Liability to Hydrophobic and Charge Interaction of Smooth ...

INFECTION AND IMMUNITY, Nov. 1977, p. 261-265

Vol. 18, No. 2 Printed in U.S.A.

Copyright © 1977 American Society for Microbiology

Liability to Hydrophobic and Charge Interaction of Smooth Salmonella typhimurium 395 MS Sensitized with Anti-MS Immunoglobulin G and Complement INGER STJERNSTROM, KARL-ERIC MAGNUSSON,* OLLE STENDAHL, AND CHRISTER TAGESSON Department of Medical Microbiology, University of Linkoping, S-581 85 Linkoping, Sweden Received for publication 7 July 1977

Sensitization of smooth Salmonella typhimurium 395 MS bacteria with hyperimmune anti-MS immunoglobulin G (IgG) antibodies increased the liability to hydrophobic interaction as assessed by the affinity for a column of Octyl-Sepharose. After sensitization, the material originally eluted with 1 M (NH4)SO4 in a 0.01 M phosphate buffer (pH 6.8) was not desorbed until the ionic strength was reduced to nil, and 0.1% (vol/vol) Triton X-100 in the 0.01 M phosphate buffer was used as eluant. Furthermore, by including positively charged bis-trimeth-

ylamino-polyethylene glycol (PEG) or negatively charged bis-sulfoamino-PEG in an aqueous two-phase system of dextran T500 and PEG 6000, the partition of the IgG-sensitized bacteria was affected by either of the polymers, whereas that of the parent bacteria was not. The hydrophobic effect of IgG binding was enhanced by complement. With heat-inactivated complement, the effect of IgG was diminished. The F(ab')2 fragment showed a much lower capacity to promote a hydrophobic interaction than the complete IgG molecule. The reaction between serum factors, such as immunoglobulins and complement, and microorganisms in general confers upon the latter the character of being more rapidly taken up by professional phagocytes, such as polymorphonuclear granulocytes (14, 21), or nonprofessional phagocytes, such as amoebae (6). This has been regarded as being due to a specific interaction between an activated part of the immunoglobulin (13, 17) or complement (03) (12, 21) and the phagocytic cell membrane, or as a consequence of altered general physicochemical surface properties of the bacteria such as charge and hydrophobicity (3, 14, 19, 20, 22-25). To further challenge the latter hypothesis, we have studied the effect of sensitizing smooth Salmonella typhimurium 395 MS bacteria, using hydrophobic interaction chromatography (HIC) (8, 16) and aqueous biphasic partitioning (1). We have found that S. typhimurium 395 MS, being hydrophilic and uncharged (11), gained a liability to hydrophobic and charge interaction by reacting with immunoglobulin G (IgG) and that the hydrophobic effect was amplified by complement. MATERIALS AND METHODS Preparation of bacteria. The smooth S. typhimurium 395 MS was maintained at 40C on agar slants before use. The bacteria were grown overnight (16 h)

at 37°C in 10 ml of the following enriched glucose medium: glucose, 1 g; Na2HPO4 .2H20, 0.6 g; NaCl, 1.8 g; peptone (Difco), 10 g; beef extract (Difco), 5 g; and deionized water, 1,000 ml; pH 7.4. They were metabolically labeled by introducing 10 or 25 pl of Lleucine (NET-460, L-[3,4,5-3H]leucine; NEN Chemical GmbH, Dreieichenheim, West Germany) into 10 ml of the cultivation medium. After heat inactivation at 56°C for 1 h, they were washed four times (1,100 x g, 15 min) in phosphate-buffered saline, pH 7.3. The radioactivity was measured in a beta-scintillation counter (Isocap 300, Searle-Nuclear, Chicago, Ill.). Alternatively, the bacteria were heat killed directly after cultivation in the absence of radioactive compounds, washed twice in phosphate-buffered saline, and labeled with Na25"CrO4 as described elsewhere (18). They were quantitated by their optical density at 650 nm, which was calibrated with viable count and microscopic counting in a Biirker chamber (depth, 0.02 mm).

