Microbial Pathogenesis 1990; 8: 393-402

Lysogenization of Salmonella choleraesuis by phage14 increases average length of 0-antigen chains,serum resistance and intraperitoneal mouse virulence

Ndubisi A. Nnalue,' ,2 * Salete Newton' and B. A. D . Stocker'

'Department of Microbiology and Immunology, Stanford University School of Medicine,Stanford, California 94305, U.S.A . and 2Karolinska Institute, Department of Clinical Bacteriology,Huddinge University Hospital, S-14186, Huddinge, Sweden

(Received March 9, 1990 ; accepted March 15, 1990)

Nnalue, N. A. (Dept of Microbiology and Immunology, Stanford University School of Medicine,Stanford, California 94305, U.S .A.), S. Newton and B. A. D . Stocker. Lysogenization ofSalmonella choleraesuis by phage 14 increases average length of 0-antigen chains, serumresistance and intraperitoneal mouse virulence . Microbial Pathogenesis 1990; 8: 393-402 .

Three clones from a strain of Salmonella choleraesuis (serogroup C,) were lysogenized withphage 14 (P14) which converts the 0-antigen of serogroup C, salmonellae from 0-6,7 to 0-6,7,14. The lysogens were compared with their parental non-lysogenic clones with respect tothe following properties : average length of 0-antigen polysaccharide chains, sensitivity tonormal human serum, and mouse-virulence . SDS-polyacrylamide gel electrophoresis of lipo-polysaccharides extracted from these bacteria showed that samples from lysogens consistedmainly of long-chained molecules whereas those from non-lysogens contained mainly short-chained molecules . The 0-antigen polysaccharide from a lysogen was estimated by chemicalanalysis to be six times as long as that from a non-lysogen . Lysogens were serum-resistantwhereas non-lysogens were serum-sensitive . About 10 times more colony forming units of alysogen than of a non-lysogen were recovered from the livers and spleens of mice on day 1and 3 after intraperitoneal inoculation of equal doses . By comparison with S. choleraesuis,lysogenization of S. typhimurium with phage P22 or phage A4 did not affect the chain-lengthdistribution of 0-antigen polysaccharide . Our data suggest that phage 14-coded determinantsincrease efficiency of O-antigen biosynthesis in S . choleraesuis leading to increase in averagelength of 0-polysaccharide chains . Increased serum resistance and mouse virulence are logicalconsequences of increase in average length of C-polysaccharide chains and represent phage-conferred selective advantage not previously described in Salmonella.

Key words: Salmonella lipopolysaccharide ; phage-conferred selective advantage ; mouse viru-lence; serum resistance ; 0-antigen increase .

Introduction

The lipopolysaccharide forms a protective layer on the surface of Gram-negativebacteria and is involved in the interaction of the organism with its environment . Amongsalmonellae the outer segment of the lipopolysaccharide (LPS), the 0-antigen, is the

basis for classification into serogroups . Individual serogroups are defined by specificdeterminants or factors in the 0-antigen that elicit agglutinating antibodies in animals .

Determinants in the 0-antigen also serve as receptors for bacteriophages . Several

`Author to whom correspondence should be addressed . Present address : Department of MedicalMicrobiology, Faculty of Medicine and Health Sciences, Tawam Hospital, P .O. Box 15258, Al Ain, UnitedArab Emirates .

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© 1990 Academic Press Limited

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temperate phages of Salmonella can alter the antigenic properties of their host organism,usually with the appearance of new factors in the 0-antigen . This phenomenon, knownas lysogenic conversion (or antigenic conversion), has been recognized for at least 30years and generally involves either alteration of existing glycosidic linkages orintroduction of new ones by phage-specified enzymes .` It is usually accompanied byloss of receptor function for the same phage by the 0-antigen .Among the best-known converting phages is P22 which lysogenizes Salmonella

species of serogroup B with the appearance of factor 1, 3 and phage 14 whichlysogenizes salmonellae of serogroup C, (e.g . S. choleraesuis) with the appearance offactor 14 . 4 Lysogeny, however, is not always accompanied by detectable changes inserological specificity . Lysogenic conversion by phages A3 or A4 greatly reduces theadsorption of the same phages or P22 to the bacterium but does not cause changes inthe serological character of 0-antigen .' Antigenic conversion by these phages is by0-acetylation of 0-2 and/or 0-3 of the L-rhamnose of the repeating unit but theresulting LPS is immunologically unaltered in the rabbit .'

