rickettsial hemolysis: adsorption, desorption, readsorption,

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  • INFECTION AND IMMUNITY, Sept. 1977, p. 607-612Copyright C) 1977 American Society for Microbiology

    Vol. 17, No. 3Printed in U.S.A.

    Rickettsial Hemolysis: Adsorption, Desorption, Readsorption,and Hemagglutination

    HERBERT H. WINKLER

    Department ofMicrobiology, University of Virginia Medical School, Charlottesville, Virginia 22901

    Received for publication 5 April 1977

    The energy-dependent adsorption of radioiodinated rickettsiae to sheep eryth-rocytes was demonstrated. The iodination procedure, however, decreased thehemolytic activity of the rickettsiae. No desorption of rickettsiae from isolatedrickettsia-erythrocyte complexes (prevented from lysing by NaF) could be mea-sured. On the other hand, rickettsiae desorbed from this complex during or afterlysis and readsorbed and lysed other erythrocytes. Thus, the usual hemolyticassay measures multiple rounds of adsorption and lysis. Although lysis of therickettsia-erythrocyte complex was insensitive to anti-rickettsial rabbit serum,adsorption and readsorption were completely inhibited by such antiserum. He-magglutination of erythrocytes by rickettsiae was observed (in the presence ofNaF to prevent lysis) and was sensitive to the same inhibitors as adsorption.

    Bacterial cell envelope interaction with eu-caryotic cell membrane is a cardinal feature ofany mechanism of bacterial invasiveness andhost defense. Rickettsia prowazeki, an obligateintracellular parasite with a morphologicallytypical gram-negative cell envelope (3, 4, 7), isan excellent (although specialized) model forsuch studies because of the extraordinary evolu-tionary pressures on this organism to perfect itsinteraction with its host.

    Lysis of an erythrocyte by a rickettsia is acomplex interaction involving adsorptive andlytic steps that can be experimentally distin-guished (8, 9). Such a model system has manyof the properties involved in initiation of para-sitism of a host cell, and its simplicity is a defi-nite experimental advantage. We now knowthat adsorption is not passive but is dependentupon the energy of the rickettsiae, most likely aprotonmotive force (9, 12). Cholesterol is a keypart of the receptor on the erythrocyte (10).Rickettsiae are able to adsorb to erythrocyteghosts and to both inside-out and right-side-outvesicles derived from ghosts (14). Rickettsia-erythrocyte complexes can be prepared basedon the fact that lysis but not adsorption is in-hibited by fluoride ions (9).

    In the present study I investigated the follow-ing questions. (i) Can adsorption of radioiodi-nated rickettsiae to erythrocytes be measureddirectly? (ii) Is there a measurable desorption ofrickettsiae from the erythrocyte in the absenceof lysis? (iii) In the presence of NaF to preventlysis, can a rickettsia interact with more thanone erythrocyte so as to effect cross-bridging oferythrocytes and resultant hemagglutination?

    (iv) Is an adsorbed rickettsia able to dissociatefrom the erythrocyte after (or during) lysis andthen lyse another erythrocyte?

    MATERIALS AND METHODSRickettsiae preparation and growth. R. prowa-

    zeki, Madrid E strain, was propagated in embryo-nated, antibiotic-free hen eggs by inoculation with 0.2ml of a 10-5 dilution of a seed pool (yolk sac passageno. 273 and 274). Rickettsial suspensions were pre-pared from heavily infected yolk sacs by a modifica-tion of the methods of Bovarnick et al. and Wissemanet al. (2, 15) as previously described (13). Only fresh,unfrozen rickettsiae were used.The diluent for the rickettsial inoculum and rick-

    ettsial suspension in the purification procedure was asucrose-phosphate-glutamate (SPG) solution origi-nally devised by Bovarnick et al. (1). The diluent forthe sheep erythrocytes and hemolysis activity assayswas SPG-Mg containing 0.01 M MgC12.Adsorption and hemolysis. The hemolysis tests

    used were modifications of the method of Snyder etal. (11) as previously described (12). Adsorption wasmeasured after the free unadsorbed rickettsiae wereseparated from rickettsia-erythrocyte complexes bycentrifugation for 450 x g for 7 min at 4C as previ-ously described (8). The supernatant fluid (unad-sorbed rickettsiae) was then assayed for rickettsial he-molytic activity by using additional erythrocytes.Rickettsia-erythrocyte complexes were prepared bywashing the sedimented fraction.

    Labeling of rickettsiae. To prepare 'nI-labeledrickettsiae, the organisms were suspended in 1 ml ofsodium phosphate buffer (0.12 M, pH 7.4) to whichwas added: glucose, 100 umol; Na1sI, 20 jtCi; lactoper-oxidase, 2 U; and glucose oxidase, 1 U. The mixturewas incubated at room temperature for 20 min, di-luted with 10 ml of sodium phosphate buffer contain-ing NaASSI3 (10 pM), and centrifuged at 12,000 x g for

