INFECTION AND IMMUNITY, Apr. 1978, p. 267-2730019-9567/78/0020-0267$02.00/0Copyright © 1978 American Society for Microbiology

Vol. 20, No. 1

Printed in U.S.A.

Neisseria gonorrhoeae Acquire a New Principal Outer-Membrane Protein When Transformed to Resistance to

Serum Bactericidal ActivityJ. F. HILDEBRANDT,t L. W. MAYER,tt S. P. WANG,' AND T. M. BUCHANAN2*

Immunology Research Laboratory, U.S. Public Health Hospital, Seattle, Washington 98114,2 and theDepartments ofMedicine2 and Pathobiology,' 2 University of Washington, Seattle, Washington 98195'

Received for publication 16 December 1977

Resistance to the complement-dependent bactericidal activity of normal humanserum is found in nearly all Neisseria gonorrhoeae strains causing disseminatedgonococcal infection. Transformation of serum-sensitive gonococcal strain NRL7189 to serum resistance using deoxyribonucleic acid from three separate dissem-inated-infection gonococci was accompanied by simultaneous structural and an-tigenic changes in the principal outer-membrane protein (POMP) of the trans-formants. In each of 10 separate transformations, there was a reduction in subunitmolecular weight of the POMP from that in the recipient (39,000) to that in thedeoxyribonucleic acid donors (36,500). Also, in each instance the POMP antigenictype, as measured by enzyme-linked immunosorbent assay, converted from thatof the recipient to an antigenic type common to each DGI donor strain. Thisconversion of POMP antigen was reflected in part by changes in the surfacefluorescence of the transformed gonococci to the microimmunofluorescence pat-tern of the donor strains. These results suggested that serum resistance ofgonococci is related to the possession of a POMP of characteristic subunitmolecular weight and antigenicity.

Neisseria gonorrhoeae strains resistant to thecomplement-dependent bactericidal activity ofnormal human serum were first observed bySpink and Keefer (19) in 1937 and subsequentlyfurther characterized by Schoolnik et al. (17).To further characterize this property, we trans-formed serum-sensitive gonococci to serum re-sistance using deoxyribonucleic acid (DNA)from serum-resistant organisms, then evaluatedthe associated characteristics of the transform-ants. The transformation to serum resistancerequired two steps. The first transformation pro-duced gonococci with intermediate resistance tobactericidal activity. Repeat transformation ofthese intermediate transformants yielded sec-ond-step transformants that were equally as re-sistant to killing by normal human serum as theDNA donor. The outer-membrane protein com-positions of both the first- and second-step trans-formants were compared with those of the DNAdonor and recipient strains. These changes inouter-membrane proteins (4, 7, 9) were furthercorrelated with alterations in antigenicity ofouter-membrane proteins (T. M. Buchanan,

t Present address: Sandoz Forschunginstitut, Vienna, Aus-tria.

tt Present address: Department of Microbiology, Univer-sity of Rochester School of Medicine and Dentistry, Roches-ter, NY 14642.

in press; T. M. Buchanan and J. F. Hildebrandt,submitted for publication) and surface immu-nofluorescence (21).

MATERIALS AND METHODSBacteriological techniques and transforma-

tion procedure. Three strains of N. gonorrhoeae,NRL 1384, 2031, and 6305, were isolated from patientswith disseminated gonococcal infection (DGI). TheDNA from these strains was prepared as describedpreviously (5) and used to transform a single recipientstrain, NRL 7189, using 10 jig of DNA per ml, asdescribed previously (14, 18). Serum-resistant trans-formants were selected by testing transformed cellsuspensions in the serum bactericidal system (below;12, 17). Four first-step transformants (NRL 7220, 7652,7657, and 8032) with intermediate resistance to serumbactericidal activity were again transformed withDNA from the donor strains to yield 10 second-steptransformants (NRL 7221, 7653-7657, 8075-8077,8080) that were completely resistant to killing bynormal human serum (Table 1).AU isolates were identified as N. gonorrhoeae by

oxidase reaction, Gram stain, and sugar fermentationreactions (2). Gonococcal strains were preserved at-70'C in a medium consisting of 5% (wt/vol) bovineserum albumin and 5% (wt/vol) monosodium gluta-mate (6).

In preparation for extraction of outer-membraneantigens, organisms were first subcultured twice onGC agar base medium (Difco Laboratories, Detroit,

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268 HILDEBRANDT ET AL.

