Microbial Pathogenesis 1997; 22: 315–320

PATHOGENESISMICROBIAL

SHORT COMMUNICATION

Complement resistance of capsulated strainsof Aeromonas salmonicidaSusana Merinoa, Alicia Aguilara, Juan M. Tomasa∗,Ramon Bonetb, Maria Jose Martinezb, Dolores Simon-Pujolb

& Francisco Congregadob

aDepartamento de Microbiologıa, Universidad de Barcelona, Diagonal 645, 08071 Barcelona, Spain,bDepartmento de Microbiologıa y Parasitologıa Sanitarias, Faculted de Farmacia, Universidad deBarcelona, Barcelona, Spain

(Received June 24, 1996; accepted in revised form December 2, 1996)

The complement resistance of Aeromonas salmonicida strains grown under conditions promotingcapsule formation was investigated using well characterized strains and their isogenic mutants.Complement resistance was previously studied using the same strains growing under non-capsulating conditions. The serum resistant strains were found to activate complement, but rapidlydegrade C3b preventing productive formation of the lytic complex C5b-9. Isogenic lipo-polysaccharide rough mutants grown under non-capsulating conditions were serum sensitive,binding a large amount of C3b and leading to productive formation of C5b-9. When grown underconditions promoting capsule formation, these mutants were partially resistant to complementbecause less C3b is bound to them and also partially degraded, with a concomitant reduction inlytic C5b-9. 1997 Academic Press Limited

Key words: Aeromonas salmonicida, surface molecules, capsule, complement-mediated killing.

49.000-molecular-weight (MW)] array, tetheredIntroductionto the cell by LPS [1]. Under some conditionsA. salmonicida is able to form a capsule in vitro

Aeromonas salmonicida is an important pathogen [2] and in vivo [3]. This capsule in vivo, has beenof salmonid fishes, producing the systemic dis- previously described as another LPS form [4],ease furunculosis. A major virulence factor of is now recognized as a capsule [2].this pathogen appears to be an S-layer (A-layer), The complement system plays a crucial roleprincipally comprised of a two-dimensional in the humoral defense against microbial patho-paracrystalline tetragonal protein [A-protein of gens [9]. This series of serum proteins is se-

quentially activated and leads to the deposition∗Author for correspondence. of C3b protein onto the microbial surface to

0882–4010/97/050315+06 $25.00/0/ 1997 Academic Press Limitedmi960121

S. Merino et al.316

produce two major effect functions (i) op-sonization for C3b receptor-bearing phagocytes,and (ii) through its activity as a stable C5 con-vertase, formation of a membrane attack com-plex (C5b-9) capable of lysing susceptiblebacteria.

Complement activation may take place byeither of two pathways (classical or alternative,CPC and APC), both resulting in activation ofthe vital third component of complement, C3.Bacterial resistance to complement-mediatedkilling may result from a failure or limitationof complement activation by either of the twopathways or by a failure of activated com-plement to exert its effect. Various surface com-ponents which render bacterial cells resistant tocomplement-mediated killing have been iden-tified, including lipopolysaccharide (LPS), outer

3

100

Hours%

Su

rviv

al

10

1

1 20

membrane proteins, and capsules [6, 7, 8]. We Figure 1. The bactericidal effects of human NIS on A.recently demonstrated that A. salmonicida is able salmonicida A450-1 (O−:A−) strain grown in differentto activate the CPC but not the APC, and A. media. (Χ), grown on TSA (non-capsulating con-salmonicida strains with smooth LPS (O+) are ditions); (Β), grown on YPGS (Φ), and grown onresistant to complement-mediated killing be- FVM (conditions promoting capsule formation). (Ε),

control of serum resistant strain A450 (O+:A+) growncause C3b is rapidly degraded without the for-on FVM (conditions promoting capsule formation).mation of C5b-9 whereas rough strains (O−) areThe results are the average of at least three in-sensitive (the C3b binding is higher than independent experiments.smooth strains and there is formation of C5b-

9). The strains were always grown on non-capsulating conditions [9]. their ability to resist the bactericidal activity of