The effects of phages on the properties of their host is of basic as well as clinicalinterest . There are several examples among pathogenic bacteria where lysogeny witha bacteriophage converts a non-virulent to a virulent strain .' The 0-antigens ofSalmonella species are important virulence determinants . We have investigated thechanges in the LPS of S. choleraesuis caused by lysogeny with phage 14 and theirconsequences for bacterial sensitivity to serum and mouse virulence .

Results

LysogenizationSeveral colonies from an overnight L-agar plate of S . cho/eraesuis 38, were char-acterized with phages and 0-specific antisera . All the colonies tested were resistantto the rough-specific phages BR60, 6SR, C21 and Ffm; they were sensitive to thecomplete core phage, FO and to phage 14 which specifically adsorbs to the 0-6,7antigen of serogroup C, salmonellae (data not shown) . Similarly, all the coloniesagglutinated in anti-0-6,7 serum but not in anti-0-4,12 serum or saline . These resultsconfirmed that the colonies were smooth and of 0-6,7 specificity . Three colonies werechosen and numbered SL4387a, SL4387b and SL4387c . Each clone was grownovernight in L-broth and treated with a lysate of phage 14 to isolate a lysogen . Thethree resulting pairs of unlysogenic and lysogenic clones were SL4387a/SL4388a,SL4387b/SL4388b and SL4387c/SL4388c . The efficiency of lysogenic conversionwas at least 95% in each case (data not shown) . We similarly tested and lysogenizedsingle colonies of S . typhimurium Q1 (SL1 292) with phage P22 or A4. The efficienciesof lysogenic conversion observed were 95% for P22 and 66% for A4 . Treatment of S.choleraesuis or S. typhimurium with these phages resulted in a two- to four-foldreduction in viable counts . Thus 25-50% of bacteria survived phage treatment andwith phages 14 and P22 ; nearly all the survivors were lysogenic . These results meanthat clones which became lysogenic after treatment with these phages were notnormally minor components of the bacterial population which survived killing or otherform of selection by phage .

Effect of lysogenic conversion on average chain length of O-antigenLPS extracted from the three pairs of phage 14-lysogenic and unlysogenic strains of S .choleraesuis were analysed by SDS-polyacrylamide gel electrophoresis . Representative

Phage-conferred selective advantage in Salmonella 395

56

Fig. 1. SDS-PAGE of LPS from a non-lysogenic strain of S. choleraesuis (lanes 1 and 5) and the same strain lysogenized with phage 14 (lanes 2 and 6). Lanes 3 and 4 contain LPS from S. typhimurium for comparison.

data are presented in Fig. 1. The LPS extracted from SL4387a, an unlysogenized clone (lane 1) consisted mainly of short chains while that of its phage-14 lysogenized derivative, SL4388a (lane 2) consisted mainly of long chains. Results obtained from the other two pairs of clones SL4387b/SL4388b and SL4387cfSL4388c were essentially identical to this (data not shown). Lanes 3 and 4 contain LPS from S. typhimurium Ql (SL1292) and a commercial preparation of S. typhimurium LPS (Sigma). It is evident by visual inspection that SL4388a made O-antigen of comparable chain-length distribution with these samples and that SL4387a (hence S. choleraesuis 38,) made abnormally-short O-antigen chains. The extent of the difference in chain- length distribution between lysogenic and non-lysogenic clones was further visualized by extended electrophoresis of LPS from such a pair (SL4387b/SL4388b (Fig. 1, lanes 5 and 6). This allowed better resolution of high molecular weight LPS but resulted in loss of several bands of low molecular weight from lower end of the gel. In another experiment an overnight broth culture of SL2824 was treated with phage and the resulting uncloned population was used for LPS isolation. Comparison of this LPS with that of SL2824 (data not shown) showed the same results as described for the cloned lysogenic/non-lysogenic pairs described above. In contrast to the above results, lysogenization of S. typhimurium 01 with P22 or A4 did not visibly alter the average chain length of LPS (Fig. 2).

