FEMS Microbiology Letters 68 (1990) 239-2,t0, 239 Published by Elsevier

Isolation and characterization of bacteriophage PM2 from Aeromonas hydrophila

Susana Mer ino , Si lvia C a m p r u b i a n d J u a n M. T~n::_~s

Deparlamento dc Mwrohiologia. UnH'ersidad de Barcelona. Barcelona. Spare

Received 27 November 1989 Accepted 7 December 1989

Key words: Aeromonas; Bacteriophage PM2; Lipopolysaccharide

1. SUMMARY

PM2 is an Aeromonas-specific bacteriophage isolated on A. hydrophila strain AH-3. The bacteriophage receptor for this phage was found to be the lipopolysaecharide (LPS), specifically a low-molecular weight LPS fraction (LPS-¢ore oligosaecharides). Mutants resistant ro this phage were isolated and found to be devoid of LPS O-antigen and altered in the LPS-~:ore. No other outer-membrane (OM) molecules appeared to be involved in phage binding.

2. INTRODUCTION

r,lotile Aeromonas spp. are an ubiquitous com- ponent of the aquatic environment [1] and are also considered to be normal in habitants of the in- testinal tract of fish [2]. Aeromonas hydrophda is an opportunistic, a~ well as primary pathogen of a variety of aquatic and terrestrial an'~mals as v.ell as humans [3]. The clinical manifestation of A. hy- drophila infections range from gastroenteritis to

Corresptmdence to: J.M. Tom/ts. Departamento de Mi~to~,ic~ Iogia. Universidad de Bercelona. Diagonal 645. 08071 Barcelona. Spain.

soft tissue infections, septicemia, and meningitis 14.51. The surface characteristics of A. hydrophila correlate with changes in virulence and can be used to classify strains 16,7], We have r~oently described a group of A ~;.g-c, phda strains belong- ing to ~ ro type 034 with heterogeneous LPS O- polysaccharide chains, which previously were re- ported to be modest in their virulence for fish and mice 18.91.

In this paper we report on the isolation and characterization of bacteriophage PM2. isolated on A. t~vdrophila AH-3 (serotype 034t which is able to form plaques on strains of this serot?.1~e and many other A. l~1"drophtta. A. sobrta and A. salmonicida strains. Spontaneous phage PM2 re- sistant mutants lack the LPS O-antigen polysac- charide chains and we demonstrate that LPS alone is the bacter ial surface receptor for this bacteriophage. Furthermore. we show that a low- molecular weight LPS (which is enriched in LPS- core ol igosaecharides) is involved in PM2 bacteriophage reception.

3. MATERIALS A N D METHODS

3.1. Bacteria, bacteriophages and media The strains, their relevant properties, origirL~,

and phage sensitivities are listed in Table 1.

0378-1097/qO/$03.50 q 1990 FederaTion of European Microbiological Socieues

240

Bacteriophage Aeh-I was kindly provided by Dr. H.W. Ackermann [10l. Tryptone-soya-broth (TSB) was used for bacterial growth and phage propa- gation. TSB was supplemented with 1.5% agar (w/v) (TSA) and TSA soft-agar with 0.6% agar. For titration and inactivation assays, phage sus- pensions were diluted in phage buffer [11].

Spontaneous phage resistant mutants of A. hy- drophila AH*3 or Ba5 were isolated, indepen- dently for each strain, by spreading a mixture containing l0 s bacteria and 109 pfu of the phages on TSA. After 48 h at 20oC individual colonies were purified by streaking and cross-streaked against the bacteriophage to confirm resistance. Resistance to other bacteriophages was assayed by spot test.

3.Z Bacteriophage isolation Freshwater samples were centriluged and the

superuatant was incubated at 20~C :vith an ex- ponential growth-phase culture of A. hydrophila AH-3. After overnight incubation, the bacteria were removed by centrifugation and filtration, and the supematant was plated en ,4. hydrophila AH-3 by using the double agar layered method of Adams [12]. Plaques which 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.

3.3. General phage ~eLAn;q,es and production of phage lysates

The methods of Adams [12] were use, d. The incubation temperature was initially 200C, and the plates were incubated 24 h. The bacteriophage host range was assayed by spot test. One-step growth experiments were performed using A. hy- drophila AH-3 as host at a multiplicity of infec- tion of 1. Solvent inactivation (ether and chloro- form) of phages, as well a~ phage particle purifica- tion, determination of the buoyant density, de- termination of the nucleic acid type or the number of polypeptides were carried out as previously described [131.