In the present study, we investigated the role non-immune serum (NIS) from different sourcesof the capsule in the susceptibility of A. sal- (rabbit, human and trout sera). The results ob-monicida strains, specially the rough strains, to tained when the strains were grown under non-the bactericidial activity of non-immune serum. capsulating conditions have been described

previously [9] and are summarized in Table 1.When the same strains were grown in YPGS orFVM that promoted capsule formation, O+:A+Results and discussionand O+:A− strains were resistant to human andrabbit NIS (data not shown) and the O−:A−

All the A. salmonicida strains tested (O+:A+, O+: strains showed a partial resistance to NIS (Fig.A− and O−:A−), when grown in YPGS, showed 1).a capsular polysaccharide, either by negativelystained EM of whole cells or using EM of thinsections labelled with polycationic ferritin, as Inhibition of serum bactericidal activity and

complement consumption by whole cellswe previously described [10]. Also, the samestrains grown on fish viscera medium (FVM) or purified surface molecules[11] showed a capsular polysaccharide using thesame techniques, while the same strains grown Preincubation of trout or human NIS with whole

cells of A. salmonicida strains (O−:A−) grownon TSA were unencapsulated as we previouslydemonstrated [2]. The A. salmonicida strains used under conditions promoting or not capsule for-

mation inhibited serum bactericidal activity.showing identical LPS profiles irrespective ofthe growth media (TSB, YPGS or FVM) and the Whole cells of A. salmonicida strains (O−:A−)

inhibited complement-mediated hemolysis ofpresence or absence of the A-protein (data notshown). sensitized sheep erythrocytes and depleted com-

plement components Clq and C3 from humanA. salmonicida strains grown under conditionspromoting capsule formation were tested for NIS.

Complement resistance of capsulated strains of Aeromonas salmonicida 317

Table 1. Interaction of complement components C3b, C5b and C5b-9 with A. salmonicida (O−:A−) whole cells grown under differentconditions.

Relative concentration

Strain C3b C5b C5b-9

Grown on TSB (non-capsulating condition)A450-1 1.72+/−0.18 1.51+/−0.19 1.49+/−0.15A449-3R 1.69+/−0.16 1.56+/−0.16 1.52+/−0.13

Grown on YPGS (condition promoting capsule formation)A450-1 0.88+/−0.10 0.25+/−0.06 0.27+/−0.04A449-3R 0.79+/−0.11 0.27+/−0.08 0.25+/−0.09

Grown on FVM (condition promoting capsule formation)A450-1 0.75+/−0.12 0.29+/−0.05 0.22+/−0.07A449-3R 0.77+/−0.09 0.28+/−0.06 0.24+/−0.05

Results are given in arbitrary A405 units from ELISAs done in triplicate atleast twice +/− SD. When control cells were incubated in the absence ofspecific antibodies, the concentrations of C3b, C5b and C5b-9 were always<0.1 A405 units.

Purified LPS of O− strains was able to com- found to bind less C3b than the same strainsgrown under non-capsulating conditions (Tablepletely inhibit the bactericidal activity of human

NIS at 0.1 mg/ml against strain A450-I (O−A−). 1). The O−:A− strains grown under non-capsulating conditions (serum-sensitive)The complement absorbing activity of purified

LPS from all the strains grown on both con- showed a high level of binding of complementcomponents C5b and C5b-9 ([9], Table 1). How-ditions was dose dependent. Also, the C3 con-

centration was depleted when human NIS was ever, these O−:A− strains showed a significantreduction of C5b or C5b-9 binding when growntreated with these purified LPSs.

Purified capsular polysaccharide (with LPS under conditions promoting capsule formation,like YPGS or FVM media (Table 1). All thesecontamination of <3%) was obtained from

strains A450-1 and A449-3R (O−:A−) [2]. These results were also confirmed by immunogoldelectron microscopy using specific antiseracapsules were not able, even at high con-

centrations (up to 0.4 mg/ml), to inhibit the against C3b or C5b-9 on strain A450-1 (O−:A−)grown on both conditions.bacterial activity of trout or human NIS for

serum-sensitive strains of A. salmonicida or toinhibit complement-mediated hemolysis of sens-itized sheep erythrocytes or to deplete C3 from

Analysis of bound C3 fragmentshuman NIS.Thus, we clearly demonstrated that all the A.