The results of sugar analysis show that the mannose (present only in the O-antigen) content per milligram of LPS is much greater in SL4388b than in SL4387b whereas the galactose (present only in the core) contents are quite similar (Table 1). The relative amounts of mannose and galactose in an LPS sample could be used to calculate the averable number of 0 repeat-units per chain. Assuming that the core of S. choleraesuis contain two galactose units per chain as in S. typhimurium’ and that the O-repeat-unit contains four mannose residues as in that of S. thompson,’ another

396 N. A. Nnalue et al.

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Fig. 2. SDS-PAGE of LPS from S. typhimurium Ql (SL1292) (lane I), its P22-lysogenic derivatives (lanes 2 and 3) and its phage A4-lysogenic derwatives (lanes 4 and 5). Lane 6 contains a commercial preparation of LPS from S. typhimurium.

member of serogroup C,, we calculate an average of two repeat units per LPS molecule for SL4387b and 12 for SL4388b. Thus the phage caused a six-fold increase in the average chain length of the O-antigen.

Analysis of l ipopolysaccharides by densitometry A semi-quantitative comparison of the distribution of bands in the LPS of one pair of lysogenic and non-lysogenic clones (SL4388a/SL4387a) was performed by spectrophotometric scanning of gel slices after electrophoresis of 10 pg of each sample. The results (Fig. 3) showed that the amounts of core oligosaccharide not substituted with O-repeat-units (horizontal arrow) was similar in the two strains. The difference was that the LPS from SL4387a contained a lot more molecules with short O-chains (region L) than that from SL4388a while the opposite was the case for long-chained molecules (region H). This result indicates that phage 14 did not significantly alter the efficiency of core substitution with O-antigen but rather converted short O-antigen chains to longer ones. Also a comparison of the positions of peaks in

Table 1 Sugar compositions of lipo- polysaccharides from a pair of lysogenic and non-lysogenic clones

Amount in /Lmoles per mg of LPS

Sugar SL4387a SL4388a

Mannose 0.129 0.347 Galactose 0.027 0.015 Glucose 0.048 0.119

Phage-conferred selective advantage in Salmonella

397

0

0u 1 .2d

I1I1IIIIII

5

10

Relative position in gel (cm)Fig . 3 . Densitometric scans of gel slices after electrophoresis of LPS samples from S . choleraesuis

SL4387a and its phage 14-lysogenic derivative SL4388a . See text for details .

the low molecular weight region showed that while the cores from the two strainshad migrated the same distance in the gel, those consisting of the core plus one ormore 0-repeat-units had migrated more slowly in the LPS from SL4388. This differencewas especially evident in the peaks which we identify as the core plus one 0-repeat-unit (vertical arrows) . We believe this to indicate the presence of an a 1 -~ 3 linkedglucose residue in the 0-6,7,14 repeat-units of SL4388a but not in the 0-6 2,7 repeat-units of SL4387a . Such a residue has been found in all polysaccharide chains of phage14-lysogenized S. thompson (0-6,7,14) but only in some chains of its 0-6 1 ,6 2,7 non-lysogenic parent s (Fig . 4) .

(a)

- . 2f3ManI -2 aMan1 ---3~, 2aManI --32/3ManI--~3GlcNAcI3

TR

R = H or aGIc

(b)

- 2 /3Man I -2 aMan I --e 2 aMan I --->2 f3Man I-3 GIcNAcI-3

TI

a Glc

2.4

F

yy

15

Ii

Fig . 4 . Structure of the 0-repeat-unit (0-6,7) of Salmonella serogroup C,, The figure shows the structuredetermined for a non-lysogenic strain of Salmonella thompson (a) and that of the same strain lysogenizedwith phage 14 (b) . 9

398

Numbers represent the average of duplicate determinations in thesame experiment . Results obtained with diluted serum (1 :5 innormal saline) are given in parenthesis .