3.4. Phage inactivation experiments Bacteriophages (103 pfu) were incubated with

either 107 bacterial cells. 200 pg of 1% (w/v)

deoxycholate (DOC)-solubilized OM proteins with or without treatment wit~ 1• , g of proteinase K [14]. 100/tg of purified I,PS. 100 ttg of purified LPS with equal volume of anti-LPS serum (diluted 1/10 in PBS), or 50 /zg of pooled high or low- molecular weight LPS fractions. Chloroform (2-3 drops) was added and the suspension was mixed for 1 rain. The mixture was centrifuged at 12 bOO × g for !0 rain at 4°C. The supernatants were assayed for phage aclivity directly on A. hydra- phila AH-3.

3.5. Cell surface isolation and analyses Cell envelopes and outer membranes were pre-

pared as previously described and analyzed by SDS-PAGE [14].

LPS was purified by the method of Westphal and Jann [15l as modified by Osbom [16]. Sub- fractionation of purified LPS by column chro- matography was performed as we described previ- ously [17].

Fractions were extensively dialyzed against dis- tilled water, first at room temperature and then at 4°C, analyzed by SDS-PAGE and silver stained by the method of Tsai and Fraseh [18].

For chemical analysis, purified LPS was hydra a,ysed with 1 M HCI for 2 h at 105oC. Colorimet- tic a.-~lyses of the deoxy-D-mannooctulosonic acid (KDO) content of LPS was performed by the method of Karkhanis et al. [19]. Monosaccharides were also analysed as their alditol z_eetate deriva- tives by gas-liquid chromatography (GLC) on a 3% SP-~840 column (Supelco) at 225°C and 180oC. Alditol acetate _-'~rhohvdrate standards were either purchased from Supclco or prc},,t,e,d by us.

3.6. Anti-LPS serum Immune serum was obtained from adult New

Zealand white rabbits injected with purified LPS from ,4 hydrot, hi!a AH-3 as previously describe~ I141.

4. RESULTS AND DISCUSSION

4 I. Bacteriophage isolation and characterization Bacteriophage PM2 was isolated from one of

three freshwater samples tested. Phage PM2 gave

Table 1

Bacterial strains used

241

Strains of Relevant characteristics

Sensitivity to

PM2 Aehl

Sollrc~

,4. hydrophila AH-3 0M,, S-layer - + AH-35 isogenic mutant derived

from AH-3. rough Ba5 034, S-layer- + AH-54 isogenic mutant derived

from Ba5, rough AH-I01 034. S-layer - , AH-199 isogenic mutant derived

from AH-101, rough - ALL50 022. S-layer- + AH-10 rough, S-layer- + ATCC 7¢66 not serotyped. S-layer - + 24830 not serotyped, S-layer- * 24963 not serotyped. S.-laye,- I 25616 not ~roG',qped, S-layer + TF7 011, S-layer + - AH-45 isogenic mutant derived

from TF7:011, S-layer + A|I-21 isogenic mutant derived

from TF7: rough, S-layer- +

A ,'rd~rtJ AS-28 0l 1. S-layer + -

AH-, 198 isogenic mutant derived from AS-28; 01 l, S-layer +

19975 rough, S-layer- +

A. salmt~mclda A 450 smooth, A-layer + + A 450-3 isogenic mutant derived

from A 450; smooth. A-layer - +

A 450-1 isogenic mutant derived from A 450: rough. A-layer - +

+ IS]

- thi~ work + 19]

- thi~, v. t)rk + [81

- this ~ork + our lab + our lab 4- ATCC + clinical -- ~.iiail.al - clinical - 191

- o u r lab

- our lab

- 161

- our lab - clinical

+ 1241

+ 1241

+ i241

! . c,mshive: -,resistant. Note: All the Aeromona~ >,~;.~ ~'-t ~rotvped do not belong to serolypes 011 or 0.~4.

clear plaques, bo th at 2 0 ° C and 370C, wi th a

range of sizes up to 3 m m with diffuse edges.