We have previously reported [9] that serumsalmonicida strains tested (grown under bothresistant strains show upon incubation a largeconditions) are able to activate complement, thatdecrease in the C3 fragment characteristic ofthe activation is LPS and not the capsule. TheC3b, as well as a large amount of the C3 fragmentresistance of the serum-resistant strains to com-characteristic of iC3b, a degraded form of C3b;plement-mediated killing is therefore due to thethey also showed other C3 degradation frag-failure of complement to exert its effect on them.ments, like C3c, indicating that serum resistantstrains are able to degrade the deposited C3b toiC3b. By 20 min practically no C3b was observedBinding of C3b, C5b and C5b-9 to whole

cells on serum resistant cells [9].The serum-sensitive strain A450-1 (O−:A−)

grown under non-capsulating conditions (inThe A. salmonicida strains (O−:A−) grown underconditions promoting capsule formation were TSB), did not show on incubation any decrease

S. Merino et al.318

Table 2. C3 fragments linked to the bacterial cell surface ofstrain A450-1 (O−:A−) grown on different media after humanNIS opsonization as described in Materials and methods.

Relative amounts of C3fragments (bands in kDa)a

Incubation timeMediab (min) 105 75 68 43

TSB 5 17.2 47.1 13.9 15.410 20.6 48.2 11.7 14.920 25.7 48.6 11.7 12.1

YPGS 5 7.1 34.6 29.2 28.610 5.3 37.8 29.7 28.420 2.7 39.1 30.2 27.9

FVM 5 6.8 33.0 30.1 28.910 4.2 34.1 29.9 29.220 2.3 35.7 30.3 29.3

a The 105 kDa band represents C3b. The 68 and 43 kDa bands representiC3b and C3c, respectively. The 75 kDa band is a common band forC3b and iC3b [9].a TSB, non-capsulating conditions; YPGS or FVM, conditions pro-moting capsule formation.The results shown are the average of three independent experiments.

in the C3b concentration, and the relative When A. salmonicida strains were grown invivo they exhibited a capsular polysaccharideamounts of the iC3b or C3c degradation prod-

ucts were lower than those observed for serum (with some differences from in vitro capsulation[3]), and some cases of furunculosis have beenresistant strains ([9] and Table 2). These results

indicated that in strain A450-1 grown under associated with rough strains [12]; we suggestedthat the capsular polysaccharide may protectnon-capsulating conditions some C3b de-

gradation (to iC3b and more) occurred but to a these rough strains from complement-mediatedkilling also in vivo, which could allow theselesser extent than in the serum-resistant strains

and bound C3b was still present at 20 min ([9] rough strains to survive in the blood and causesepticemic furunculosis.and Table 2). Since not all the C3b was degraded,

a large amount of C5b and C5b-9 could be easilyobserved on the cell membrane of the serumsensitive strains (rough LPS) [9]. However, whenthe same strain was grown under conditions Materials and methodspromoting capsule formation (YPGS or FVMmedia), the amount of C3b was decreased and Bacterial strains and mediathe amount of C3b degradation products (iC3band C3c) increased (Table 1 and 2). This may The A. salmonicida strains used in this study are

previously described [9] and were a gift from W.explain that less C5b or C5b-9 (lytic complex)was bound to the rough strains when grown on W. Kay (University of Victoria, British Columbia,

Canada). Strains A450 and A449 showed andthese conditions (Table 1). Because there wasless C5b-9 bound to these rough strains when A-layer (A+) and a complete LPS (with O−

antigen, O+). Strains A450-3 and A449-3 weregrown under conditions promoting formation.They showed a partial resistance to the bacterial isogenic mutants from A450 and A449 re-

spectively, unable to maintain an A-layer (A−)activity of serum; while they were serum sens-itive under non-capsulating conditions because with a complete LPS (O+) and at least strain