Preparation and labeling of IgG and F(ab')2. The production of rabbit hyperimmune sera against S. typhimurium 395 MS was performed as described by Edebo and Normann (4). The serum was fractionated on a column with diethylaminoethyl-celiulose and eluted with 0.01 M phosphate buffer, pH 7.6 (20). IgG was quantitated by the optical density at 280 nm in a Beckman DU-2 spectrophotometer (Beckman Instruments Inc., Fullerton, Calif.) after calibration with standard protein. Only one precipitation line against goat anti-rabbit serum was obtained. A reaction of identity with goat anti-rabbit IgG at immunodiffusion appeared. The pepsin digestion of IgG to 261

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prepare F(ab')2 was performed, with slight modifications, according to Forsgren and Sjoquist (5). The procedure has been described in detail elsewhere (20). The digested material was fractionated on a G-200 column (Pharmacia Fine Chemicals, Uppsala, Sweden). It was quantitated by the absorption at 280 nm, as with the IgG. Immunodiffusion gave one precipitation line with goat anti-rabbit serum. Partial identity with an IgG precipitation line was obtained. Radioactive labeling of IgG and F(ab')2 was made with 125I as described by Stendahl et al. (20). Sensitization of bacteria. For the analysis with HIC, 0.1 ml of the bacterial suspension was mixed with 0.10 ml of the IgG or F(ab')2 dilutions and, in some experiments, with complement (guinea pig normal serum, 1/10). The concentrations are given in context with the results. The mixtures were incubated for 30 min at 37°C and centrifuged for 15 min at 1,100 x g, and the pellet was resuspended in a 0.01 M phosphate buffer, pH 6.8, containing 1 M (NH4)2SO4. For partitioning in the two-phase system, 0.5 ml of each IgG dilution was incubated for 30 min at 37°C with a pellet of washed (4x), 5"Cr-labeled bacteria. Before partitioning, the sensitized bacteria were washed in phosphate-buffered saline. HIC (8, 16). The HIC procedure was performed essentially as described by the manufacturer of OctylSepharose (15). However, ordinary Pasteur pipettes were used as columns. The tip of the pipette was filled with glass wool, on which was laid a nylon net (70,um pores) and about 1 ml of the gel. Thus, about 1 ml of space was left for the eluant. The flow rate (22°C) was 20 ml/h. The columns were equilibrated with the starting buffer, 1 M (NH4)SO4 in 0.01 M phosphate buffer (pH 6.8), whereupon a 0.1- to 0.5ml sample was added. Elution was made by decreasing stepwise the concentration of (NH4)2SO4 to nil and at the same time increasing the concentration of Triton X-100 from 0 to 0.1% (vol/vol). Finally, distilled water and 95% ethanol were used. All eluants except the latter two were 0.01 M with respect to phosphate and had a pH of 6.8. Two-phase partitioning. The two-phase systems used (1) contained 4.4% (wt/wt) polyethylene glycol (PEG) 6000 (Carbowax 6000; Union Carbide, New York, N.Y.) and 6.2% (wt/wt) dextran T500 (Pharmacia Fine Chemicals AB, Uppsala, Sweden) in 0.03 M tris(hydroxymethyl)aminomethane buffer, pH 7.0. It was equilibrated at 4°C overnight in a separation funnel. Then the bottom phase (rich in dextran) and the top phase (rich in PEG) were collected and stored

charged PEG, bis-trimethylamino [(CH3)3N+-]-PEG (TMA-PEG), or negatively charged PEG, bis-sulpho (-SO3 )-PEG (S-PEG) (10).