    607

  • 608 WINKLER

    10 min. The rickettsiae were washed in sodium phos-phate buffer and then in SPG, resuspended in SPG,layered on top of 10 ml of Renografin-76 (20%[vol/vol] in SPG), and centrifuged at 27,000 x g for 20min. The rickettsial pellet was then washed and sus-pended in SPG-Mg.Chromium labeling. Erythrocytes were labeled

    with 5'Cr by incubating them at 50% (vol/vol) in NaCl(0.85%) with 5'Cr (40 uCi/ml) for 30 min at 230C. Thecells were then washed three times in NaCl and twicein SPG-Mg.Antiserum. Rabbits were injected intradermally

    at multiple sites with rickettsiae (1 mg of protein)sonically oscillated with complete Freund adjuvant.They were subcutaneously boosted weekly for 3weeks with rickettsiae (1 mg of protein) sonically os-cillated with incomplete Freund adjuvant. The serumfrom three rabbits was pooled, heat inactivated at56C for 1 h, and stored at -20C. The serum wastested for antihemolytic activity by incubating serialdilutions of serum (0.1 ml) with 0.1 ml of rickettsiae(about 0.2 mg of protein per ml) for 10 min at 40C,adding 0.4 ml of sheep erythrocytes (25%), and deter-mining hemolysis after incubation at 340C for 1 h.Inhibition was complete at a 1:20 dilution. Proteinswere determined by the method of Lowry et al. (6).

    RESULTSAdsorption of radioiodinated rickettsiae.

    Table 1 shows the results of experiments inwhich the adsorption of 125I-labeled rickettsiaewas monitored either indirectly by measuringhemolytic activity remaining in the supernatantfluid after sedimenting the '25I-labeled rickett-sia-erythrocyte complexes or directly by count-ing the unadsorbed radioactivity in the superna-tant fluid after sedimentation of complexes. Al-though adsorption could be readily demon-strated by either method, both methods yieldedvalues lower than routinely obtained with un-treated rickettsiae (in which adsorption wasgreater than 90% [8]). This indicated that theiodination process had impaired the ability ofthe rickettsiae to adsorb. In addition, the totalhemolytic activity of the rickettsiae was alwaysdecreased 50 to 70% by iodination. The additionof NaF to the incubation did not inhibit adsorp-tion, whereas KCN almost completely abolishedit. The effect ofKCN could not be examined bythe hemolytic assay because the KCN-treatedrickettsiae were unable to lyse erythrocytes inthe second incubation. However, as previouslydescribed, adenosine 5'-triphosphate was able torestore the ability of KCN-poisoned rickettsiaeto adsorb to and lyse erythrocytes (12).The time of incubation was varied to further

    characterize the impaired ability of the iodi-nated rickettsiae to adsorb to erythrocytes (Fig.1). Panels A and B show adsorption measuredby radioactivity and residual hemolytic activity,respectively. Both assays confirmed that at 00C

    TABLE 1. Adsorption of iodinated rickettsiae tosheep erythrocytes

    Addition to incubation Adsorptionb (mediUMa A,. 125I

    None (control) 58 43NaF (l0mM) 67 59KCN (1 mM) ND 10KCN (1 mM) plus ATP 48 38

    (1 mM)a 125I-labeled rickettsiae (0.2 mg ofprotein per ml, 30

    to 50 kcpm/ml) were incubated with 2 volumes oferythrocytes (25%, [vol/vol]) for 10 min at 340C withthe indicated additions. ATP, Adenosine 5'-triphos-phate.

    bThe erythrocyte-rickettsial complexes and freeerythrocytes were separated from the unadsorbedrickettsiae by low-speed centrifugation. Percent ad-sorption was measured by determining either the he-molytic activity of the unadsorbed rickettsiae or theradioactivity of the unadsorbed rickettsiae relative tothat at 00C. The average of two experiments is shown.ND, Not determinable.

    8 16 24 32MINUTES MINUTES

    FIG. 1. Kinetics of adsorption of iodinated rickett-siae to sheep erythrocytes. (A) Radioactive rickettsiaeremaining in the supernatant fluid after removal oferythrocytes and erythrocyte-rickettsial complexes bylow-speed centrifugation at the incubation time andtemperature indicated. (B) Residual hemolytic activ-ity in these supernatant fluids. The arrow in (A)indicates the initial radioactivity. For the assay ofhemolytic activity in (B), 1 volume of supernatantfluid was added to 10 volumes of erythrocytes (5%,vol/vol) in SPG plus MgC42 (20 mM) to dilute theinhibitory effects of NaF. Symbols: 0, 0C; *, 0Cplus 10 mM NaF; EO, 34C; U, 34C plus NaF; A0C plus 1 mM KCN; A, 34C plus KCN. BKG,Background radioactivity.

    the rickettsiae did not adsorb (8, 9). At 00C (Fig.1B), adsorption in the presence of fluoride ap-peared greater than in its absence because afterdilution some inhibitor remained in the secondincubation. In the presence of NaF at 340C(where adsorption could occur without lysis en-suing [9]), almost complete adsorption occurredin the hemolytic assay but not in the isotopic

    INFECT. IMMUN.

  • RICKETTSIAL HEMOLYSIS 609

    assay. This is because the isotopic assay mea-sured both metabolically intact and dead rick-ettsiae, whereas the hemolytic assay measuredonly those able to hemolyze erythrocytes. Theradioactive assay (Fig. 1A) showed that KCNinhibited adsorption, whereas in the hemolyticassay (Fig. 1B) the curve KCN only represents abackgr

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