A450-3 also unable to synthesize A protein.of a large quantity of C5b-9 was bound to thesecells [9]. Strains A450-1 and A449-3R were rough LPS

Complement resistance of capsulated strains of Aeromonas salmonicida 319

mutants (O−) derived from strains A450 and bactericidal reaction was studied as we pre-viously described [9]. For purified capsularA449 respectively, and unable to assemble an

A-layer (A−). Cultures were maintained and polysaccharide we used the same technique de-scribed for purified LPS, but the final con-grown as previously described on non-cap-

sulating conditions [9]. The medium used to centration was always 5 mg/ml.Controls consisting of NIS incubated for 1 hobtain conditions promoting capsule formation

was yeast extract-peptone-glucose-mineral salts at 37°C in PBS without cells, LPS or purifiedcapsular polysaccharide showed no inhibition(YPGS) previously described [2]. The strains

were also grown on an autolysate of fish viscera of serum bactericidal activity.medium (FVM) prepared as described byClausen et al. [11].

Measurement of the anticomplementactivity of whole cells or purified molecules

LPS and capsule isolationThe anticomplement activity of whole cells, puri-fied LPS, or purified capsular polysaccharideLPS from A. salmonicida strains was purified by

the method of Westphal and Jann [13] as modi- was measured by the method of Shafer et al.[18]. The positive control consisted of sensitizedfied by Osborn [14]. Capsular polysaccharide

were isolated from different A. salmonicida sheep erythrocytes plus NIS alone, and the neg-ative control consisted of cells or purifiedstrains as previously described [2]. SDS-PAGE of

LPS was performed by the procedure of Laemmli molecules without added NIS.Concentrations of C1q and C3 complement[15] and detected by the silver stain method of

Tsai and Frasch [16]. components were measured as previously de-scribed [9].

AntiseraBinding of C3b, C5b and C5b-9 to wholecellsAnti-capsule serum was obtained after im-

munization of New Zealand White rabbits withpurified capsular polysaccharide following the The interaction between whole A. salmonicida

cells and complement components C3b, C5b andprocedure previously described [17]. Anti-cap-sule serum was always adsorbed with the same C5b-9 was quantified by an enzyme immuno-

assay and observed by immunogold electronA. salmonicida strain used grown under non-capsulating conditions in order to render the microscopy [9].

Controls consisted of cells treated with proteinantiserum specific. The antisera were tested byELISA using whole cells as antigens as we pre- A-alkaline phosphatase for the immunoassay or

protein A conjugated to 10-nm gold particlesviously described [17].for the immunogold electron microscopy in theabsence of specific antibodies.

Bacterial survival in fresh non-immuneserum

Analysis of bound C3 fragmentsThe survival of exponential-phase bacteria infresh NIS was measured as previously described An A. salmonicida suspension (2×108 cfu/ml)

was opsonized with NIS diluted in PBS (25%[9]. Control measurements with bacteria in phos-phate-buffered saline (PBS) (containing 0.15 M final concentration). Opsonization was carried

out at 37°C for 0 to 90 min and the reaction wassodium chloride and 0.15 M sodium phosphate,pH 7.2) or heat-inactivated NIS (56°C for 30 min) stopped by adding ice-cold PBS. Serum-sensitive

strains were only opsonized for 0 to 20 min.were performed.Analysis of bound C3 fragments was performedas previously described by us [9]. Two C3 puri-fied preparations were used as controls (kindlyInhibition of serum bactericidal activityprovided by F. Vivanco, Fundacion JimenezDiaz, Madrid, Spain), one containing C3dThe effect of treating the serum with bacterial

cells or purified cell components in the serum (33kDa band), and the other containing C3c (43

S. Merino et al.320

5 Taylor PW. Bacterial resistance to complement. In: Rothand 27 kDa bands) and the 75 kDa band commonJA (ed), Virulence mechanisms of bacterial pathogens.to C3b and iC3b. The Western blots were scannedWashington, WA: ASM Press; 1988: 107–20.using a Bio-Image densitometer (Millipore). 6 Hildebrant JF, Mayer LW, Wang SP, Buchanan TM.Neisseria gonorrhoeae acquire a new principal outer-mechanism protein when transformed to resistanceto serum bactericidal activity. Infect Immun 1978; 20:Electron microscopy 267–73.

