FEMS Microbiology Letters 118 (1994) 163-166 © 1994 Federation of European Microbiological Societies 0378-1097/94/$07.00 Published by Elsevier

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FEMSLE 05931

Effect of growth temperature on complement- mediated killing of mesophilic Aeromonas spp. serotype 0 : 3 4

Susana Merino a, Dolores Alvarez b, Santiago Hernfindez-All6s b and Juan M. Tomfis ,,a

a Departamento de Microbiolog(a, Facultad de Biologfa, Universidad de Barcelona, Diagonal 645, 08071 Barcelona, Spain b Laboratorio de Microbiolog[a, Departamento de Biolog[a Ambienta~ Universidad de los lslas Baleares, Crta. de valldemossa Km 7.5, Palma de Mallorca, Spain

(Received 21 December 1993; revision received 14 February 1994; accepted 19 February 1994)

Abstract: Mesophilic Aeromonas spp. strains (serotype O:34) showed sensitivity to complement-mediated killing when they were cultivated at 37~C (serum-sensitive) but not when they were cultivated at 20°C (serum-resistant). These strains produced smooth lipopolysaccharide when they were grown at 20°C and rough lipolysaccharide when cultivated at 37°C. The reason for the resistance to complement-mediated killing could be that C3b is rapidly degraded (possibly because it is bound far from the cell membrane), consequently the lyric complex (C5b-9) is not formed.

Key words: Aeromonas; Serotype 0:34; Complement-mediated killing; Growth temperature

Introduction

Mesophilic Aeromonas spp. are both oppor- tunistic and primary pathogens of a variety of aquatic and terrestrial animals, including humans [1]. The clinical manifestation of mesophilic Aeromonas spp. infection ranges from gastroen- teritis to soft-tissue infections, septicemia and meningitis [2]. We recently described a group of mesophilic Aeromonas spp. strains belonging to serotype 0 : 3 4 producing heterogeneous O-poly-

* Corresponding author. Tel: 34-3-4021486; Fax: 34-3- 4110592.

saccharide chains in their lipopolysaccharide (LPS) when grown at 20°C but rough LPS after growth at 37°C, and always without an S-layer [3]. Serotype 0 : 34 has been recently reported as one of the most frequently encountered among mesophilic Aeromonas species [4]. This serotype comprises the species hydrophila, caviae and veronii biovar sobria (formerly A. sobria) and biovar veronii [5]. Strains from this serotype are frequently associated with gastroenteritis in hu- mans and septicemia in fish [5,6].

In the present report we associated the growth temperature-induced changes in the LPS of these strains with their complement resistance.

SSDI 0378-1097(94)00077-5

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Materials and Methods

Bacteria and media A. hydrophila strains AH-3 and Ba5 were iso-

lated from moribund fish [7]. A. hydrophila strains AH-101, AH-102 and AH-103, A. veronii biovar sobria (AS-176) and A. caviae (AC-279) were isolated from gastroenteritic human patients [5,7]. All the strains belong to serotype 0 :34 . Strains were usually cultured on tryptic soy broth (TSB) or agar (TSB plus 1.5% agar).

Bacterial survival in fresh non-immune serum The survival of exponential-phase bacteria, ei-

ther growing at 20 or 37"C, in fresh non-immune human serum (NHS) was measured at 37°C as previously described [8]. Control measurements with bacteria in phosphate buffered saline (PBS) (containing 0.15 M sodium chloride and 0.15 M sodium phosphate, pH 7.2) or heat-inactivated NHS (560C for 30 min) were performed.

Electrophoretic techniques Sodium dodecyl sulphate-polyacrylamide gel

electrophoresis (SDS-PAGE) was performed as previously described [3].

Binding of C3b and C5b-9 to whole cells The interaction between whole mesophilic

Aeromonas cells and complement components C3b and C5b-9 was quantified by an enzyme immunoassay as previously described [9]. Con- trois consisted of cells treated with protein A-al- kaline phosphatase in the absence of specific antibodies.