Increase in serum resistance associated with lysogenization with phage 14The survival and multiplication of two pairs of lysogenic and non-lysogenic strains innormal human serum was compared (Table 2) . The non-lysogenic strains, SL4387aand SL4387b, were serum-sensitive, with a 10-fold reduction in number in nomal ordiluted serum after 1 h treatment . However, more bacteria were recovered aftertreatment for 3 h than for 1 h showing that clones which survived initial killing byserum later multiplied . The data would suggest that these strains contained a minorserum-resistant population which later multiplied . An alternative explanation that thesame serum-sensitive population multiplied after exhaustion of complement is notplausible since data obtained with diluted and undiluted serum were quite similar .With the lysogenic strains at least a 1 .5-fold increase was observed after 1 h and atleast a seven-fold increase after 3 h . Since these experiments allowed bacterialmultiplication which compounded analysis of the data we also did one experiment inthe presence of the bacteriostatic agent, chloramphenicol (10 ©g/ml) . As expected ofa bacteriostatic agent this antibiotic (at 20 ug/ml) did not affect the viability of any ofthese strains even after 6 h of contact (data not shown) . The result of a serum-sensitivity test in its presence was 8% survival for SL4347a and 93% for SL4388a after3 h of incubation in serum . We deduce from the overall data that S. choleraesuis strain38, is serum-sensitive and is converted to serum-resistant by phage 14 .

I n vivo survival and multiplication of bacteriaAs serum resistance is associated with bacterial virulence it was of interest to comparethe mouse virulence of lysogenic and non-lysogenic strains . We therefore tested thesurvival and multiplication of SL4387a and SL4388a by intraperitoneal (i .p .) inocu-lation of mice with an equal dose (2X10 4 cfu) of either strain . Table 3 shows that anaverage of approximately 10 times more SL4388a than SL4387a was recovered fromlivers and spleens of these mice on both days 1 and 3 after infection . We believe thisto reflect difference in the ability of the two strains to survive complement-mediatedphagocytosis and intracellular killing by peritoneal macrophages rather than in theirrates of in vivo multiplication .

Discussion

The data presented here clearly show that the average chain length of S . choleraesuislipopolysaccharide increases as a consequence of lysogenization with phage 14 and

N. A . Nnalue et at.

Table 2 Ability of bacteria to survive and multiplyin normal human serum

Percent of inoculum surviving after time (h)

Strain 0 1 3

Without chloramphenicol6.5 (10) 46 (65)SL4387a 100

SL4388a 100 167 (150) 727 (889)SL4387b 100 9 (14) 38 (50)SL4388b 100 121 (179) 714 (1142)

With chloramphenicol (10 ©g/ml)16 8SL4387a 100

SL4388a 100 116 93

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399

Table 3 Survival and multiplication of SL4387a andSL4388a in the liver and spleen of BALB/c mice afteri .p . inoculation

cfu in liver and spleen

Day post-infection

SL4387a

SL4388a

1

5, 5, 15

35, 90, 1403

2 x 10 3 , 2x10 3 , 7 X103 104 , 2 x 105, 6x105

Each value represents cfu recovered from one mouse .

that this results in increased resistance to killing by serum and increased ability tosurvive and multiply in the mouse . As mobile and assessory genomes, phages cancontribute useful genes to bacteria . Survival advantage to a Salmonella strain as aconsequence of lysogenization has, however, not been shown previously . Some studieshave shown that lysogenic conversion of S . typhimurium by phage P22 or P27 hadno effect on mouse virulence 10 and that lysogenic conversion of S . anatum to S .newington by phage el5 is associated with increased serum sensitivity ." Our datashowing that P22 did not alter the chain length distribution of S. typhimurium LPSwould be consistent with the observed lack of effect of lysogenization with this phageon virulence . Phages are known to encode virulence factors for many bacteria such asEscherichia coli, Corynebacterium diphtheriae, Clostridium botulinum, Staphylococcusaureus and Streptococcus pyogenes.' These examples however differ from thatdescribed here because they involve conversion to toxigenicity and do not readilyreveal selective advantage to the bacterium under natural conditions . Our findingtherefore may be an example of a unique class of symbiotic relationship between abacteriophage and a bacterium that results in increased ability of the bacterium tosurvive in adverse environment . Evidence that phages can confer survival advantageon S . choleraesuis include the facts that most natural isolates of this organism arelysogens' 1,13 and that lysogeny for phage 6, can increase its virulence ." The mechanismfor such increased virulence was, however, not investigated .