7 Mushel LH, Larsen LJ. The sensitivity of smooth andNegatively stained EM of whole cells and rough gram-negative bacteria to the immune bac-

tericidal reaction. Proc Soc Exp Biol Med 1970; 133: 345–8.labelled thin sections with polycationic ferritin8 Sutton A, Schneerson R, Kendall-Morris S, Robbins JB.was performed as previously described [10].

Differential complement resistance mediates virulenceImmunoelectron microscopy using whole cells of Haemophilus influenzae type b. Infect Immun 1982; 35:with anti-C3b and anti-C5b-9 serum were also 95–104.

9 Merino S, Albertı S, Tomas JM. Aeromonas salmonicidapreviously described [9].resistance to complement mediated killing. Infect Immun1994; 62: 5483–90.

10 Martinez MJ, Simon-Pujol D, Congregado F, MerinoS, Rubires X, Tomas JM. The presence of capsularAcknowledgements polysaccharide in mesophilic Aeromonas hydrophila sero-types O:11 and O:34. FEMS Microbiol Lett 1995; 128:69–74.

This study was supported by grant PB94-0906 to 11 Clausen E, Gildberg A, Raa J. Preparation and testingJ.M.T. from DGICYT (Ministerio de Educacion y Ci- of an autolysate of fish viscera as growth substrate forencia) and grant 1995SGR00414 from Generalitat de bacteria. Appl Environ Microbiol 1995; 50: 1556–7.

12 McCarthy DH, Rawle CT. The rapid serological dia-Catalunya. A.A. is a recipient fellowship from Min-gnosis of fish furunculosis caused by “smooth” andisterio de Educacion y Ciencia. We thank Maite Polo“rough” strains of Aeromonas salmonicida. J Gen Microbiolfor her technical assistance.1975; 86: 185–7.

13 Westphal O, Jann K. Bacterial lipopolysaccharides: ex-traction with phenol-water and further applications ofthe procedure. Carbohydr Chem 1965; 5: 83–91.

References 14 Osborn MJ. Preparation of lipopolysaccharide frommutant strains of Salmonella. Methods Enzymol 1966; 8:161–4.

1 Belland RJ, Trust TJ. Synthesis, export and assembly of 15 Laemmli UK. Cleavage of structural proteins duringAeromonas salmonicida A-layer analyzed by transposon the assembly of the head of bacteriophage T4. Naturemutagenesis. J. Bacteriol 1985; 163: 877–81. (London) 1970; 227: 680–5.

2 Garrote A, Bonet R, Merino S, Simon-Pujol MD, Con- 16 Tsai CM, Frasch CE. A sensitive silver stain for detectinggregado F. Occurrence of a capsule in Aeromonas sal- lipopolysaccharide in polyacrylamide gels. Anal Bio-monicida. FEMS Microbiol Lett 1992; 95: 127–32. chem 1982; 119: 115–9.

3 Garduno RA, Kay WW. Capsulated cells of Aeromonas 17 Benedi VJ, Ciurana B, Tomas JM. Isolation and charac-salmonicida grown in vitro have different functional terization of Klebsiella pneumoniae unencapsulated mut-properties than capsulated cells grown in vivo. Can J ants. J Clin Microbiol 1989; 27: 82–7.Microbiol 1995; 41: 941–5. 18 Shafer WM, Joiner K, Guyman LF, Cohen MS, Sparling

4 Garduno RA, Thornton JC, Kay WW. Aeromonas sal- PF. Serum sensitivity of Neisseria gonorrhoeae: the roleof lipopolysaccharide. J Infect Dis 1984; 149: 175–83.monicida grown in vivo. Infect Immun 1993; 61: 3854–62.