells from each of two monolayers were counted. The assays were performed (at least) in triplicate.

3. Results and discussion

2.4. Antiserum

Polyclonal antiserum against purified LPS of strain AH-3 (0:34) grown at 20°C was raised in adult New Zealand White rabbits as previously described by us, and also rendered specific for 0:34 antigen LPS by extensive adsorption using LPS mutant AH-22 (only devoid of O-antigen LPS but with a complete LPS

core> [ 181.

2.5. Hep-2 cell adhesion assay

Adhesion of bacteria to Hep-2 cell monolayers was tested as described previously [19]. Briefly,

bacterial cells (lo6 cfu ml- ‘) in PBS with 0.5% Dulbecco’s mineral salts solution were added to monolayers of Hep-2 cells grown for 24 h on glass coverslips in 24-well trays. After incubation for 90

In order to enable Tn5 mutagenesis, we obtained rifampicin-resistant mutants from A. hydrophila

strains from serogroup 0:34. Spontaneous mutants were found at a frequency of l-3 X lo-‘. These mutants (AH-405 and AH-406) showed identical sur-

face properties as their wild-type strains. No changes in bacteriophage sensitivity were detected (both were sensitive to bacteriophages PM1 (a bacteriophage specific for the 0:3Cantigen LPS [lo]) or PM2 (a

bacteriophage specific for the LPS core [ll]). No changes in the outer membrane protein profile or LPS with cells grown at different temperatures were

observed when we compared the wild-type strains with their isogenic rifampicin-resistant mutants

(SDS-PAGE, data not shown). As we have previ- ously described for A. hydrophila [ 181, these strains also expressed smooth LPS (with 0:3Cantigen LPS)

when they were grown at 20°C and rough LPS

Table 2

Main chemical comnosition of uurified LPS from A. hvdroohila 0:34 strains

Strain fimol/mg of LPS

AH-3 (20°C) ’

AH-3 (37’0

AH-457 (20°C)

Ba5 (20°C)

Ba5 (37°C)

AH-459 (20°C)

AH-22 (20°C)

AH-35 (20°C)

Kdo a 1 -r+Heptose b Glucose b Hexosamines b

0.018 0.31 1.51 0.41

0.039 0.99 1.67 < 0.05

0.040 0.98 1.69 < 0.05

0.019 0.3 1 1.55 0.43

0.042 0.96 1.68 < 0.05

0.041 0.97 1.66 < 0.05

0.038 0.96 1.64 < 0.05

0.05 1 1.25 1.32 < 0.05

a Assayed by calorimetric method as previously described by us [18]. b Assayed by gas-liquid chromatography as described in Section 2. Only residual content of glucosamine from the lipid A is obtained

because the hydrolysis procedure used is too weak to completely cleave this sugar.

’ Growth temperature. LPS from wild-type strains AH-3 and Ba5 is smooth when they grow at 20°C and rough when they grow at 37°C. LPS from strains AH-457,

AH-459 and AH-22 is rough, and LPS from strain AH-35 is deeply rough at both growth temperatures.

1234567 Fig. 1. SDS-PAGE of LPS from A. /mirophi/u stralnb serogroup

0:34. Lanes: I. AH-405 grown at 20°C; 2. AH-405 grown at

37°C; 3, AH-457 grown at 20°C: 4. AH-35 (LPS mutant with

incomplete LPS core [I I]) grown at 20°C: 5, AH-406 grown at

20°C; 6, AH-406 grown at 37°C: 7, AH-459 grown at 20°C.

(without 0:34-antigen LPS) when they were grown

at 37°C (Fig. 1). In order to demonstrate the role of the 0:34-anti-

gen LPS, we isolated mutants devoid of the 0:34-an- tigen LPS from strains AH-405 and AH-406 by Tn.5 transposon mutagenesis and immunoscreening as de- scribed in Section 2. We obtained different mutant colonies, strains AH-457 and AH-459 (respectively from AH-405 and AH-406) being representatives of them. These mutants exhibited rough LPS when they were grown at either 20” or 37°C (Fig. 1). They were resistant to bacteriophage PM1 and sensitive to bac- teriophage PM2, consistent with the pattern of rough mutants with complete LPS core previously isolated by us [IO]. LPS of these Tn5 mutants also showed identical chemical composition to that of the wild-

Table 3

1234567

The adhesion of different A. hy/mphila strain& (aenrgroup 034) to Hep-2 cell>

Fig. 2. SDS-PAGE of LPS core oligosaccharides [ 16.171 from A.

