Characterization of an 0-antigen bacteriophage from Aeromonas hydrophila SUSANA MERINO,SILVIA CAMPRUBI, AND JUANM.
TOMAS'
Departarnento de Microbiologlh, Facultad de Biologia, Universidad de Barcelona, Diagonal 645, 08071 Barcelona, Spain Received May 28, 1991 Revision received September 9, 1991
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Accepted September 10, 1991 MERINO,S., CAMPRUBI, S., and TOMAS,J. M. 1992. Characterization of an 0-antigen bacteriophage from Aerornonas hydrophila. Can. J. Microbiol. 38: 235-240. A unique bacteriophage of Aerornonas hydrophila serotype 0:34 was isolated, purified, and characterized. The bacterial surface receptor was shown to be the 0-antigen polysaccharide component of lipopolysaccharide specific to serotype 0:34, which was chemically characterized. The high molecular weight lipopolysaccharide fraction (a fraction enriched in 0 antigen) was fully able to inactivate bacteriophage PMI. Phage-resistant mutants of A. hydrophila 0:34 were isolated and found to be specifically devoid of lipopolysaccharide 0 antigen. No other cell-surface molecules were involved in phage binding. The host range of bacteriophage PMl was found to be very narrow, producing plaques only on A. hydrophila strains from serotype 0:34. Key words:' Aerornonas hydrophila, serotype 0:34, bacteriophage P M l , lipopolysaccharide. MERINO,S., CAMPRUBI, S., et TOMAS,J. M. 1992. Characteri~ationof an 0-antigen bacteriophage from Aerornonas hydrophila. Can. J. Microbiol. 38 : 235-240. Nous avons isole, purifie et caracterise un bacteriophage unique d'Aerornonas hydrophila serotype 0:34. La caracterisation chimique a confirme que le recepteur de surface etait l'antigene polysaccharidique 0 associe au lipopolysaccharide specifique du serotype 0:34. La fraction lipopolysaccharidique qui a un poids moleculaire eleve (une fraction riche en antigene 0 ) pouvait inactiver completement le bacteriophage PMl. Nous avons isole des mutants d'A. hydrophilia 0:34 resistants au bacteriophage et confirme que ceux-ci etaient depourvus de I'antigene lipopolysaccharidique 0. Aucune autre molecule de la surface cellulaire n'etait impliquee dans la liaison avec le bacteriophage. Les bacteries-h8tes du bacteriophage PMl sont peu nombreuses. Ce phage produit des plages seulement en presence de souches d'A. hydrophila du serotype 0:34. Mots clks : Aerornonas hydrophila, serotype 0:34, bacteriophage PM1, lipopolysaccharide. [Traduit par la redaction]
Introduction Motile Aeromonas species are ubiquitous inhabitants of the aquatic environment and are also considered to be part of the normal microflora of the intestinal tract of fish (Trust and Sparrow 1974). Aeromonas hydrophila is an opportunistic as well as primary pathogen of a variety of aquatic and terrestrial animals, including humans; the clinical manifestations range from gastroenteritis to soft-tissue infections, including septicemia, and meningitis (Freij 1984). Aeromonas hydrophila strains are classified on the basis of virulence related to cell-surface components (Lallier et al. 1984; Janda et al. 1985). Strains with homogeneous 0 polysaccharide chains ( 0 antigen) both in their lipopolysaccharide (LPS) as well as S layers are highly virulent toward fish and mice (Lallier et al. 1984; Janda et al. 1985), whereas strains with heterogeneous LPS are considered avirulent (Dooley et al. 1985). We recently described A . hydrophila species belonging to serotype 0 : 3 4 with heterogeneous 0 polysaccharide chains, similar to strain Ba5, previously reported to be moderately virulent for fish (Lallier et al. 1984) and mice (Merino et al. 1989). It would be diagnostically beneficial to have a phage typing system for A . hydrophila species. In this regard we isolated and characterized bacteriophage PM 1, specific for A . hydrophila strains belonging to serotype 0:34, and we demonstrated that LPS 0 antigen is the specific receptor. We also characterized LPS from this serotype (0:34) as well as phage-resistant mutants useful for the study of virulence mechanisms in A . hydrophila 0 : 3 4 . ' ~ u t h o rto whom all correspondence should be addressed. Pr~ntedin Canada / lmprime au Canada
Materials and methods Bacteria, bacteriophages, and media The strains, their relevant properties and origins, as well as their sensitivity for bacteriophages P M l , PM2 (Merino et al. 1990a), and 18 (Merino et al. 1990b) are listed in Table 1. The A. hydrophila strains belonging to serotype 0:34 and their respective PM1-resistant mutants were also sensitive to bacteriophages 24, 69, and 145 (Trust et al. 1980), which were kindly provided by T. J. Trust. Tryptone-soya broth (TSB) was used for bacterial growth and phage propagation. TSB was supplemented with either 1.5% agar (w/v) (TSA) or 0.6% agar (TSA soft agar). For titration and inactivation assays, phage suspensions were diluted in phage buffer (10 mM MgSO,, 20 mM NaC1, and 50 mM Tris-HC1, pH 7.5). Spontaneous mutants of A. hydrophila strain AH-3 and strain Ba5 resistant to bacteriophage PMl were isolated by spreading a mixture containing about lo8 bacteria and lo9 phage plaqueforming units on TSA. After 48 h at 20°C, colonies of phageresistant mutants were picked and purified by streaking and were cross streaked against PMl to confirm resistance. Bacteriophage isolation Freshwater samples were centrifuged and the supernatant was incubated at 20°C with an exponential growth phase culture of A. hydrophila AH-3 on double-concentrated TSB. After overnight incubation, the bacteria were removed by centrifugation and filtration, and the supernatant was plated on A. hydrophila AH-3 by using the double agar layer method of Adams (1959). Plaques that formed on the plates were stabbed with a needle and eluted with a small volume of phage buffer. Each phage suspension was serially propagated twice on the same strain. General phase techniques and production of phage lysates The methods of Adams (1959) were used. The incubation temperature was 20°C, and the plates were incubated for 24 h. The
CAN. J . MICROBIOL. VOL. 38, 1992
TABLE1. Aeromonas hydrophila strains, their relevant characteristics, source, and bacteriophage sensitivity
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Sensitivity to: Strain
Relevant characteristics
AH-102 AH-103 TF7 LL1 AH-50 ATCC 7966 43014 17577 24963 31075 8973 31075 291 1 1480 38589
Serotype 0:34, smooth Isogenic PM1-resistant mutant from AH-3; rough Serotype 0:34, smooth Isogenic PM1-resistant mutant from Ba5; rough Serotype 0:34, smooth Isogenic PM1-resistant mutant from AH-101; rough Serotype 0:34, smooth Serotype 0:34, smooth Serotype 0:11, S layer Serotype 0:11, S layer Serotype 0:22, smooth Not serotyped, smooth Not serotyped, smooth Not serotyped, smooth Not serotyped, smooth Not serotyped, smooth Not serotyped, smooth Not serotyped, smooth Not serotyped, smooth Not serotyped, rough Not serotyped, rough
PM1
PM2
18
Source Our laboratory This work Our laboratory This work Our laboratory This work Our laboratory Our laboratory T.J. Trust T.J. Trust Our laboratory ATCC Clinical Clinical Clinical Clinical Clinical Clinical Clinical Clinical Clinical
NOTE:All the A. hydrophila strains not serotyped give a negative reaction against anti-0:34 or anti-0:ll serum. All the strains from clinical sources are from human origin. S, sensitive; R, resistant.