TABLE 1. Transformation sequences by strainDNA do- Intermedi-

Sequence nor .reint ate trans- Furans-strains formats formats

I 6305 7189 7220 7221II 6305 7189 7220 7655

III 6305 7189 7220 7657IV 6305 7189 7220 7653V 6305 7189 7652 7656VI 1384 7189 7657 7654VII 2031 7189 8032 8075VIII 2031 7189 8032 8076IX 2031 7189 8032 8077X 2031 7189 8032 8080

Mich.) containing 1% defined supplement (23) andwere then inoculated onto 15-cm-diameter plates ofthe same medium composition for mass production.After incubation for 18 to 22 h at 360C in an atmos-phere with 5% C02, organisms were harvested into 0.1M NaCl and processed as described below for analysisof their outer-membrane proteins.

Transformation and selection for increased resist-ance to serum killing were performed as follows. Re-cipient organisms were exposed to DNA from donorstrains at 370C in a previously described broth (15).Samples were removed at 1- to 2-h intervals for up to22 h and assayed for transformants to serum resistanceto determine the time required for phenotypic expres-sion of this trait. After we learned that transformationto serum resistance was not detected until after 10 hof incubation. and that it reached a plateau at 11 to 12h, all subsequent transformation incubations were con-tinued for 12 hi Aliquots of the transformation suspen-sion were diluted to 5 x 103 colony-forming units perml, and 50 pl of this suspension was added 50 ILI ofheat-inactivated, pooled human serum (diluted 1:20 inmedium 199) together with 50 Il of a 1:2 dilution of acomplement source consisting of serum from a patientwith agammaglobulinemia. The viable count was de-termined for each experiment by including wells thatcontained 50,ul of bacterial suspension and 100 1d ofmedium 199. The contents of each well were plated,incubated, and enumerated. An equivalent portion ofthe cell suspension not treated with DNA was run inparallel as a control along with the serum and comple-ment controls. In addition, a parallel transformationexperiment, with another equivalent portion of thecell suspension, was performed using DNA from theserum-sensitive strain NRL 6777. This transformationmixture served as a control for possible nonspecificeffects of DNA on the expression of serum resistanceby recipient cell suspensions. Control and transfor-mation mixtures were assayed at least in triplicate.The number of colonies from each well was enumer-ated after 24 h of incubation. Alternatively, serum-resistant transformants were selected by a procedurebased upon the bactericidal assay of McCutchan et al.(12). After incubation for 12 h, the cells that had beenexposed to DNA, and the control cell suspensions,were diluted in medium 199 to 2 x 104 to 105 colony-forming units per ml. Twofold serial dilutions of eachcell suspension were prepared, and 50-1il amounts ofeach dilution were placed in microtiter trays. Freshnormal human serum (50 til of a 1:4 dilution in medium

199) from an individual with no history of gonorrheawas added to each well. Determinations were done intriplicate. Incubation and plating was identical to theprevious selection procedure. Serum-resistant trans-formants were isolated from a point in the twofoldserial dilution of the cell suspension at which nogrowth was present on the controls but -5 colonieswere present from the transformed mixture. Bothmethods used for the selection of serum-resistanttransformants gave equivalent results.Serum bactericidal assay. Gonococci were mea-

sured for their resistance to complement-dependentkilling by normal human sera by the method of School-nik et al. (17). The bactericidal test was performed ina microtiter system using disposable U-well trays and50-ml diluters (Cooke Lab Products Div., Alexandria,Va.). Serum from 10 people with no history of gonor-rhea was pooled, filter sterilized, and heat inactivatedbefore use. The source of complement was a personwith x-linked agammaglobulinemia whose serumlacked bactericidal activity for N. gonorrhoeae.The reactive mixture contained 50 ,tl of bacteria

suspension (600 colony-forming units harvested in logphase), 50 ll of serum in different dilutions, and 50,Ilof complement in medium 199 (activity, 50% hemolyticcomplement units per ml). The incubation time at37°C in a shaking incubator was 60 min before 1 dropof the suspension from each well was seeded ontoprewarmed plates. Growth medium was GC base, sup-plemented with 1% Kellogg supplement (23). Plateswere incubated for 24 h at 37°C in a 5% CO2 atmos-phere. The colonies were counted, and the reciprocalof the serum dilutions producing 75% reduction incolony-forming units compared with the controls wasdefined as the bactericidal titer.ELISA for POMP antigen. Gonococcal principal