I~~&op/ri/a strains herogroup 035. Lanes: I. AH-3 grown at

37°C: 2. AH-405 grown at 37°C: 3. AH-457 grown at 20°C: 1.

AH-35 (LPS mutant with incomplete LPS core [I I]) grown at

20°C: 5. AH-22 grown at 20°C (LPS mutant with complete LPS

core [IO]); 6. AH-406 grown at 37°C: 7. AH-459 grown at 20°C.

type strains grown at 37°C (rough LPS) or LPS mutants with a complete LPS core, like AH-22 [IO] (Table 2). Furthermore. in SDS-PAGE with tricine buffer in order to visualize the LPS core oligosac- charides [ 171, LPS of the Tn5 mutants showed the same relative electrophoretic mobility as LPS of wild-type strains grown at 37°C (rough LPS) or of the mutant AH-22 with a complete LPS core, while the LPS of mutant AH-35 with an incomplete LPS core showed a faster migration (Fig. 2). We can

conclude that mutants AH-457 and AH-459 are clearly $Y mutants. i.e. the Tn5 is inserted in the DNA region coding for the genes responsible of the

0:34-antigen biosynthesis. The wild-type strains or their isogenic rifampicin-

Strain Growth temperature

AH-3 (smooth) 20°c

AH-3 (rough) 37°C

AH-405 (smooth) 20°C

AH-405 (rough) 37°C

AH-457 (rough) 20°C

Ba5 (smooth) 20°C

Ba5 (rough) 37°C

AH-406 (smooth) 20°C

AH-406 (rough) 37°C

AH-459 (rough) 20°C

Mean number of bacteria per Hep-2 cell

18.3 * 4. I 19.4 i 2.4

16.5 k 3.7 18.3 + 2.Y

19.1 i- 2.3

46.9 f 4.4

IX.6 + 2.7 17.3 * 4. I 19.1 * 3.1 IS.9 + 2.2

% reduction in adhesion of 0

59.9 ,’

60.7

59 .o

60.3 _ 59.1

60.1

’ Student’s r-k%. P < 0.001

S. Merino et al./ FEMS Microbiology Letters 139 (1996) 97-101 101

resistant mutants grown at 20°C (smooth LPS) showed a high level of adhesion to Hep-2 cells (Table 3). By contrast, the same strains grown at 37°C (LPS without the 0:3Cantigen) or the fl mutants obtained by transposon mutagenesis (AH- 457 and AH-4591 grown at 20°C showed a reduction of approximately 60% in their adhesion to Hep-2 cells, under the same conditions in which the strains with smooth LPS were able to do it.

All the strains grown at 20°C (with smooth or

rough LPS) were agglutinated by a specific anti- serum against the fimbriae (antiserum kindly pro- vided by M. Sato [20]). Thus, all of them were able

to produce fimbriae (an important adhesin as previ- ously described in Aeromonas [21,22]).

This study has demonstrated an important role for the 0:3Cantigen LPS in the adhesion to Hep-2 cells by A. hydrophila 0:34 strains. Together with the

previous report of Francki and Chang [9] about the role of LPS from one non-serotyped strain of A.

sobria as an adhesin, the results suggests that the LPS of mesophilic aeromonads could be an impor- tant factor of adhesion and gut colonization, indicat-

ing that some Aeromonas spp. are important primary pathogens in gastroenteritis.

Acknowledgements

This work has been supported by a research grant

from DGICYT (PB94-0906) and a fellowship to X.R. We thank M. Sato for antiserum against fim- briae. We thank Maite Polo for her technical assis- tance.

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