Antigenic conversion generally confers immunity to the same and related phagesbecause the altered LPS no longer serves as a good receptor for phage absorbtion .Some phages such as A4, however, can confer immunity without altering the antigeniccomposition of the LPS . While phage-mediated antigenic conversion of bacterial LPShas been recognized for a long time and, in some cases well-understood at thestructural level, the molecular and regulatory mechanisms involved remain largelyunknown . The 0-antigen of salmonellae consists of repeat units synthesized byenzymes coded by the rfb genes and polymerized by an enzyme specified by rfc. Thestructural basis for the serological changes associated with lysogenic conversion bysome phages have been well investigated . Phages P22 and E34 respectively convertfor factors 1 and 34 by attachment of glucosyl branches to galactosyl residues withinthe 0-repeat-unit' while P27 converts for factor 27 by changing the linkage betweensuccessive repeat units . 2 Thus with P22 and E34 phage coded transferases modify the0-repeat-units while with P27, a phage coded polymerase must replace or modify thebacterial enzymes . Three phages are involved in lysogenic conversion of serogroup C,Salmonellae but only that mediated by P14 is well understood at the structural level .The 0-antigen of S. thompson (a member of serogroup C,) is known to consist of apolymer of four mannose and one N-acetyl glucosamine residues, with a glucosyl sidebranch attached to one of the mannose residues in some chains but not others . 9Lysogenic conversion of S . thompson by P1 4 results both in glucosylation of previously

400

non-glucosylated chains as well as in change in the glucosylated mannose residue inchains which previously had glucosyl units .' Our data would at present suggest threepossibilities with respect to mechanism of conversion . The phage may have introduceda polymerase where none existed ; replaced or complemented the native bacterial 0-polymerase with a more-efficient one ; or the phage-specified glucosylation per seresults in more-efficient 0-antigen assembly by increasing the affinity of the 0-repeatunit for a bacterial polymerase . A determination of which alternative is the case cannotbe made at this time .

A high density of long-chain LPS is the characteristic most clearly associated withserum resistance in Salmonella and other enteric bacteria.'"' The mechanism for thisis that long-chain LPS preferentially bind complement components 17,18 and hinderaccess of the membrane attack complex to the outer membrane .""' Resistance to lysisby complement is however not likely to be a mechanism of increased mouse-virulencein Salmonella because mouse complement is normally poorly lytic for bacteria .Complement nonetheless plays a major role in mouse salmonellosis because itpromotes phagocytosis and intracellular killing . The rate at which the 0-antigenpolysaccharide of Salmonella species activate complement by the alternate pathwayhas been inversely related to mouse virulence . 19 µ2 0 The 0-6,7 antigen of S. cho/eraesuisis especially proficient at complement activation via the alternative pathway andconsequently promotes rapid phagocytosis and killing of the bacteria in vivo . It hasbeen shown21 that complement components prevented from anchoring hydrophobicallyin the outer membrane by long-chain LPS are also readily released from the bacterialsurface . A mechanism such as this which decreases deposition of complementcomponents at the bacterial surface should reduce the rate of phagocytosis of S .choleraesuis and consequently increases the mouse virulence of this species .

Materials and methods

Bacterial strains, phages and antisera . S. choleraesuis strain SL282422 is a wild-type, mousevirulent isolate of serogroup C, with 0-antigen factors 6 2 and 7. SL1292 is wild-type S .typhimurium Q1 of serogroup B with 0-antigen factors 4 and 12 . These strains are not lysogenicfor any known converting phages . Phages P22 and A4 were available in this laboratory . PhageP22 is active on salmonellae of serogroups B and D and converts for factor 1 . Phage A4 hasthe same host range but is non-converting . Phage 14 was obtained from Dr L. LeMinor ofInstitut Pasteur, Paris, France . It is active on serogroup C, salmonellae and converts for factor14. Phages FO, 131360, 6SR and Ffm used only for determination of LPS character of cloneswere also available in this laboratory. Antisera to factor 14 was from Dr L. LeMinor. Otherantisera were purchased from Difco Laboratories, Detroit, Michigan .