Phage PM2 incorpora ted 3H- thymid ine in to its

nucleic acid, which is therefore D N A . Fur ther - more, us ing Bradley 's me thod [20] we conf i rmed

tha t phage PM2 has doub le s : randed D N A . Also,

certain restr ict ion enzymes ( E c o R l , Sa i l , H i n d l l l

and B a m H l ) were able :o cleave PM2 bacterio-

phage D N A . Bacter iophage PM2 produced 15

polypept ides , there be ing three major pro te ins of

56.0. 42.0 and 18.> K ~ , . N~itFcr ch loroform nor

ether caused any loss of F'+J2 inlecfi+~: 7 Also,

bac te r iophage PM2 showed a buoyant densi ty o f

1.51 g / c m ~, a la tent per iod o f 30 rain, a rise

per iod of 20 rain and a burst size o f 290 pfu.

Bacteri¢ h~ge PM2 shows a polyhedric head with

a collar, • .ntractile tail and a base plate with at

least sL': spikes (Fig. 1 ). This bacter iophage seems

very s imi lar to the well characterized T-even E.

coli bacter iophages. Bacteriophage PM2 has a

242

Fig. l. Electron micrography of purified bacteriophage PM2 negatively stained with 17o phosphotungstic acid (bar = 100

nm).

broad host range in that it is able to produce plaques on different Aeromonas strains. For ex- ample PM2 is able to produce plaques on differ- ent serotypes of A. hydJophila strains, on A. sobria and on A. salmonicida strains (Table 1). Vibrio anguillarum (serotypes 01 and 02) strains and all Enterobacteriaceae tested were resistant to this bacteriophage.

Thus, PM2 is a bacteriophage that can be clas- sifted in the Myoviridae family by Matthews [21] and in group A2 of Ackermaan [22] according to their morphological physico-chemical and bio- logical characteristics.

4.2. Bacteriophage surface zeceptor Mutants resistant to bacteriophage PM2 oc-

cuffed at a frequency of about 3 x 10 ~ and fell into a s in~e class based on their phage sensitivi- ties and LPS profiles. All mutants (20 tested for A. hydr~phila strains AH-3 and Ba5) lacked the O-antigen polysaccharide chains, showed a higher electrophoresis migration rate of their LPS-core oligosaccharides and they were resistant to bacteriophage Ach-1.

Phage PM2 adsorbed readily to A. hydrophila AH-3 or Ba5 but not to the PM2-resistant mutants AH-53 and AH-54, respectively (Table 2) suggest- ing that phage resistance was due to an altered phage receptor. IX)C-solubilized OM fro~.a A. hydrophila AH-3 or Ba5 (wildqypes) and from PM2-resistant mutants showed similar phage ad- sorption, as did the corresponding whole cells (Table 2). Proteinase K digestion did not alter the ability of DOC-solubilized OM from the wild type strains to inactivate bacteriophage PM2 (Table 2). Furthermore, if we treated the whole cells of the wild t )pe strains (AH-3 or Ba5) with s~ecific anti-LPS serum for 1 h at 3 7 ° C and remo,,ed the serum by centrifugation, the treated cells were no longer able to inactivate bacteriophage PM2.

Table 2

Inactivation of bac|criophage PM2 by whole cells and OM components

.4. hydrophila % bacteriophage inactivation by

strains Whole OM " OM with LPS c HMW-LPS u LMW-LPS a cells proteinase K

treatment b

AH-3 98.3 97.6 %.8 77.2 < 2.0 75.8 AH-53 < 1.0 < 1.0 NT • < 1.0 NT NT Ba5 97.8 97.2 96.8 78.3 < 2.0 76.9 AH-54 < 1.0 < 1.0 NT < 1.0 NT NT

* OM solubilized with 19~ DO(7 and 2 mM EDTA [14]. h Treatment with proteinase K (10/+g/rid) for 2 h at 45°C. ¢ Purified LPS (100 p.g) [15,16]. d Pooled high-molecular weight LPS fractions and pooled low-molecular weight LPS fracP.ons (50 pg) as it is shown in Fig. 2. e NT, not tested.