bacteriophage host range was assayed by spot tests. One-step Membrane proteins were analyzed by sodium dodecyl sulfate growth experiments were done using A . hydrophila AH-3 as the polyacrylamide gel electrophoresis (SDS-PAGE), using a modification (Ames et al. 1974) of the Laemmli procedure. Protein gels host and a multiplicity of infection of 1. Solvent inactivation (ether were routinely stained with Coomassie blue or silver reagent, and and chloroform), phage particle purification, buoyant densities, protein concentrations were determined by the Lowry procedure nucleic acid determination, and polypeptide analyses were performed as previously described (Merino et al. 1989). [ 3 ~ ~ h y r n i d i n e (Lowry et al. 1951), with bovine serum albumin as the standard. incorporation on the purified phage particles was measured as LPS was purified by the method of Westphal and Jann (1965), described by ReguC et al. (198 1). as modified by Osborn (1966). Subfractionation of purified LPS by column chromatography was performed according to Ciurana and Tomas (1987). Fractions were extensively dialyzed against Bacteriophage inactivation experiments distilled water, first at room temperature and then at 4"C, and Bacteriophages (lo3 pfu) were incubated for 20 min at 20°C analyzed by SDS-PAGE and silver stained by the method of Tsai with one of the following: 10' cells, 200 pg of 1% deoxycholate and Frasch (1982). (DOC) solubilized outer membrane (OM), 200 pg of 1% DOC For chemical analyses, purified LPS or LPS fractions were solubilized OM treated with phenol or with proteinase K (Tomas hydrolyzed with 1 M HCl for 2 h at 100°C. 2-Keto-3-deoxyand Jofre 1985), 200 pg of purified LPS (unless otherwise octulosonic acid (KDO) was released by hydrolysis with 4 M HCl indicated), or 100 pg of high molecular weight LPS (HMW-LPS) at 100°C for 30 min as described by Brade (1985), and the KDO ( 0 antigen enriched fraction) or 100 pg of low molecular weight released from LPS was assayed by the colorimetric method of LPS (LMW-LPS) (LPS core and lipid A enriched fraction). After Karkhanis et al. (1978) and by gas-liquid chromatography accordeach of the different treatments, the OM was extensively dialyzed ing to Brade (1985). Monosaccharides were also analyzed as their against distilled water for 24 h at room temperature. Chloroform alditol acetate derivatives by gas-liquid chromatography on a 3% (three or four drops) was added and mixed for 60 s, and the mixSP-3840 column (Supelco Inc.) at 225 and 180°C. Alditol acetate ture was centrifuged at 12 000 x g for 10 min at 4°C. The supermonosaccharides were obtained by the following derivatization pronatants were assayed directly on A . hydrophila AH-3. cedure (bulletin 774A, Supelco Inc., Cras, Switzerland). Samples (50 mg) were neutralized with an equal volume of 0.5 M NaBH, Cell-surface isolation and analyses for 60 min at room temperature and acetylated by the dropwise Cell envelopes were prepared by French pressure cell lysis at addition of glacial acetic acid until bubbling stopped. Dried samples 16 000 lb/in2 (1 lb/in2 = 6.9 kPa) of whole cells followed by the were washed twice with 0.1 mL of concentrated HCl per 100 mL removal of unbroken cells at 10 000 x g for 10 min and by of methanol and with ethanol. The supernatant was dried under sedimentation of the membrane fraction at 100 000 x g for 2 h. N,, suspended in equal volumes of pyridine and acetic anhydride, Cytoplasmic membranes were solubilized twice with sodium lauryl and heated at 100°C for 15 min, then analyzed by gas chromatogsarcosinate (Filip et al. 1973), and the OM fraction was sedimented raphy. Alditol acetate carbohydrate standards were either purtwice at 100 000 x g for 2 h. OM proteins were solubilized in 1% chased from Supelco or prepared by us. DOC - 2 mM EDTA (Tomas and Jofre 1985). To remove the LPS from the solubilized OM proteins, samples were treated with 88% Purified LPS or LPS fractions were further analyzed by SDSPAGE and silver stained by the method of Tsai and Frash (1982). phenol at 70°C, as described by Hancock and Nikaido (1978).
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MERINO ET AL.
FIG. 1. Characteristics of phage PM1. (A) Phage DNA was digested with different restriction enzymes. The resulting fragments were separated by electrophoresis through a 1% agarose gel for 6 h at 5 V/cm. Lane 0, X phage DNA digested with HindIII; lane 1, HindIII; lane 2, PstI; lane 3, Sari; lane 4, BgnI; lane 5, EcoRI; lane 6, BamHI; lane 7, EcoRV. (B) SDS-PAGE of bacteriophage PM1. ~ a n 1, e molecular size standards (14.4,21.5,31 .O, 45.0; 66.2, Fm. 2. Morphology of bacteriophage PMl negatively stained and 91.0 kDa) from Bio-Rad Laboratories; lane 2; ~ o l ~ ~ e ~ t i d e s and observed with an electron microscope Hitachi MT800. Bar of bacteriophage P M 1. represents 50 nm. Anti-LPS serum Immune serum was obtained as previously described using purified LPS from A. hydrophila AH-3 (serotype 0:34) or A. hydrophila TF7 (serotype 0 : 11) (Tomas et al. 1986). Molecular biological methods Standard techniques for manipulation of DNA were as described previously (Maniatis et al. 1982). Electron m icroscopy Purified bacteriophage were negatively stained with 1To phosphotungstic acid (pH 7.2) for 30 s, and examined using an Hitachi MT800 electron microscope.
Bacteriophage isolation and characterization We isolated bacteriophage PM 1 from one of five different freshwater samples. Phage PM1 gave clear plaques characteristically varying in size from 2 to 3 mm, with diffuse edges. Phage PM 1 incorporated ['HI thymidine in their nucleic acid and, therefore, are DNA based. Furthermore, Bradley's method (Bradley 1966) confirmed that phage PM 1 contained double-stranded DNA. Restriction enzymes PstI, SalI, and EcoRI were able to cleave bacteriophage PM1 DNA, whereas HindIII, BglII, BamHI, and EcoRV were without effect (Fig. 1A). Fourteen polypeptides were observed (108, 79, 51,48,45, 33, 32, 31,29,28,26, 24, 18, and 16 kDa) on SDS-PAGE; the three major ones were
33.0, 18.0, and 16.0 kDa (Fig. 1B). Neither chloroform nor ether caused any loss of infectivity. Bacteriophage PM1 had a buoyant density of 1.51 g/cm3, a latent period of 50 min, a rise period of 30 min, and a burst size of 170 pfu at 20°C. These phage presented a polyhedric contractile tail and a base plate with some tail fibers but no collar (Fig. 2). The host range of bacteriophage PM 1 was found to be very narrow, producing plaques only on A. hydrophila strains belonging to serotype 0:34 (Table 1). Furthermore, Aeromonas salmonicida A450 and A450- 1 (Kay et al. 198 1) and Vibrio anguillarum (serotype 01) strains were resistant. None of 20 strains of the Enterobacteriaceae genera tested, encompassing Enterobacter, Providencia, Proteus, Citrobacter, Serratia, Yersinia, Klebsiella, Escherichia, and Salmonella, was sensitive to bacteriophage PM1. P M I bacteriophage surface receptor Mutants resistant to bacteriophage PM 1 occurred at a frequency of 2 x 10 and fell into a single class on the basis of their LPS profile (Fig. 3). All of them (20 tested for strains AH-3 and Ba5) lacked the 0-antigen polysaccharide chains and were sensitive to bacteriophages PM2, 18, 24, 69, and 145 (data given for bacteriophages PM2 and 18 in Table 1). Phage PM1 adsorbed readily to A. hydrophila AH-3, Ba5, and AH-101 but not to the PM1-resistant mutants AH-22, AH-35, and AH-161, respectively (Table 2),
CAN. J. MICROBIOL. VOL. 38, 1992
TABLE 2. Inactivation of bacteriophage PM1 by whole cells and OM components of different A. hydrophila strains -
% of bacteriophage inactivation by:
LPS~
OMa
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Strain AH-3 (Se) AH-22 (R) Ba5 (S) AH-35 (R) AH-101 (S) AH-161 (R)
whole cells
Untreatedc
Treatedc 93.4 ntf 92.1 nt 92.6 nt
97.8
93.2
< 1.0
< 1.0
96.5 < 1.0 97.1 < 1.0
92.4 < 1.0 92.6 < 1.0
untreatedd 76.8
~reated~
< 1.0
< 1.0
nt
75.3 < 1.0 76.4 < 1.0
< 1.0 nt
< 1.0 nt
-
'OM solubilized with 1% DOC and 2 mM EDTA (Tomhs and Jofre 1985). b ~ u r i f i e dLPS (100 pg) (Osborn 1966). 'OM treated or untreated with proteinase K (10 pg/mL) for 2 h at 45°C. d ~ u r i f i e dLPS (100 pg) treated with equal volume of anti-LPS (serotype 0:34) serum for 1 h at 37OC. No phage inactivation was observed with anti-LPS serum treatment using other bacteriophages. eS and R, sensitive and resistant to PMI phage, respectively. 'nt, not tested.