Lysogenization of bacteria with phages . Overnight broth cultures of bacteria were mixed withphage at a multiplicity of infection (moi) of 50-100 and stood for 1 h at room temperature . Themixtures were diluted in saline and plated in nutrient agar to isolate single clones . Lysogenswere identified by immunity to superinfection with the same phage and by agglutinability in 0-factor specific antiserum. The efficiency of lysogenization in a bacterial culture treated withphage was determined by testing 20 single clones as above . To lysogenize an entire populationof bacteria, 0 .9 ml of overnight broth culture was mixed with 0 .1 ml of phage (moi c. 100) andadsorption allowed to take place for 2 h at room temperature . The mixture was diluted to 5 mlwith fresh broth, grown overnight and frozen .

LPS isolation and analysis by SDSpolyacrylamide gel electrophoresis . Small samples oflipopolysaccharides used initially were prepared by the method of Darveau and Hancock 23 andquantified by assay for 3-deoxy-D-manno-octulosonic acid (KDO) as described . 24 Subsequentlylipopolysaccharides were prepared by the phenol-water procedure 25 in sufficient amounts toallow quantitation by analytical balance . LPS prepared by the two methods were indis-tinguishable in polyacrylamide gels . For analysis by SDS-polyacrylamide gel electrophoresis a

N . A . Nnalue et al.

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sample of LPS (10 ug) was heated at 100°C for 5 min with an equal volume of sample buffer(0 .1 M Tris-HCI, pH 6 .8, containing 2% SDS, 20% sucrose, 1% 2-mercaptoethanol and 0 .001%bromophenol blue) and electrophoresed at 20 mA in a discontinuous system incorporating 4 Murea in an 18% separating gel .26 On termination of electrophoresis, gels were fixed and stainedwith silver as described .26

Densitometry by spectrophotometric scanning . A Beckman Du-8 spectrophotometer equippedwith an adapter for gel scanning was used for quantitative densitometry to trace size distributionof bands in the LPS after electrophoresis . A desired lane was cut out from a silver-stained gel,wrapped in Saran wrap and inserted in the adapter . Scanning was done at a wavelength of 600nm .

Sugar analyses. Lipid-free polysaccharides were prepared from lipopolysaccharides bytreatment with 2% aqueous acetic acid at 100°C for 1 h ; this cleaves the acid-labile 3-deoxy-D-manno-octulosonyl linkages . The insoluble lipid was removed by centrifugation at 12000xg at4°C for 15 min and the polysaccharide purified by gel chromatography on Biogel P4 columnand lyophilized . The polysaccharides were hydrolysed in 0 .5 M trifluoroacetic acid at 100°C for16 h and the resulting mixtures of aldoses reduced to the corresponding alditols with sodiumborohydride. The alditols were converted to more-volatile alditol acetates by treatment withacetic anhydride : pyridine (1 : 1 v/v) at 100°C for 1 h . After removal of excess acetic anhydrideby repeated addition of water and concentration to dryness the resulting mixture of fullyacetylated alditol acetates was dissolved in dichloromethane and analysed by gas-liquidchromatography in a Hewlet-Packard model 5890A gas chromatograph .

Tests of serum-sensitivity . Sera from four healthy North American adults with no previoushistory of salmonella infection were pooled and frozen at -20°C . Aliquots were thawed whenneeded and used immediately . Bacteria were grown unshaken overnight in nutrient broth,washed once in saline and resuspended in saline to three times the original culture volume .Bacterial suspension (0 .1 ml containing 1-2x 10' cfu) was added to 0 .9 ml of normal or dilutedserum (one part serum to four parts normal saline) and incubated in a water bath at 37°C .Aliquots (0 .1 ml) of the mixture were removed at intervals, diluted in saline and plated on L-agar for determination of viable counts .

Bacterial survival in mice . Male BALB/c mice were purchased from the Department ofRadiology, Stanford University. Separate groups of mice were inoculated i .p. with 2x10° cfuof either SL4387a, or its phage 14-lysogenized derivative, SL4388a . Three mice per group weresacrificed on days 1 and 3 after inoculation for determination of bacterial numbers in the liverand spleen, as described previously . 22

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