outer-membrane protein (POMP) antigen was purifiedas previously described (Buchanan and Hildebrandt,submitted for publication). Briefly, the 130,000 x gouter-membrane pellet material (10) was chromato-graphed over a Sepharose 6B column (1.5 by 90 cm)containing 1.5% sodium deoxycholate and 5 mM gly-cine buffer (pH 9.0). The POMP eluted at the voidvolume free of contaminating lipopolysaccharide andother proteins. This protein in 2-,ug/ml concentrationswas used to coat polystyrene tubes for an enzyme-linked immunosorbent assay (ELISA) (Buchanan,in press, Buchanan and Hildebrandt, submitted forpublication). The enzyme conjugate was horseradishperoxidase linked to goat anti-rabbit immunoglobulin(Cappell Laboratories, Cochranville, Pa.), and thechromagen was o-phenylenediamine. Serotyping ofgonococci based specifically on POMP antigen wasperformed by using gonococci to inhibit the binding ofantibody to purified POMP antigen coated to tubes.Organisms containing POMP of the same antigenictype as the POMP coated to tubes inhibited theELISA test color development, whereas organismswith heterologous POMP antigens did not (Buchananand Hildebrandt, submitted for publication). For thisstudy, strains were tested against only one POMPserotype. This POMP antigen was obtained fromstrain 7122, a strain with the characteristics associatedwith an ability to cause DGI (11, 17, 24). This POMPantigenic type was chosen because each of the three

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GONOCOCCAL PROTEIN RELATED TO SERUM RESISTANCE

DGI DNA donor strains inhibited antibody binding tostrain 7122 POMP antigen in the ELISA test, but didnot inhibit antibody binding to any of five other puri-fied POMP antigenic types (Buchanan and Hilde-brandt, submitted for publication).

Extraction of POMP. Bacteria were suspended in0.15M NaCl (40C) and centrifuged for 10 min at 12,000x g. The bacterial pellet was suspended in a buffercontaining 0.2 M lithium acetate and 0.01 M ethylene-diaminetetraacetic acid adjusted to pH 5.9 in a pro-portion of 1:10 (weight of organisms per volume ofbuffer) and then shaken vigorously at 450C for 2 h inthe presence of glass beads (6 mm OD). The fluid wasthen centrifuged for 10 minutes at 12,000 x g and thesupernatant was retained and centrifuged for 40 minat 30,000 X g. This supernatant was then spun at130,000 x g for 1 h. The pellet obtained from thisprocedure consisted of a crude outer-membrane prep-aration (10). This pellet was suspended in 1 ml ofbuffer [0.01 M tris(hydroxymethyl)aminomethane(pH 7.6), 0.15 M sodium chloride, 0.02% sodium azide]and preserved at -70'C until used.

Polyacrylamide gel electrophoresis. Sodiumdodecyl sulfate-10% polyacrylamide slab gel electro-phoresis (13, 20) was performed for comparative anal-ysis of the partially purified gonococcal POMP prep-arations. A 50-pl volume the POMP material wasmixed with an equal volume of 2% sodium dodecylsulfate and 2% 2-mercaptoethanol (pH 7) and heatedin boiling water for 5 min. After cooling, 50 pl ofbromophenol blue in 80% (wt/vol) sucrose was added,and a 50-1il sample of the mixture was applied to thegel.The gel (15 by 11 cm) was subjected to electropho-

resis at 90 mA for 6 h and then fixed in 5% trichloro-acetic acid overnight. The fixed gel was then stainedfor 2 h in 0.25% Coomassie brilliant blue R (SigmaChemical Co., St. Louis, Mo.) dissolved in 50% meth-anol-10% acetic acid. Destaining was performed in10% acetic acid-20% methanol containing Dowex 1 x8 anion exchange and Dowex 50 W-X8 cation exchangeresins (BioRad Laboratories, Richmond, Calif.).

Micro-IF immunotyping. Microimmunofluores-cence (micro-IF), immunotyping was performed asdescribed by Wang et al. (21). Briefly, Formalinizedwhole gonococci were used as test antigen and asimmunogen for the preparation of antisera in mice.By using pen points, dots of homologous and heterol-ogous strain antigens (each mixed with normal yolksac fluid) were placed on the microscope slide so thata single bacteriological loop of serum could cover agroup of antigens. By using a template, nine groups ofan identical set of antigens were placed on a singleslide so that serial dilutions of an antiserum could betested on the same slide. An indirect fluorescent anti-body technique was utilized. The highest dilution ofserum resulting in a definite fluorescence of the rim ofthe bacterial cell was considered the serum titer forthat particular strain. The titers obtained for immuneserum to heterologous strains were expressed as thepercentage of the titer obtained for that serum pro-duced to the homologous strains. Antigenic similaritieswere based upon cross-reaction patterns. By extensivecross-testing, strains have been classified into types A,B, and C, and further to Al, A2, A3, B1, B2, B3, C1, orC2.