1 2 3 4 FIG. 3. SDS-PAGE from A. hydrophila strains. Purified LPS Was assayed by the method of Tsai and Frasch (1982). Lane 1, strain AH-3 (S); lane 2, strain AH-22 (R); lane 3, strain Ba5 (S): lane 4, strain AH-35 (R). S and R, sensitive and resistant to phage PMl , respectively.
suggesting that phage resistance was due to an altered phage receptor. DOC-solubilized OM from A. hydrophila AH-3, Ba5, and AH-101 (wild types) was able to inactivate phage PM1, but DOC-solubilized OM from PM1-resistant mutants was unable to inactivate the phage (Table 2). DOC-solubilized OM always contained both LPS as well as OM proteins. When LPS was removed, DOC-solubilized OM no longer inactivated bacteriophage PM1; however, these conditions also denatured the OM proteins. When we removed OM proteins by proteinase K digestion, no differences were found in the ability to inactivate bacteriophage PM1 (Table 2). Furthermore, lo7 cells of A. hydrophila AH-3, Ba5, and AH-101 treated with specific anti-LPS serum (1:5 in PBS
pH = 7.4) for 1 h at 37°C were no longer able to inactivate bacteriophage P M l . Control experiments were performed treating the A. hydrophila cells with nonimmune serum, using the same conditions mentioned, and the cells were able to inactivate bacteriophage PM1. Finally, purified LPS from A. hydrophila AH-3, Ba5, and AH-101 inactivated bacteriophage PM1, but purified LPS from AH-22, AH-35, and AH- 161 (spontaneous PM 1-resistant mutants) was wi.thout effect. The degree of phage inactivation was proportional to the LPS concentration; 12.5 pg of LPS was required to reduce the phage titer to 50070 (Fig. 4). Furthermore, when LPS of A. hydrophila AH-3 or Ba5 was fractionated (Fig. 5), we found that the HMW-LPS fractions ( 0 antigen enriched) were able to fully inactivate bacteriophage P M l , while the LMW-LPS fractions (lipid A and LPS core enriched) were unable to do it. HMW-LPS fractions A, B, and C (see Fig. 5) were able to inactivate 70% of PM1 phages as compared with the complete LPS, while LMW-LPS fractions G to L (see Fig. 5) were completely unable to inactivate this bacteriophage. Fractions D, E, and F showed decreasing PM1 bacteriophage inactivation activity: 62, 45, and 25%, respectively. NO differences were found in the protein profile of the OM of A . hydrophila AH-3 and Ba5 (wild types) and their p ~ 1 - ~ ~mutants. ~ i ~ when t ~ examined ~ t by the method of Tsai and Frasch (1982), there was an apparent complete loss of the 0 antigen (HMW-LPS) in the purified LPS from the PM1-resistant mutants (AH-22 and AH-35) (Fig. 3), confirming the inactivation data of Table 2 and indicating that the 0 antigen was actually lost rather than chemically altered. Chemical analyses of purified LPS from A. hydrophila AH-3 and Ba5 (wild types) and one of their respective PM1-resistant mutants (Table 3) showed that LPS from the PM 1-resistant mutants (AH-22 and AH-35) was devoid of hexosamines. However, LPS from the PM 1-resistant mutants contained rhamnose, not normally found in the LPS of wild-type strains, as these strains are from the LPS core serotype 2 (Merino et al. 1989; Shaw and Hodder 1978). Also, LPS from the PM1-resistant mutants showed a decrease in the glucose content in comparison with the LPS from the wild-type strains, No D-heptose, galactose, or
MERINO ET AL.
TABLE3. Chemical composition of purified LPS from A . hydrophila strains (pmol/mg LPS)
Strain
K D O " . ~ ~ - ~ e ~ t o s Cilucoseb e
AH-3 (Sc) AH-22 (Rc) Ba5 (S) AH-35 (R)
hamn nose
~exosamines
0.018
0.31
1.57
0
0.41
0.038 0.019 0.037
0.96 0.31 0.97
1.46 1.55 1.47
0.45 0 0.39
0 0.43 0
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NOTE:The hexosamine content is mainly N-acetylgalactosamine. 'Assayed by colorimetric method. b ~ s s a y e dby gas-liquid chromatography. 'S, sensitive to bacteriophage PMI; R, resistant to bacteriophage PMI.
0
25
50
75
100
125
150
175
200
LPS, pg/mL FIG. 4. Inactivation of phage PM1 with purified LPS from A. hydrophila AH-3 (wild type), as determined by the method described in the text.
pentoses were detected on the LPS of the wild-type strains or the PM1 -resistant mutants. To identify the normal components of the 0-antigen repeating units, purified HMW-LPS as well as LMW-LPS fractions of the wild-type strains were analyzed. In all cases glucose and hexosamines (mainly N-acetylgalactosamine) were found as the main components of HMW-LPS fractions (a fraction enriched in 0-antigen polysaccharide chains), while no hexosamines were found in LMW-LPS fractions, the main components being KDO, ~-heptose,and glucose. Glucose appears to be a normal component of the HMW-LPS and LMW-LPS.
Discussion PM1 is a bacteriophage that can be classified in the Myoviridae family by Matthews (1980) and in group A2 by Ackermann (1973), according to morphological, physicochemical, and biological characteristics. The PM1-resistant mutants of A. hydrophila were unable to bind phage PM1 when assayed directly by binding to whole cells. The phage inactivation component was found in DOC-solubilized OM from the wild-type strains, indicating that the phage receptor is likely an OM component. Insensitivity to proteinase K digestion excluded OM proteins as PM1 receptors, and inactivation by anti-LPS serum implicated LPS as the receptor. Furthermore, purified LPS from wild-type strains was able to inactivate bacteriophage PM1, whereas LPS from strain AH-3, Ba5, and AH-101 PM1-resistant mutants (AH-22, AH-35, and AH- 161, respectively) could not.
ABCDE FGHIJKL FIG. 5. SDS-PAGE of A . hydrophila AH-3 LPS fractionation as described in Materials and methods. Lanes A , B, and C contained only HMW-LPS and were fully able to inactivate bacteriophage PM1; lanes G to L contained only LMW-LPS and were completely unable to inactivate bacteriophage PM1; and lanes D, E, and F showed both HMW-LPS and LMW-LPS, with increasing amounts of LMW-LPS and decreasing amounts of HMW-LPS showing decreasing PM1 bacteriophage inactivation activity.