A B ! '

1 2 3 4 5 6 I 2 3 Fig. 2. SDS-PAGE of LPS from ,4 hydrophda strains. (A) Lanes: I, strain AH-3: 2, strain AH-53: 3, strain BaS: 4. strain AH-54: 5. strain AH-IOI: and 6, strain AH-199 (B) Lanes: ]. LPS from strain AH-3: 2. Iowmolecular weight LPS from strain AH-3 subfractionated according to materials and meth-

ods: and 3. high-molecular weight LPS from strain AH-3.

Purified LPS f rom .4. hydrophila AH-3 or Ba5 was able to inactivate bacter iophage PE'2 , but purif ied LPS from AH-53 or AH-54 (spontaneous PM2-resistant mutants ) was not. The degree of phage inactivat ion was directly related to the LPS concent ra t ion in the incubat ion mixture (1.5 fig of LPS was required to reduce the phage titer in 50%). LPS fractions conta in ing low-molecular weight AH-3 LPS (enriched on LPS-core oligosac- charide~XFig. 2) were able to inactivate bacterio- phage PM2, while LPS fractions from the wild type strains conta in ing high-molecular ,-'eight LPS (enriched on O-antigen chains) (Fig. 2) were not inhibitory (phage inactivation in all ca:,=.s < 1%).

Table 3

Chemical compo.~iti~;, of purified LPS from A. hvdrophda strains

243

We examined the protein composi t ion of the OM~ ~f ,I. h)J, uyhila stratus AH-3 and BaS. and their respective PM2-re~istant mutants . N o main difference were found in the O M protein profiles (da ta not shown). We also examined purified LPS from the wild type strains and the phage resistant mutan t s by the method of Tsai and Frasch [18]. There was an apparen t complete loss of the H M W - L P S (O-ant igen) and a change on the rela- tive electrophoretic mobili ty of the LMW-LPS (LPS-c.ore) on the purified LPS from the PM2-re- sistant mutan t s (Fig. 2), conf i rming the inactiva- tion da ta of Table 2.

Chemical analyses of purified LPS from A. hydrophila AH-3 and BaS, and their respective phage-resis tant mutan t s is shown on Table 3. A reduct ion of glucose and a complete loss of hexosamines in the purif ied LPSs of the mutan t strains was observed, which can be correlated with the changes observed on SDS-PAGE. This reduc- tion in glucose may explain the alteration in the electrophoret ic moiety of the LMW-LPS on the mutants , because these strains belong to type 2 accord ing to Shaw and Hodder [23). and in this type glucose is a componen t of the LPS core. Also. it seems clear that hexosamines are components of the O-ant igen LPS in these strains because t h ~ were lost on the LPSs of the mutan t strains lack- ing HMW-LPS.

Thus it is apparen t that the I.~w-molecu!C.~ weight LPS (LPS core oligosa,~,:harides) alone is the receptor for bacter iophage P~'2. and sponta- neous phage-resistant mutan ts unable to bind this phage iack the O-antigen poly-sacchande chains. As this bacter iophage ~howed a broad host range

LPS from Amount ( #tool/rag of LPS) of: A. [(vdrophda KDO a L-hep- D-hep- Pen- Glu- Galac- Rham- Hexo~.,~- strain tosa b rosa h lOSe b co~ h lose b no~ ~ mlne~ ~

AH-3 0.018 0.31 0 0 1.57 0 0 041 AH-53 0.051 1.25 0 0 1 32 0 0 0 Ba5 0.019 0.31 0 0 1.55 0 0 043 AH-54 0.050 1.26 0 0 1.29 0 0 0

a Assayed by colorimetric method [19]. b Assayed by ggs-liqaid chromatography.

244

on Aeromonas spp., the isolat ion of PM2- re s i s t an t m u tan t s f r o m d i f fe ren t Aeromonas s t ra ins is a useful tool for o b t a i n i n g isogenic L P S m u t a n t s l ack ing an O-ant igen . T h e avai lab i l i ty o f such isogenic m u t a n t s will al low us to s t udy the cont r i - bu t ion of the O-an t igen to the v i ru lence of .4 eromonas.

A C K N O W L E D G E M E N T S

W e thank H . W . A c k e r m a n n for bac t e r i ophage A a k 1 • ' . . . . . . . ~o.,~,, J~ a J tx ip ien t o f a s tuden t g ran t f r o m C. I .R . I .T . (Gene ra l i t a t de Ca ta lunya) .

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