RESULTS AND DISCUSSIONIt was of interest that transformation to serum

resistance required two separate steps, and re-quired 10 to 12 h for each step. This suggeststhat two separate genetic events may be re-quired to produce strains that possess serumresistance equal to strains isolated from patientswith the DGI syndrome. The transformation offirst-step transformants to full serum resistanceoccurred at a frequency of approximately 0.2%.The prolonged time required to produce serum-resistant transformants contrasts with transfor-mation of most previously reported gonococcalgenes, which required 2 to 4 h for phenotypicexpression (14, 18). Some traits, for exampleresistance to penicillin or loss of a growth re-quirement for uracil, have required up to 6 h forfull phenotypic expression (14). Inoue et al. (8)have presented evidence to show that the one-hit theory of immune hemolysis could be ex-tended to the immune bactericidal reaction. Thiscould explain the unusually long time requiredfor phenotypic expression. If the entire outerlayer of the gonococcus must be replaced toeliminate even a single site of bactericidal actionbefore we could detect any increase in resistanceto serum, several generations would be required.Under the growth conditions used, about 10generations would take place during incubationbefore serum-resistant gonococci could be de-tected.The gonococcal serum resistance selected by

passage in serum and reported by McCutchanet al. (12) was unstable and probably did notrepresent DNA-mediated transformation orother genetic alteration at two loci. Instead, itmay have represented a phenotypic change stim-ulated by growth conditions, which revertedupon transfer to other media. The fully serum-resistant transformants in this study have re-tained their strain 7122 POMP antigenic type,serum resistance, and micro-IF characteristicsdespite multiple passages on artificial solid agarmedia.As shown in Fig. 1 and 2, the transformation

of serum-sensitive gonococci to serum resistance(Tables 1 to 3) was accompanied by simultane-ous change in one or more proteins of the 130,000x g pellet material. As shown previously byJohnston and Gotschlich (9), and confirmed byBuchanan and Arko (4) and by Heckels (7), atleast one of these proteins, one with a subunitmolecular weight in the rage of 36,000 to 39,000was an outer-membrane protein. This proteinhas been termed POMP. A consistently ob-served change in each of the 10 separate trans-formations analyzed in these studies was a re-

duction in subunit molecular weight by approx-imately 2,500 in the POMP for each of the fully

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270 HILDEBRANDT ET AL.- ,~.......FIG. 1. Sodium dodecyl sulfate-10% polyacrylamide slab gel ofpartially purified outer-membrane prepa-

rations (130,000 x g pellet material) or gonococci. Sequence of transformation: DNA donor-recipient-inter-mediate transformant-full transformant. Sequence (i) 6305- 7189- 7220- 7221; (ii) 6305- 7189- 7220- 7655; (iii) 6305-7189- 7220- 7657; (iv) 6305- 7189- 7220- 7653; (v) 6305- 7189- 7652- 7656; (vi) 1384- 7189- 7651- 7654.

resistant transformants, as compared to gono-cocci of intermediate or extreme serum sensitiv-ity (Fig. 1 and 2, Tables 1 to 3). Furthermore, allfully serum-resistant strains (donors and fullyserum-resistant transformants) contained

TABLE 2. Biological and immunological propertiesof N. gonorrhoeae strains listed in Fig. I

SDS- ELISAPAGEa well Strain Bacteri- Micro-IF (strain 7122

no. cidal titer serotype POMP)

1 6305 s2 A2 +2 7189 256 B2 -3 7220 32 B2 -4 7221 C2 A2 +5 7655 C2 A2 +6 7657 52 A2 +7 7653 C2 A2 +8 7652 16 B2 -9 7656 s2 A2 +10 7654 s2 A2 +11 7651 16 B2 -12 7189 256 B2 -13 1384 s2 A3 +

a SDS-PAGE, Sodium dodecyl sulfate-10% poly-acrylamide slab gel electrophoresis.