In addition, HMW-LPS fractions from the wild-type strains (enriched in 0-antigen polysaccharide chains) inactivated bacteriophage PMI, whereas LMW-LPS fractions from the wild-type strains (devoid of 0-antigen repeating units) could not. The observation that LPS from the PM1-resistant mutants lacks 0 antigen is completely in agreement with the 0-antigen chains being the bacteriophage receptor; however; we cannot explain the higher relative electrophoretic moiety of the LPS core on these mutants versus the wild type despite the fact that chemically the LPS core seems to be rather complete (they only lack hexosamines from their LPS in comparison with the LPS from the wildtype strains). It appears that LPS 0-antigen oligosaccharides are the bacterial receptor sites for bacteriophage PM1. A similar situation was found for other bacteriophages, suchas FC3-1 from Klebsiella pneumoniae and P22 from Salmonella typhirnurium (Benedi et al. 1988), whose bacterial receptor is the 0-antigen polysaccharide fraction of the LPS. Hexosamines (mainly N-acetylgalactosamine) and glucose were shown to be components of the 0 antigen in A. hydrophila strains of serotype 0:34. The hexosamine component was absent and glucose was reduced from LPS of PM1-
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CAN. J. MICROBIOL. VOL. 38, 1992
resistant mutants concomitant with the disappearance of HMW-LPS. Glucose is also a component of the LPS oligosaccharide core of A. hydrophila strains from serotype 0:34 (Merino et al. 1989; Shaw and Hodder 1978). The LPS of PMl-resistant mutants appears to have gained rhamnose as part of its remaining core oligosaccharides, since rhamnose is not present in LPS from the wild-type strains. It may be that the addition of rhamnose prevents LPS completion with 0-antigen oligosaccharides in PM1-resistant mutants by preventing the insertion of 0 antigen to the LPS core. This explanation has been previously suggested for Klebsiella pneumoniae (Tomas and Jofre 1985). The PM1-resistant mutants still contained a LPS core oligosaccharide with all the components of LMW-LPS from the wild-type strains able to inactivate core LPS specific bacteriophages PM2 and 18. LPS is normally a potent endotoxin. It is interesting that PM1-resistant mutants, which were devoid of the 0 antigen, showed a LDSoin mice 100-fold higher than their respective wild types (data not shown). These phage-resistant mutants could also be useful for finding out more about the role of the 0 antigen in the pathogenesis of serotype 0:34 of A. hydrophila. Because of the narrow host range of bacteriophage PM1 (able to produce plaques only on strains of serotype 0:34), typical of bacteriophages in which the bacterial receptor is an 0 antigen (Makela 1985; Tomas et al. 1986), it would be of additional interest to use this phage as a diagnostic tool for determining clinical importance of A. hydrophila strains of serotype 0:34. Acknowledgements We thank T.J. Trust for bacteriophages 18, 24, 69, and 145. S.M. was supported by a fellowship from CIRIT (Generalitat de Catalunya). Ackermann, H. W. 1973. The morphology of bacteriophage. In Handbook of microbiology, CRC. Vol. 1. Edited by A.I. Laskin and H. Lechevalier. CRC Press, Boca Raton, FL. Adams, M.H. 1959. Bacteriophages. Interscience, New York. Ames, G.F.L., Spudich, E.N., and Nikaido, H. 1974. Protein composition of the outer-membrane of Salmonella typhimurium: effect of lipopolysaccharide mutations. J. Bacteriol. 117: 406-416. Benedi, V.J., Ciurana, B., and Tomas, J.M. 1988. A high molecular weight lipopolysaccharide specific bacteriophage for Klebsiella pneumoniae. Can. J. Microbiol. 34: 918-921. Brade, H. 1985. Occurrence of 2-keto-deoxyoctonic acid 5-phosphate in lipopolysaccharides of Vibrio cholerae Ogawa and Inaba. J . Bacteriol. 117: 795-798. Bradley, D.E. 1966. The fluorescent staining of bacteriophage nucleic acids. J. Gen. Microbiol. 44: 383-391. Ciurana, B., and Tomas, J.M. 1987. Role of lipopolysaccharide and complement in susceptibility of Klebsiella pneumoniae to non-immune serum. Infect. Immun. 55: 2741-2746. Dooley, J.S.G., Lallier, R., Shaw, D.H., and Trust, T.J. 1985. Electrophoretic and immunochemical analyses of the lipopolysaccharides from various strains of Aeromonas hydrophila. J. Bacteriol. 164: 263-269. Filip, C., Fletcher, G., Wulff, L.J., and Earhart, F. 1973. Solubilization of the cytoplasmatic membrane of Escherichia coli by the detergent sodium lauryl sarcosinate. J . Bacteriol. 115: 717-722. Freij, B. J . 1984. Aeromonas: biology of the organism and diseases in children. Pediatr. Infect. Dis. 3: 164-175.
Hancock, R.E.W., and Nikaido, H. 1978. Outer membranes of gram-negative bacteria. XIX. Isolation from Pseudomonas aeruginosa P A 0 1 and use in reconstitution and definition of the permeability barrier. J . Bacteriol. 136: 38 1-390. Janda, J.M., Clark, R.B., and Brenden, R. 1985. Virulence of Aeromonas species as assessed through mouse lethality. Curr. Microbiol. 12: 163-168. Karkhanis, Y.D., Zeltner, J.Y., Jackson, J.J., and Carlo, D.J. 1978. A new and improved microassay to determine 2-keto-3deoxyoctonate lipopolysaccharide of Gram-negative bacteria. Anal. Biochem. 85: 595-601. Kay, W.W., Buckley, J.T., Ishiguro, E.E., et al. 1981. Purification and disposition of a surface protein associated with virulence of Aeromonas salmonicida. J. Bacteriol. 147: 1077-1084. Lallier, R., Bernard, F., and Lalonde, G. 1984. Difference in the extracellular products of two strains of Aeromonas hydrophila virulent and weakly virulent for fish. Can. J. Microbiol. 30: 900-904. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. 1951. Protein measurement with the Folin phenol reagent. J . Biol. Chem. 193: 265-275. Makela, P.M. 1985. The use of bacteriophages and bacteriocins in the study of bacterial cell surface structures. In Enterobacterial surface antigens. Edited by T.K. Korhonen, E.A. Dawes, and P.H. Makela. Elsevier, New York. Maniatis, T., Fritsch, E.F., and Sambrook, J . 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Mattews, R.E.F. 1980. Classification de nomenclature des virus. Troisieme Report du Comite International de Taxonomies des Virus. Nasson, Paris. Merino, S., Benedi, V.J., and Tomas, J.M. 1989. Aeromonas hydrophila strains with moderate virulence. Microbios, 59: 165-173. Merino, S., Camprubi, S., and Tomas, J.M. 1990a. Isolation and characterization of bacteriophage PM2 from Aeromonas hydrophila. FEMS Microbiol. Lett. 68: 239-244. Merino, S., Camprubi, S., and Tomas, J.M. 1990b. Identification of the cell surface receptor for 18 bacteriophage from Aeromonas hydrophila. Res. Microbiol. 141: 173-180. Osborn, M.J. 1966. Preparation of lipopolysaccharide from mutant strains of Salmonella. Methods Enzymol. 8: 161-164. Regue, M., Tomas, J., Pares, R., and Jofre, J. 1981. Isolation and partial characterization of phages infecting Citrobacter intermedius C3. Curr. Microbiol. 5: 151-1 54. Shaw, D.H., and Hodder, J.A. 1978. Lipopolysaccharide of the motile aeromonads: core oligosaccharide analysis as an aid to taxonomic classification. Can. J. Microbiol. 24: 864-868. Tomas, J .M., and Jofre, J . 1985. Lipopolysaccharide-specific bacteriophage for Klebsiella pneumoniae C3. J. Bacteriol. 162: 1276- 1279. Tomas, J.M., Benedi, V.J., Ciurana, B., and Jofre, J. 1986. Role of the capsule and the 0-antigen in resistance of K. pneumoniae to serum bactericidal activity. Infect. Immun. 54: 85-89. Trust, T.J., and Sparrow, R.A.H. 1974. The bactericidal flora in the alimentary tract of fresh-water salmonid fish. Can. J. Microbiol. 20: 1219-1228. Trust, T.J., Ishiguro, E.E., and Atkinson, H.M. 1980. Relationship between Haemophilus piscium and Aeromonas salmonicida revealed by Aeromonas hydrophila bacteriophages. FEMS Microbiol. Lett. 9: 199-201. Tsai, C.M., and Frasch, C.E. 1982. A sensitive silver stain for detecting lipopolysaccharide in polyacrylamide gels. Anal. Biochem. 119: 115-119. Westphal, O., and Jann, K. 1965. Bacterial lipopolysaccharides: extraction with phenol-water and further applications of the procedure. Methods Carbohydr. Chem. 5: 83-91.