RESULTS HIC. (i) Sensitization of S. typhimurium 395 MS with IgG. The elution pattern on HIC of untreated and treated S. typhimurium 395 MS is shown in Fig. 1. With untreated bacteria most of the material (75%) came off with the void volume. After sensitization three peaks appeared, i.e., the original, a second weak one eluted with 0.25 M (NH4)2SO4-0.075% (vol/vol) Triton X-100 and 0.1 M (NH4)2SO4-0.09% (vol/vol) Triton X-100, and a third eluted with 0.10% (vol/vol) Triton X-100, distilled water, and 95% ethanol. Figure 2 shows the relative percentage of material in the three regions for different concentrations of IgG. A gradual transfer of material from the first to the second and third areas was observed. The recovery of bacteria varied between 95 and 107% for IgG concentrations up to 150 Ag/ml. With 300, 600, and 1,200 jig/ml it was decreased to 86, 87, and 69%, respectively. In I cn z

x 0

0

1.0 0.5

0.5 0.05

ur 0

D.25 0.075

E

0.10 0.09

0.

0

0.10

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Aq.dest. Ethanol

separately. For the partitioning studies, 2 ml of the top and 2 ml of the bottom phase were pipetted into graded test tubes together with 0.1 ml of the sample containing 5tCr-labeled bacteria. After the material was mixed, the tubes were kept at 4°C for 45 min to separate the phases. Then 0.5-ml samples were withdrawn from the top and bottom phases, and the radioactivity of these and that remaining in the tube was measured in an automatic gamma spectrometer (CG 30; Intertechnique SA, Plaisir, France). The distribution of bacteria was calculated from the counts and volumes of the phases. To assess charge on the bacteria, 6.25 or 12.5% of PEG was exchanged with either positively

0

5

10

El ution volume ( ml )

FIG. 1. Elution pattern after HIC of S. typhimurium 395 MS (2.1 x 109/ml; PH]leucine labeled) unsensitized ( ) and sensitized with hyperimmune IgG (75 ug/ml [- - -] and 1,200 ug/ml [-----). The ) were 0.01 M with respect to elution buffers ( phosphate and of pH 6.8, except for ethanol (95%o) and distilled water.

SMOOTH S. TYPHIMURIUM SENSITIZED WITH IgG

VOL. 18, 1977

the microscope the bacteria appeared slightly aggregated at the highest IgG concentration. A separate experiment with '25I-labeled IgG showed that a minimum of around 2,700 IgG molecules per bacterium was required to achieve a detectable transfer of material from region I to II in the chromatogram. The amount of IgG bound to the bacteria was directly proportional to the concentration at sensitization. A negligible proportion, less than 5%, of the "I activity was obtained from the nylon net, whereas a larger proportion, up to 10 to 15%, was found in the glass wool of the Pasteur pipette. (ii) F(ab')2. The effect of the F(ab')2 fragment on the physicochemical character of S. typhimurium 395 MS was tested by using HIC in the same way as with IgG. At equal quantities of bacteria, the concentration of F(ab')2 had to be four to five times greater to achieve a hydrophobic effect compatible with that of IgG (not shown by figure). (iii) Complement. The effect of complement was tested in HIC as before by comparing the elution patterns of bacteria, bacteria sensitized 100

with IgG, and bacteria sensitized with IgG and fresh or heat-inactivated complement (Table 1). It was evident that fresh complement potentiated the effect of IgG such that less material was eluted in high salt concentrations, i.e., in region I as defined by Fig. 1. In contrast, heatinactivated complement diminished the effect of IgG. Two-phase partitioning. Charge effects at IgG binding were evaluated by two-phase partitioning in a dextran-PEG system with either positively or negatively charged PEG derivatives. Table 2 shows that sensitization with IgG tended to move the bacteria from the top phase towards the bottom phase in the system without charged polymers (20). At greater IgG concentration interaction was observed with both SPEG and, to a lesser extent, TMA-PEG.

DISCUSSION Specific IgG antibodies increase the phagocytosis of smooth S. typhimurium 395 MS by polymorphonuclear leukocytes possibly by augmenting the hydrophobicity of the bacteria (3, 19, 20). This explanation is consistent with the finding that the unopsonized bacteria are apparTABLE 1. Effect of complement on S. typhimurium 395 MS presensitized with hyperimmune IgG, as assessed by HIC

0O.

Bacteria (%) in region:

0

50

/0