Analysis of bound C3 fragments An Aeromonas spp. suspension (2 × 10 s

CFU/ml) were opsonized with NHS (25% final concentration in PBS) at 37°C for 0 to 20 min and the reaction was stopped by adding ice-cold PBS. The tubes were centrifuged and the pellets were washed three times with PBS. The pellets were resuspended in 1 M hydroxylamine in 50 mM carbonate-bicarbonate buffer (pH 9.0) to disrupt ester bonds between complement fragments and the bacterial surface [10]. After 2 h at 37°C, the C3 fragment suspension was reduced with 10 mM

dithiothreitol in 1% SDS at 37°C for 1 h, and then alkylated with 22 raM iodoacetamide at 37°C for 1 h [10]. Aliquots of the C3 fragment suspen- sion were diluted 1 : 1 in sample denaturing buffer and subjected to SDS-PAGE. After electrophore- sis, the gels were electroblotted to nitrocellulose membranes [11], and t he membranes were blocked overnight with PBS plus 1% BSA, washed, incubated with anti-C3 serum for 2 h, washed again, and reincubated with protein A-al- kaline phosphatase for 1 h. The C3 fragments were visualized on the membrane blots with BCIP-NBT (5-bromo-4-chloro-3-indolylphosphate and disodium-nitroblue tetrazolium) [12]. The Western blots were scanned using a Bio-Image densitometer (Millipore). Controls of C3 and C3 degradation fragments were kindly provided by F. Vivanco (Fundaci6n Jimenez Diaz, Madrid, Spain).

Results and Discussion

The complement system plays a crucial role in humoral defence against microbial pathogens [13,14]. This series of serum proteins which are sequentially activated produces two major effects in terms of host defence: (i) deposition onto the microbial surface of an opsonic protein (C3b or iC3b), which activates the C5 cascade, and (ii) the result of the C5 cascade, the formation and as- sembly of the membrane attack complex (C5b-9) capable of lysing susceptible bacteria. Any failure of activated complement to exert its effect would render the cell resistant to complement-mediated killing.

All the mesophilic Aeromonas spp. strains from serotype 0 : 3 4 were resistant to the bactericidal action of NHS when they were grown at 20°C on TSB, but all of them were sensitive when they were grown at 37°C on the same medium (Table 1). No decrease in cell viability (strains grown at 37°C) was observed when the NHS was treated for 30 min at 56°C or by adding 20 mM EDTA to NHS for 1 h at 37°C. As we previously reported [3], the LPS extracted from these cells cultivated at 20°C was smooth (with heterogeneous polysac- charide chains), while the LPS extracted from the

Table 1

Survival of mesophilie Aeromonas spp. strains (serotype O: 34) in non-immune human serum (NHS) after being cultivated at different temperatures

Strain Growth Percentage of inoculum temperature surviving on NHS

l h 2h 3h

AH-3 20"C 96 99 101 AH-3 37"C 1.7 0.2 < 0.1 Ba5 20"C 98 102 105 Ba5 37"C 1.9 0.2 < 0.1 AH-101 20"C 97 100 108 AH-101 37"C 1.8 < 0.1 < 0.1 AH-102 20"C 96 99 103 AH-102 37"C 1.5 0.2 < 0.1 AH-103 20"C 99 101 104 AH-103 37*{2 1.7 0.3 < 0.1 AS-176 20"C 97 101 103 AS-176 370C 1.8 < 0.1 < 0.1 AC-279 20"C 98 99 101 AC-279 370C 1.6 0.2 < 0.1

The results shown are the average of three independent experiments.

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Table 2

Interaction of complement components C3b and C5b-9 with mesophilic Aeromonas spp. whole cells (serotype 0:34) grown at different temperatures, measured as A405 in an ELISA assay (see Materials and Methods)

Strain Growth temperature

Relative concentration (A4o5)

C3b C5b-9

AH-3 20"C 0.32 + 0.06 0.08 + 0.03 AH-3 37*(2 1.54+0.13 1.33+0.14 Ba5 20"C 0.30 + 0.05 0.09 + 0.04 Ba5 37"C 1.53+0.14 1.38+0.15 AH-101 20"C 0.36+0.07 0.09+0.03 AH-101 37"C 1.59 + 0.14 1.35 + 0.12 AH-102 20"C 0.33 + 0.07 0.08 + 0.04 AH-102 370C 1.55 + 0.13 1.36 + 0.14 AH-103 20"C 0.35 + 0.06 0.09 + 0.03 AH-103 37"C 1.51-t-0.12 1.34+0.11 AS-176 20°C 0.36+0.09 1.37+0.15 AS-176 37°C 1.52+0.12 1.40+0.12 AC-279 20"C 0.34 + 0.08 0.07 + 0.04 AC-279 370C 1.54+0.13 1.36+0.11