TOMAS'
Departarnento de Microbiologlh, Facultad de Biologia, Universidad de Barcelona, Diagonal 645, 08071 Barcelona, Spain Received May 28, 1991 Revision received September 9, 1991
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Accepted September 10, 1991 MERINO,S., CAMPRUBI, S., and TOMAS,J. M. 1992. Characterization of an 0-antigen bacteriophage from Aerornonas hydrophila. Can. J. Microbiol. 38: 235-240. A unique bacteriophage of Aerornonas hydrophila serotype 0:34 was isolated, purified, and characterized. The bacterial surface receptor was shown to be the 0-antigen polysaccharide component of lipopolysaccharide specific to serotype 0:34, which was chemically characterized. The high molecular weight lipopolysaccharide fraction (a fraction enriched in 0 antigen) was fully able to inactivate bacteriophage PMI. Phage-resistant mutants of A. hydrophila 0:34 were isolated and found to be specifically devoid of lipopolysaccharide 0 antigen. No other cell-surface molecules were involved in phage binding. The host range of bacteriophage PMl was found to be very narrow, producing plaques only on A. hydrophila strains from serotype 0:34. Key words:' Aerornonas hydrophila, serotype 0:34, bacteriophage P M l , lipopolysaccharide. MERINO,S., CAMPRUBI, S., et TOMAS,J. M. 1992. Characteri~ationof an 0-antigen bacteriophage from Aerornonas hydrophila. Can. J. Microbiol. 38 : 235-240. Nous avons isole, purifie et caracterise un bacteriophage unique d'Aerornonas hydrophila serotype 0:34. La caracterisation chimique a confirme que le recepteur de surface etait l'antigene polysaccharidique 0 associe au lipopolysaccharide specifique du serotype 0:34. La fraction lipopolysaccharidique qui a un poids moleculaire eleve (une fraction riche en antigene 0 ) pouvait inactiver completement le bacteriophage PMl. Nous avons isole des mutants d'A. hydrophilia 0:34 resistants au bacteriophage et confirme que ceux-ci etaient depourvus de I'antigene lipopolysaccharidique 0. Aucune autre molecule de la surface cellulaire n'etait impliquee dans la liaison avec le bacteriophage. Les bacteries-h8tes du bacteriophage PMl sont peu nombreuses. Ce phage produit des plages seulement en presence de souches d'A. hydrophila du serotype 0:34. Mots clks : Aerornonas hydrophila, serotype 0:34, bacteriophage PM1, lipopolysaccharide. [Traduit par la redaction]
Introduction Motile Aeromonas species are ubiquitous inhabitants of the aquatic environment and are also considered to be part of the normal microflora of the intestinal tract of fish (Trust and Sparrow 1974). Aeromonas hydrophila is an opportunistic as well as primary pathogen of a variety of aquatic and terrestrial animals, including humans; the clinical manifestations range from gastroenteritis to soft-tissue infections, including septicemia, and meningitis (Freij 1984). Aeromonas hydrophila strains are classified on the basis of virulence related to cell-surface components (Lallier et al. 1984; Janda et al. 1985). Strains with homogeneous 0 polysaccharide chains ( 0 antigen) both in their lipopolysaccharide (LPS) as well as S layers are highly virulent toward fish and mice (Lallier et al. 1984; Janda et al. 1985), whereas strains with heterogeneous LPS are considered avirulent (Dooley et al. 1985). We recently described A . hydrophila species belonging to serotype 0 : 3 4 with heterogeneous 0 polysaccharide chains, similar to strain Ba5, previously reported to be moderately virulent for fish (Lallier et al. 1984) and mice (Merino et al. 1989). It would be diagnostically beneficial to have a phage typing system for A . hydrophila species. In this regard we isolated and characterized bacteriophage PM 1, specific for A . hydrophila strains belonging to serotype 0:34, and we demonstrated that LPS 0 antigen is the specific receptor. We also characterized LPS from this serotype (0:34) as well as phage-resistant mutants useful for the study of virulence mechanisms in A . hydrophila 0 : 3 4 . ' ~ u t h o rto whom all correspondence should be addressed. Pr~ntedin Canada / lmprime au Canada
Materials and methods Bacteria, bacteriophages, and media The strains, their relevant properties and origins, as well as their sensitivity for bacteriophages P M l , PM2 (Merino et al. 1990a), and 18 (Merino et al. 1990b) are listed in Table 1. The A. hydrophila strains belonging to serotype 0:34 and their respective PM1-resistant mutants were also sensitive to bacteriophages 24, 69, and 145 (Trust et al. 1980), which were kindly provided by T. J. Trust. Tryptone-soya broth (TSB) was used for bacterial growth and phage propagation. TSB was supplemented with either 1.5% agar (w/v) (TSA) or 0.6% agar (TSA soft agar). For titration and inactivation assays, phage suspensions were diluted in phage buffer (10 mM MgSO,, 20 mM NaC1, and 50 mM Tris-HC1, pH 7.5). Spontaneous mutants of A. hydrophila strain AH-3 and strain Ba5 resistant to bacteriophage PMl were isolated by spreading a mixture containing about lo8 bacteria and lo9 phage plaqueforming units on TSA. After 48 h at 20°C, colonies of phageresistant mutants were picked and purified by streaking and were cross streaked against PMl to confirm resistance. Bacteriophage isolation Freshwater samples were centrifuged and the supernatant was incubated at 20°C with an exponential growth phase culture of A. hydrophila AH-3 on double-concentrated TSB. After overnight incubation, the bacteria were removed by centrifugation and filtration, and the supernatant was plated on A. hydrophila AH-3 by using the double agar layer method of Adams (1959). Plaques that formed on the plates were stabbed with a needle and eluted with a small volume of phage buffer. Each phage suspension was serially propagated twice on the same strain. General phase techniques and production of phage lysates The methods of Adams (1959) were used. The incubation temperature was 20°C, and the plates were incubated for 24 h. The
CAN. J . MICROBIOL. VOL. 38, 1992
TABLE1. Aeromonas hydrophila strains, their relevant characteristics, source, and bacteriophage sensitivity
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Sensitivity to: Strain
Relevant characteristics
AH-102 AH-103 TF7 LL1 AH-50 ATCC 7966 43014 17577 24963 31075 8973 31075 291 1 1480 38589
Serotype 0:34, smooth Isogenic PM1-resistant mutant from AH-3; rough Serotype 0:34, smooth Isogenic PM1-resistant mutant from Ba5; rough Serotype 0:34, smooth Isogenic PM1-resistant mutant from AH-101; rough Serotype 0:34, smooth Serotype 0:34, smooth Serotype 0:11, S layer Serotype 0:11, S layer Serotype 0:22, smooth Not serotyped, smooth Not serotyped, smooth Not serotyped, smooth Not serotyped, smooth Not serotyped, smooth Not serotyped, smooth Not serotyped, smooth Not serotyped, smooth Not serotyped, rough Not serotyped, rough
PM1
PM2
18
Source Our laboratory This work Our laboratory This work Our laboratory This work Our laboratory Our laboratory T.J. Trust T.J. Trust Our laboratory ATCC Clinical Clinical Clinical Clinical Clinical Clinical Clinical Clinical Clinical
NOTE:All the A. hydrophila strains not serotyped give a negative reaction against anti-0:34 or anti-0:ll serum. All the strains from clinical sources are from human origin. S, sensitive; R, resistant.