POMPs of nearly identical subunit molecularweights (Fig. 1 and 2). This POMP subunitmolecular weight was approximately 36,500, andthis particularPOMP may be in part responsiblefor the resistance of these strains to comple-ment-dependent killing by normal human se-rum. It has been demonstrated that lipopolysac-

TABLE 3. Biological and immunological propertiesof N. gonorrhoeae strains listed in Fig. 2

SDS- Batr-McoI ELSPAGE' well Strain Bacteri- Micro-IF E 2ISA

no. cidal titer serotype POMP)

1 2031 s2 A3 +2 7189 256 B2 -3 8032 264 B2 -4 8080 s2 B2/A +5 8075 s2 B2/A +6 8076 -c2 B2/A +7 8077 s2 B2/A +8 2031 s2 A3 +9 7189 256 B2 -10 8032 264 B2 -

aSDS-PAGE, Sodium dodecyl sulfate-10% poly-acrylamide slab gel electrophoresis.

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GONOCOCCAL PROTEIN RELATED TO SERUM RESISTANCE 271I-fl re~~~~~d _ f .

FIG. 2. Sodium dodecyl sulfate-10% polyacrylamide slab gel ofpartially purified outer-membrane prepa-rations (130,000 x g pellet material) of gonococci. Sequence of transformation: DNA donor-recipient-inter-mediate transformant-full transformant. Sequence (i) 2031-7189-8032-8080; (ii) 2031-7189-8032-8075; (iii) 2031-7189-8032-8076; (iv) 2031-7189-8032-8077.

charide antigens react with bactericidal antibod-ies to result in killing of gonococci (16, 20). Onemight hypothesize, therefore, that this proteinin some way masks lipopolysaccharide surfaceantigens to prevent their recognition by bacte-ricidal antibodies in human serum (17).

Strains of N. gonorrhoeae that cause DGI arecharacteristically serum resistant (17), andWang et al. have determined that the greatmajority of strains fall into similar immuno-sub-types A2-A3 in the micro-IF test (21). For ex-ample, the strains used as DNA donors for thesestudies (NRL 1384, 2301, and 6305) were all DGIstrains, and all were classified as members ofsubtypes A2-A3 (serogroup A) by the micro-IF.Of 37 Seattle strains obtained from patients withthe DGI syndrome, 35 were subtypes A2-A3 (21).As shown in Tables 1 to 3, intermeidate trans-

formants retained the same micro-IF serogroupas the recipient (subtype B2, serogroup B),whereas transformants to full serum resistanceacquired complete (94io) or partial (4Mlo) immuno-fluorescence characteristics of immunotype A

coincident with a change in the subunit molec-ular weight of their POMP to approximately36,500. Interestingly, all 10 of the fully serum-resistant transformants also acquired strain 7122POMP antigen on their surfaces, the samePOMP antigen type expressed on the surface ofeach of the donors (Tables 2 and 3).

It is likely that the shift to immunotype A wasdue at least in part to a change in POMP antigen,since this was the only consistent protein changeobserved (Fig. 1 and 2) and since Apicella (1)has determined that lipopolysaccharide sero-types vary extensively within DGI strains (un-published collaborative data). This suggests thatthe POMP is at least one of the antigens in-volved in surface immunofluorescence of gono-cocci as measured in the micro-IF system andconfirms Heckels' (7) suggestions that this pro-tein is involved in surface immunofluorescence.The combination of both shared antigenicityand common subunit molecular weight of thePOMP for each of the donor and fully serum-resistant transformants suggests that this

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272 HILDEBRANDT ET AL.

POMP may be related to an ability to resistkilling by normal human serum, and perhaps tothe capacity to cause disseminated infections.Further ELISA typing of POMP antigens inmany strains of gonococci is needed to determinethe actual prevalence of the strain 7122 POMPantigenic type in serum-resistant as comparedwith serum-sensitive organisms, and in strainscausing disseminated as compared with localizedinfections.

ACKNOWLEDGMENTS

We are grateful to Stanley Falkow and King Holmes foradvice and criticisms regarding the manuscript, and to DeniseRose for performing the bactericidal assays.

This research was supported by Public Health Servicecontract NO 1-AI-52535 and grants RO 1-AI-13149 and Al-12192 from the National Institute of Allergy and InfectiousDiseases and grant EY-00219 from the National Eye Institute,by Federal Health Programs Service Project SEA 76-43, andby a research grant from Sandoz Forschungsinstitut, Vienna,Austria.

LITERATURE CITED1. Apicella, M. A. 1976. Serogrouping of Neisseria gonor-

rhoeae: identification of four immunologically distinctacidic polysaccharides. J. Infect. Dis. 134:377-383.

2. Bodily, H. L., E. L. Upkyke, and J. W. Mason (ed.).1970. Diagnostic procedures for bacterial, mycotic andparasitic infections, 5th ed., p. 296. American PublicHealth Association, New York.