Results are the means from experiments done at least twice in triplicate.

same cells cultivated at 37"C was rough. The level of serum sensitivity of these cells grown at 370C is similar to some isogenic rough mutants, as we previously described [15].

As shown in Table 2, the Aeromonas strains belonging to serotype 0 : 3 4 were able to bind more C3b when they were grown at 37°C than when they were grown at 20"C. The level of C3b bound to the strains cultivated at 37"C was simi- lar to that observed previously for isogenic rough mutants [10]. However, the strains grown at 20"C were unable to bind C5b-9 (serum-resistant), while the same strains grown at 37°C showed a high binding of this lytic complex (serum-sensi- tive). Similar observations were made previously for isogenic rough (serum-sensitive) mutants [15],

Table 3 shows the relative amounts of C3 degradation products produced by the strains grown at 20°C and 37°C over time. It is clear that the 105 kDa band (which represents C3b, the t~' chain of C3 [16]) is present In small amounts on the strains grown at 20°C and disappears over the incubation time. Also the 68 kDa band (which represents iC3b, the degraded form of C3b [16]) is present in large quantities over the time. Simi-

Table 3

C3 fragments linked to the bacterial cell surface after NHS opsonization as described in Materials and Methods

Strain Growth Incubation temper- time (rain) ature

Relative amounts of C3 fragments (bands in kDa) a

105 75 68 43

AH-3 20"C 5 4.8 32.0 32.0 30.0 10 2.6 33.1 33.6 30.6 20 <0.1 33.5 33.7 30.8

AH-3 37"C 5 16.0 46.1 11 .4 12.7 10 19.1 47 .5 11.2 13.1 20 21.0 47.4 11.2 13.0

AH-101 20"C 5 4.7 32.1 32.0 29.8 10 2.4 33.0 33.4 30.2 20 <0.1 33.7 33.4 30.8

AH-101 37"C 5 16.3 45.9 11.1 12.3 10 19.8 46.9 11.3 12.8 20 20.7 47.2 11.4 12.9

The results are the average of three independent experiments. Similar results were obtained for strains Ba5, AH-102, AH- 103, AS-176 and AC-279 (data not shown), a The 105 kDa band represented C3b. The 68 kDa and 43 kDa bands repre- sented iC3b and C3c, respectively. The 75 kDa band is a common band for C3b and iC3b [16].

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lar results are seen for the 43 kDa band (a marker for C3c, another C3b degradation prod- uct [16]). It appears that, in these strains grown at 20°C the bound C3b is rapidly degraded to iC3b and C3c, consequently no C5b-9 could be formed (serum-resistant). However, when the same strains were grown at 37°C, the 105 kDa band is present in large amounts. By contrast, the 68 kDa and 43 kDa bands are reduced in comparison with the same bands when the strains were grown at 20°C. It appears that, although some C3b degradation could be observed, there was still enough C3b to bind C5b and to form the C5b-9 complex (serum-sensitive).

From the results we suggest that when mesophilic Aeromonas spp. (serotype 0 :34 ) are grown at 20°C producing smooth LPS, the C3b is bound to the O-antigen chains of the LPS (away from the sensitive cell membrane) and is rapidly degraded. The cells are resistant to complement- mediated killing because no C5b-9 can be formed. When the same cells are grown at 37°C, the C3b is bound close to the cell membrane, and, despite some degradation, there is still enough C3b to bind C5b and also the final lytic complex C5b-9, making the cells sensitive to complement-media- ted killing. This may explain why Aeromonas spp. strains from serotype 0 : 3 4 produce septicemia at 20°C but usually not at 37°C.

Acknowledgement

We thank Dr. F. Vivanco for providing the C3 and C3 degradation standards.

References

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