bacteriophage host range was assayed by spot tests. One-step Membrane proteins were analyzed by sodium dodecyl sulfate growth experiments were done using A . hydrophila AH-3 as the polyacrylamide gel electrophoresis (SDS-PAGE), using a modification (Ames et al. 1974) of the Laemmli procedure. Protein gels host and a multiplicity of infection of 1. Solvent inactivation (ether were routinely stained with Coomassie blue or silver reagent, and and chloroform), phage particle purification, buoyant densities, protein concentrations were determined by the Lowry procedure nucleic acid determination, and polypeptide analyses were performed as previously described (Merino et al. 1989). [ 3 ~ ~ h y r n i d i n e (Lowry et al. 1951), with bovine serum albumin as the standard. incorporation on the purified phage particles was measured as LPS was purified by the method of Westphal and Jann (1965), described by ReguC et al. (198 1). as modified by Osborn (1966). Subfractionation of purified LPS by column chromatography was performed according to Ciurana and Tomas (1987). Fractions were extensively dialyzed against Bacteriophage inactivation experiments distilled water, first at room temperature and then at 4"C, and Bacteriophages (lo3 pfu) were incubated for 20 min at 20°C analyzed by SDS-PAGE and silver stained by the method of Tsai with one of the following: 10' cells, 200 pg of 1% deoxycholate and Frasch (1982). (DOC) solubilized outer membrane (OM), 200 pg of 1% DOC For chemical analyses, purified LPS or LPS fractions were solubilized OM treated with phenol or with proteinase K (Tomas hydrolyzed with 1 M HCl for 2 h at 100°C. 2-Keto-3-deoxyand Jofre 1985), 200 pg of purified LPS (unless otherwise octulosonic acid (KDO) was released by hydrolysis with 4 M HCl indicated), or 100 pg of high molecular weight LPS (HMW-LPS) at 100°C for 30 min as described by Brade (1985), and the KDO ( 0 antigen enriched fraction) or 100 pg of low molecular weight released from LPS was assayed by the colorimetric method of LPS (LMW-LPS) (LPS core and lipid A enriched fraction). After Karkhanis et al. (1978) and by gas-liquid chromatography accordeach of the different treatments, the OM was extensively dialyzed ing to Brade (1985). Monosaccharides were also analyzed as their against distilled water for 24 h at room temperature. Chloroform alditol acetate derivatives by gas-liquid chromatography on a 3% (three or four drops) was added and mixed for 60 s, and the mixSP-3840 column (Supelco Inc.) at 225 and 180°C. Alditol acetate ture was centrifuged at 12 000 x g for 10 min at 4°C. The supermonosaccharides were obtained by the following derivatization pronatants were assayed directly on A . hydrophila AH-3. cedure (bulletin 774A, Supelco Inc., Cras, Switzerland). Samples (50 mg) were neutralized with an equal volume of 0.5 M NaBH, Cell-surface isolation and analyses for 60 min at room temperature and acetylated by the dropwise Cell envelopes were prepared by French pressure cell lysis at addition of glacial acetic acid until bubbling stopped. Dried samples 16 000 lb/in2 (1 lb/in2 = 6.9 kPa) of whole cells followed by the were washed twice with 0.1 mL of concentrated HCl per 100 mL removal of unbroken cells at 10 000 x g for 10 min and by of methanol and with ethanol. The supernatant was dried under sedimentation of the membrane fraction at 100 000 x g for 2 h. N,, suspended in equal volumes of pyridine and acetic anhydride, Cytoplasmic membranes were solubilized twice with sodium lauryl and heated at 100°C for 15 min, then analyzed by gas chromatogsarcosinate (Filip et al. 1973), and the OM fraction was sedimented raphy. Alditol acetate carbohydrate standards were either purtwice at 100 000 x g for 2 h. OM proteins were solubilized in 1% chased from Supelco or prepared by us. DOC - 2 mM EDTA (Tomas and Jofre 1985). To remove the LPS from the solubilized OM proteins, samples were treated with 88% Purified LPS or LPS fractions were further analyzed by SDSPAGE and silver stained by the method of Tsai and Frash (1982). phenol at 70°C, as described by Hancock and Nikaido (1978).
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MERINO ET AL.
FIG. 1. Characteristics of phage PM1. (A) Phage DNA was digested with different restriction enzymes. The resulting fragments were separated by electrophoresis through a 1% agarose gel for 6 h at 5 V/cm. Lane 0, X phage DNA digested with HindIII; lane 1, HindIII; lane 2, PstI; lane 3, Sari; lane 4, BgnI; lane 5, EcoRI; lane 6, BamHI; lane 7, EcoRV. (B) SDS-PAGE of bacteriophage PM1. ~ a n 1, e molecular size standards (14.4,21.5,31 .O, 45.0; 66.2, Fm. 2. Morphology of bacteriophage PMl negatively stained and 91.0 kDa) from Bio-Rad Laboratories; lane 2; ~ o l ~ ~ e ~ t i d e s and observed with an electron microscope Hitachi MT800. Bar of bacteriophage P M 1. represents 50 nm. Anti-LPS serum Immune serum was obtained as previously described using purified LPS from A. hydrophila AH-3 (serotype 0:34) or A. hydrophila TF7 (serotype 0 : 11) (Tomas et al. 1986). Molecular biological methods Standard techniques for manipulation of DNA were as described previously (Maniatis et al. 1982). Electron m icroscopy Purified bacteriophage were negatively stained with 1To phosphotungstic acid (pH 7.2) for 30 s, and examined using an Hitachi MT800 electron microscope.
Bacteriophage isolation and characterization We isolated bacteriophage PM 1 from one of five different freshwater samples. Phage PM1 gave clear plaques characteristically varying in size from 2 to 3 mm, with diffuse edges. Phage PM 1 incorporated ['HI thymidine in their nucleic acid and, therefore, are DNA based. Furthermore, Bradley's method (Bradley 1966) confirmed that phage PM 1 contained double-stranded DNA. Restriction enzymes PstI, SalI, and EcoRI were able to cleave bacteriophage PM1 DNA, whereas HindIII, BglII, BamHI, and EcoRV were without effect (Fig. 1A). Fourteen polypeptides were observed (108, 79, 51,48,45, 33, 32, 31,29,28,26, 24, 18, and 16 kDa) on SDS-PAGE; the three major ones were
33.0, 18.0, and 16.0 kDa (Fig. 1B). Neither chloroform nor ether caused any loss of infectivity. Bacteriophage PM1 had a buoyant density of 1.51 g/cm3, a latent period of 50 min, a rise period of 30 min, and a burst size of 170 pfu at 20°C. These phage presented a polyhedric contractile tail and a base plate with some tail fibers but no collar (Fig. 2). The host range of bacteriophage PM 1 was found to be very narrow, producing plaques only on A. hydrophila strains belonging to serotype 0:34 (Table 1). Furthermore, Aeromonas salmonicida A450 and A450- 1 (Kay et al. 198 1) and Vibrio anguillarum (serotype 01) strains were resistant. None of 20 strains of the Enterobacteriaceae genera tested, encompassing Enterobacter, Providencia, Proteus, Citrobacter, Serratia, Yersinia, Klebsiella, Escherichia, and Salmonella, was sensitive to bacteriophage PM1. P M I bacteriophage surface receptor Mutants resistant to bacteriophage PM 1 occurred at a frequency of 2 x 10 and fell into a single class on the basis of their LPS profile (Fig. 3). All of them (20 tested for strains AH-3 and Ba5) lacked the 0-antigen polysaccharide chains and were sensitive to bacteriophages PM2, 18, 24, 69, and 145 (data given for bacteriophages PM2 and 18 in Table 1). Phage PM1 adsorbed readily to A. hydrophila AH-3, Ba5, and AH-101 but not to the PM1-resistant mutants AH-22, AH-35, and AH-161, respectively (Table 2),
CAN. J. MICROBIOL. VOL. 38, 1992
TABLE 2. Inactivation of bacteriophage PM1 by whole cells and OM components of different A. hydrophila strains -
% of bacteriophage inactivation by:
LPS~
OMa
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Strain AH-3 (Se) AH-22 (R) Ba5 (S) AH-35 (R) AH-101 (S) AH-161 (R)
whole cells
Untreatedc
Treatedc 93.4 ntf 92.1 nt 92.6 nt
97.8
93.2
< 1.0
< 1.0
96.5 < 1.0 97.1 < 1.0
92.4 < 1.0 92.6 < 1.0
untreatedd 76.8
~reated~
< 1.0
< 1.0
nt
75.3 < 1.0 76.4 < 1.0
< 1.0 nt
< 1.0 nt
-
'OM solubilized with 1% DOC and 2 mM EDTA (Tomhs and Jofre 1985). b ~ u r i f i e dLPS (100 pg) (Osborn 1966). 'OM treated or untreated with proteinase K (10 pg/mL) for 2 h at 45°C. d ~ u r i f i e dLPS (100 pg) treated with equal volume of anti-LPS (serotype 0:34) serum for 1 h at 37OC. No phage inactivation was observed with anti-LPS serum treatment using other bacteriophages. eS and R, sensitive and resistant to PMI phage, respectively. 'nt, not tested.