3. Buchanan, T. M. 1975. Antigenic heterogeneity of gono-coccal pili. J. Exp. Med. 141:1470-1475.

4. Buchanan, T. M., and R. J. Arko. 1977. Immunity togonococcal infection in guinea pigs induced by vacci-nation with isolated outer membranes of gonococci. J.Infect. Dis. 135:879-887.

5. Catlin, B. W. 1960. Transformation of Neisseria menin-gitidis by deoxyribonucleates from cells and from cul-ture slime. J. Bacteriol. 79:579-590.

6. Greaves, R. I. H. 1960. Preserving living cells by freeze-drying. Ann. N.Y. Acad. Sci. 85:723-728.

7. Heckels, J. E. 1977. The surface properties of Neisseriagonorrhoeae: isolations of the major components of theouter membrane. J. Gen. Microbiol. 99:333-341.

8. Inoue, K., Y. Akiyama, T. Kinoshita, Y. Higashi, andT. Amano. 1976. Evidence for a one-hit theory in theimmune bactericidal reaction and demonstration of amulti-hit response for hemolysis by streptolysin 0 andClostridium perfringens theta-toxin. Infect. Immun.13:337-344.

9. Johnston, K. H., and E. C. Gotschlich. 1974. Isolationand characterization of the outer membrane of Neis-

seria gonorrhoeae. J. Bacteriol. 119:250-257.10. Johnston, K. H., K. K. Holmes, and E. C. Gotschlich.

1976. The serological classification of Neisseria gonor-rhoeae. I. Isolation of the outer membrane complexresponsible for serotype specificity. J. Exp. Med.143:741-758.

11. Knapp, J. S., and K. K. Holmes. 1975. Disseminatedgonococcal infections caused by Neisseria gonorrhoeaewith unique nutritional requirements. J. Infect. Dis.132:204-298.

12. McCutchan, J. A., S. Levine, and A. I. Braude. 1976.Influence of colony type on susceptibility of gonococcito killing by human serum. J. Immunol. 116:1652-1655.

13. Maizel, J. G. 1971. Polyacrylamide gel electrophoresis ofviral proteins. Methods Virol. 5:179-246.

14. Mayer, L. W., G. K. Schoolnik, and S. Falkow. 1977.Genetic studies on Neisseria gonorrhoeae from dissem-inated gonococcal infections. Infect. Immun.18:165-172.

15. Mayer, L. W., K. K. Holmes, and S. Falkow. 1974.Characterization of plasmid deoxyribonucleic acid fromNeisseria gonorrhoeae. Infect. Immun. 10:712-717.

16. Rice, P. A., and D. L. Kasper. 1977. Characterization ofgonococcal antigens responsible for induction of bacte-ricidal antibody in disseminated infections. The role ofgonococcal endotoxin. J. Clin. Invest. 60:1149-1158.

17. Schoolnik, G. K., T. M. Buchanan, and K. K. Holmes.1976. Gonococci causing disseminated gonococcal infec-tion are resistant to the bactericidal action of normalhuman sera. J. Clin. Invest. 58:1163-1173.

18. Sparling, P. F. 1966. Genetic transformation of Neisseriagonorrhoeae to streptomycin resistance. J. Bacteriol.92:1364-1371.

19. Spink, W. W., and C. S. Keefer. 1937. Studies of gono-coccal infection. II. The bacteriolytic power of the wholedefibrinated blood of patients with gonococcal arthritis.J. Clin. Invest. 16:177-183.

20. Tramont, E. C., J. C. Sadoff, and C. Wilson. 1977.Variability of the lytic susceptibility of Neisseria gon-orrhoeae to human sera. J. Immunol. 118:1843-1851.

21. Wang, S. P., K. K. Holmes, J. S. Knapp, S. Ott, andD. D. Kyzer. 1977. Immunologic classification of Neis-seria gonorrhoeae with microimmunofluorescence. J.Immunol. 119:795-803.

22. Weber, K., and M. Osborn. 1969. The reliability ofmolecular weight determination by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem.244:4406-4412.

23. White, L. A., and D. S. Kellogg, Jr. 1965. Neisseriagonorrhoeae identification in direct smears by a flu-orescent antibody-counterstain method. Appl. Micro-biol. 13:171-174.

24. Wiesner, P. J., H. H. Handsfield, and K. K. Holmes.1973. Low antibiotic resistance of gonococci causingdisseminated infection. N. Engl. J. Med. 288:1221-1222.

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