1 2 3 4 FIG. 3. SDS-PAGE from A. hydrophila strains. Purified LPS Was assayed by the method of Tsai and Frasch (1982). Lane 1, strain AH-3 (S); lane 2, strain AH-22 (R); lane 3, strain Ba5 (S): lane 4, strain AH-35 (R). S and R, sensitive and resistant to phage PMl , respectively.
suggesting that phage resistance was due to an altered phage receptor. DOC-solubilized OM from A. hydrophila AH-3, Ba5, and AH-101 (wild types) was able to inactivate phage PM1, but DOC-solubilized OM from PM1-resistant mutants was unable to inactivate the phage (Table 2). DOC-solubilized OM always contained both LPS as well as OM proteins. When LPS was removed, DOC-solubilized OM no longer inactivated bacteriophage PM1; however, these conditions also denatured the OM proteins. When we removed OM proteins by proteinase K digestion, no differences were found in the ability to inactivate bacteriophage PM1 (Table 2). Furthermore, lo7 cells of A. hydrophila AH-3, Ba5, and AH-101 treated with specific anti-LPS serum (1:5 in PBS
pH = 7.4) for 1 h at 37°C were no longer able to inactivate bacteriophage P M l . Control experiments were performed treating the A. hydrophila cells with nonimmune serum, using the same conditions mentioned, and the cells were able to inactivate bacteriophage PM1. Finally, purified LPS from A. hydrophila AH-3, Ba5, and AH-101 inactivated bacteriophage PM1, but purified LPS from AH-22, AH-35, and AH- 161 (spontaneous PM 1-resistant mutants) was wi.thout effect. The degree of phage inactivation was proportional to the LPS concentration; 12.5 pg of LPS was required to reduce the phage titer to 50070 (Fig. 4). Furthermore, when LPS of A. hydrophila AH-3 or Ba5 was fractionated (Fig. 5), we found that the HMW-LPS fractions ( 0 antigen enriched) were able to fully inactivate bacteriophage P M l , while the LMW-LPS fractions (lipid A and LPS core enriched) were unable to do it. HMW-LPS fractions A, B, and C (see Fig. 5) were able to inactivate 70% of PM1 phages as compared with the complete LPS, while LMW-LPS fractions G to L (see Fig. 5) were completely unable to inactivate this bacteriophage. Fractions D, E, and F showed decreasing PM1 bacteriophage inactivation activity: 62, 45, and 25%, respectively. NO differences were found in the protein profile of the OM of A . hydrophila AH-3 and Ba5 (wild types) and their p ~ 1 - ~ ~mutants. ~ i ~ when t ~ examined ~ t by the method of Tsai and Frasch (1982), there was an apparent complete loss of the 0 antigen (HMW-LPS) in the purified LPS from the PM1-resistant mutants (AH-22 and AH-35) (Fig. 3), confirming the inactivation data of Table 2 and indicating that the 0 antigen was actually lost rather than chemically altered. Chemical analyses of purified LPS from A. hydrophila AH-3 and Ba5 (wild types) and one of their respective PM1-resistant mutants (Table 3) showed that LPS from the PM 1-resistant mutants (AH-22 and AH-35) was devoid of hexosamines. However, LPS from the PM 1-resistant mutants contained rhamnose, not normally found in the LPS of wild-type strains, as these strains are from the LPS core serotype 2 (Merino et al. 1989; Shaw and Hodder 1978). Also, LPS from the PM1-resistant mutants showed a decrease in the glucose content in comparison with the LPS from the wild-type strains, No D-heptose, galactose, or
MERINO ET AL.
TABLE3. Chemical composition of purified LPS from A . hydrophila strains (pmol/mg LPS)
Strain
K D O " . ~ ~ - ~ e ~ t o s Cilucoseb e
AH-3 (Sc) AH-22 (Rc) Ba5 (S) AH-35 (R)
hamn nose
~exosamines
0.018
0.31
1.57
0
0.41
0.038 0.019 0.037
0.96 0.31 0.97
1.46 1.55 1.47
0.45 0 0.39
0 0.43 0
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NOTE:The hexosamine content is mainly N-acetylgalactosamine. 'Assayed by colorimetric method. b ~ s s a y e dby gas-liquid chromatography. 'S, sensitive to bacteriophage PMI; R, resistant to bacteriophage PMI.
0
25
50
75
100
125
150
175
200
LPS, pg/mL FIG. 4. Inactivation of phage PM1 with purified LPS from A. hydrophila AH-3 (wild type), as determined by the method described in the text.
pentoses were detected on the LPS of the wild-type strains or the PM1 -resistant mutants. To identify the normal components of the 0-antigen repeating units, purified HMW-LPS as well as LMW-LPS fractions of the wild-type strains were analyzed. In all cases glucose and hexosamines (mainly N-acetylgalactosamine) were found as the main components of HMW-LPS fractions (a fraction enriched in 0-antigen polysaccharide chains), while no hexosamines were found in LMW-LPS fractions, the main components being KDO, ~-heptose,and glucose. Glucose appears to be a normal component of the HMW-LPS and LMW-LPS.
Discussion PM1 is a bacteriophage that can be classified in the Myoviridae family by Matthews (1980) and in group A2 by Ackermann (1973), according to morphological, physicochemical, and biological characteristics. The PM1-resistant mutants of A. hydrophila were unable to bind phage PM1 when assayed directly by binding to whole cells. The phage inactivation component was found in DOC-solubilized OM from the wild-type strains, indicating that the phage receptor is likely an OM component. Insensitivity to proteinase K digestion excluded OM proteins as PM1 receptors, and inactivation by anti-LPS serum implicated LPS as the receptor. Furthermore, purified LPS from wild-type strains was able to inactivate bacteriophage PM1, whereas LPS from strain AH-3, Ba5, and AH-101 PM1-resistant mutants (AH-22, AH-35, and AH- 161, respectively) could not.
ABCDE FGHIJKL FIG. 5. SDS-PAGE of A . hydrophila AH-3 LPS fractionation as described in Materials and methods. Lanes A , B, and C contained only HMW-LPS and were fully able to inactivate bacteriophage PM1; lanes G to L contained only LMW-LPS and were completely unable to inactivate bacteriophage PM1; and lanes D, E, and F showed both HMW-LPS and LMW-LPS, with increasing amounts of LMW-LPS and decreasing amounts of HMW-LPS showing decreasing PM1 bacteriophage inactivation activity.
In addition, HMW-LPS fractions from the wild-type strains (enriched in 0-antigen polysaccharide chains) inactivated bacteriophage PMI, whereas LMW-LPS fractions from the wild-type strains (devoid of 0-antigen repeating units) could not. The observation that LPS from the PM1-resistant mutants lacks 0 antigen is completely in agreement with the 0-antigen chains being the bacteriophage receptor; however; we cannot explain the higher relative electrophoretic moiety of the LPS core on these mutants versus the wild type despite the fact that chemically the LPS core seems to be rather complete (they only lack hexosamines from their LPS in comparison with the LPS from the wildtype strains). It appears that LPS 0-antigen oligosaccharides are the bacterial receptor sites for bacteriophage PM1. A similar situation was found for other bacteriophages, suchas FC3-1 from Klebsiella pneumoniae and P22 from Salmonella typhirnurium (Benedi et al. 1988), whose bacterial receptor is the 0-antigen polysaccharide fraction of the LPS. Hexosamines (mainly N-acetylgalactosamine) and glucose were shown to be components of the 0 antigen in A. hydrophila strains of serotype 0:34. The hexosamine component was absent and glucose was reduced from LPS of PM1-
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resistant mutants concomitant with the disappearance of HMW-LPS. Glucose is also a component of the LPS oligosaccharide core of A. hydrophila strains from serotype 0:34 (Merino et al. 1989; Shaw and Hodder 1978). The LPS of PMl-resistant mutants appears to have gained rhamnose as part of its remaining core oligosaccharides, since rhamnose is not present in LPS from the wild-type strains. It may be that the addition of rhamnose prevents LPS completion with 0-antigen oligosaccharides in PM1-resistant mutants by preventing the insertion of 0 antigen to the LPS core. This explanation has been previously suggested for Klebsiella pneumoniae (Tomas and Jofre 1985). The PM1-resistant mutants still contained a LPS core oligosaccharide with all the components of LMW-LPS from the wild-type strains able to inactivate core LPS specific bacteriophages PM2 and 18. LPS is normally a potent endotoxin. It is interesting that PM1-resistant mutants, which were devoid of the 0 antigen, showed a LDSoin mice 100-fold higher than their respective wild types (data not shown). These phage-resistant mutants could also be useful for finding out more about the role of the 0 antigen in the pathogenesis of serotype 0:34 of A. hydrophila. Because of the narrow host range of bacteriophage PM1 (able to produce plaques only on strains of serotype 0:34), typical of bacteriophages in which the bacterial receptor is an 0 antigen (Makela 1985; Tomas et al. 1986), it would be of additional interest to use this phage as a diagnostic tool for determining clinical importance of A. hydrophila strains of serotype 0:34. Acknowledgements We thank T.J. Trust for bacteriophages 18, 24, 69, and 145. S.M. was supported by a fellowship from CIRIT (Generalitat de Catalunya). Ackermann, H. W. 1973. The morphology of bacteriophage. In Handbook of microbiology, CRC. Vol. 1. Edited by A.I. Laskin and H. Lechevalier. CRC Press, Boca Raton, FL. Adams, M.H. 1959. Bacteriophages. Interscience, New York. Ames, G.F.L., Spudich, E.N., and Nikaido, H. 1974. Protein composition of the outer-membrane of Salmonella typhimurium: effect of lipopolysaccharide mutations. J. Bacteriol. 117: 406-416. Benedi, V.J., Ciurana, B., and Tomas, J.M. 1988. A high molecular weight lipopolysaccharide specific bacteriophage for Klebsiella pneumoniae. Can. J. Microbiol. 34: 918-921. Brade, H. 1985. Occurrence of 2-keto-deoxyoctonic acid 5-phosphate in lipopolysaccharides of Vibrio cholerae Ogawa and Inaba. J . Bacteriol. 117: 795-798. Bradley, D.E. 1966. The fluorescent staining of bacteriophage nucleic acids. J. Gen. Microbiol. 44: 383-391. Ciurana, B., and Tomas, J.M. 1987. Role of lipopolysaccharide and complement in susceptibility of Klebsiella pneumoniae to non-immune serum. Infect. Immun. 55: 2741-2746. Dooley, J.S.G., Lallier, R., Shaw, D.H., and Trust, T.J. 1985. Electrophoretic and immunochemical analyses of the lipopolysaccharides from various strains of Aeromonas hydrophila. J. Bacteriol. 164: 263-269. Filip, C., Fletcher, G., Wulff, L.J., and Earhart, F. 1973. Solubilization of the cytoplasmatic membrane of Escherichia coli by the detergent sodium lauryl sarcosinate. J . Bacteriol. 115: 717-722. Freij, B. J . 1984. Aeromonas: biology of the organism and diseases in children. Pediatr. Infect. Dis. 3: 164-175.
Hancock, R.E.W., and Nikaido, H. 1978. Outer membranes of gram-negative bacteria. XIX. Isolation from Pseudomonas aeruginosa P A 0 1 and use in reconstitution and definition of the permeability barrier. J . Bacteriol. 136: 38 1-390. Janda, J.M., Clark, R.B., and Brenden, R. 1985. Virulence of Aeromonas species as assessed through mouse lethality. Curr. Microbiol. 12: 163-168. Karkhanis, Y.D., Zeltner, J.Y., Jackson, J.J., and Carlo, D.J. 1978. A new and improved microassay to determine 2-keto-3deoxyoctonate lipopolysaccharide of Gram-negative bacteria. Anal. Biochem. 85: 595-601. Kay, W.W., Buckley, J.T., Ishiguro, E.E., et al. 1981. Purification and disposition of a surface protein associated with virulence of Aeromonas salmonicida. J. Bacteriol. 147: 1077-1084. Lallier, R., Bernard, F., and Lalonde, G. 1984. Difference in the extracellular products of two strains of Aeromonas hydrophila virulent and weakly virulent for fish. Can. J. Microbiol. 30: 900-904. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. 1951. Protein measurement with the Folin phenol reagent. J . Biol. Chem. 193: 265-275. Makela, P.M. 1985. The use of bacteriophages and bacteriocins in the study of bacterial cell surface structures. In Enterobacterial surface antigens. Edited by T.K. Korhonen, E.A. Dawes, and P.H. Makela. Elsevier, New York. Maniatis, T., Fritsch, E.F., and Sambrook, J . 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Mattews, R.E.F. 1980. Classification de nomenclature des virus. Troisieme Report du Comite International de Taxonomies des Virus. Nasson, Paris. Merino, S., Benedi, V.J., and Tomas, J.M. 1989. Aeromonas hydrophila strains with moderate virulence. Microbios, 59: 165-173. Merino, S., Camprubi, S., and Tomas, J.M. 1990a. Isolation and characterization of bacteriophage PM2 from Aeromonas hydrophila. FEMS Microbiol. Lett. 68: 239-244. Merino, S., Camprubi, S., and Tomas, J.M. 1990b. Identification of the cell surface receptor for 18 bacteriophage from Aeromonas hydrophila. Res. Microbiol. 141: 173-180. Osborn, M.J. 1966. Preparation of lipopolysaccharide from mutant strains of Salmonella. Methods Enzymol. 8: 161-164. Regue, M., Tomas, J., Pares, R., and Jofre, J. 1981. Isolation and partial characterization of phages infecting Citrobacter intermedius C3. Curr. Microbiol. 5: 151-1 54. Shaw, D.H., and Hodder, J.A. 1978. Lipopolysaccharide of the motile aeromonads: core oligosaccharide analysis as an aid to taxonomic classification. Can. J. Microbiol. 24: 864-868. Tomas, J .M., and Jofre, J . 1985. Lipopolysaccharide-specific bacteriophage for Klebsiella pneumoniae C3. J. Bacteriol. 162: 1276- 1279. Tomas, J.M., Benedi, V.J., Ciurana, B., and Jofre, J. 1986. Role of the capsule and the 0-antigen in resistance of K. pneumoniae to serum bactericidal activity. Infect. Immun. 54: 85-89. Trust, T.J., and Sparrow, R.A.H. 1974. The bactericidal flora in the alimentary tract of fresh-water salmonid fish. Can. J. Microbiol. 20: 1219-1228. Trust, T.J., Ishiguro, E.E., and Atkinson, H.M. 1980. Relationship between Haemophilus piscium and Aeromonas salmonicida revealed by Aeromonas hydrophila bacteriophages. FEMS Microbiol. Lett. 9: 199-201. Tsai, C.M., and Frasch, C.E. 1982. A sensitive silver stain for detecting lipopolysaccharide in polyacrylamide gels. Anal. Biochem. 119: 115-119. Westphal, O., and Jann, K. 1965. Bacterial lipopolysaccharides: extraction with phenol-water and further applications of the procedure. Methods Carbohydr. Chem. 5: 83-91.
