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S T U D I E S O N G O N O C O C C U S I N F E C T I O NIV . PI Ll : TH EIR ROLE IN ATTACHMENT OF GONOCOCCI TO

TISSUE CULTURE CELLS*BY JOHN SWANSON

(From the Department of Pathology, University of Utah College of Medicine, Salt LakeCity, Utah 84112)(Received for publicatio n 23 October 1972)

Recent s tud ies on Neisseria gonorrhoeae have yielded suggestions that the surfacesof these organisms play vital roles as pathogenetic determiners in gonococcal infec-tions. Virulence has been correlated with specif ic gonococcal colony forms by Kellogget al. (1, 2) . This f inding was established through inoculation of human volunteerswith organisms der ived f rom the var ious co lony types tha t could be morphologica l lyd if fe ren t ia ted . Colony types 1 and 2 produced typ ica l acu te venerea l in fec t ion ,whereas co lony types 3 and 4 produced only mild , t r ans ien t symptoms withoutestablishment of infection in the genital tract. This colony type-virulence correlationwas extended by the studies of Swanson et al. (3) and of Jephcott et al. (4) whodemons tra ted p i l l on the gonococc i of types 1 and 2 and the absence of p i l l o r organ-isms from types 3 and 4 colonies. These studies suggest a correlation between viru-lm ce and the presence of pill on gonococci.

I n th e s tu d y c o r r e la t in g p r e s e n c e of p i l l w i th c o lo n y mo r p h o lo g y o f g o n o c o c c ii t wa s s u g g e s te d th a t th e s e c e l l wa l l a p p e n d a g e s mig h t p la y a r o le in v i r u le n c e b yp r o m o t i n g a t t a c h m e n t o f t h e o r g a n is m s t o c e l l s o f th e p o t e n t i a l l y i n f e c t e d h o s t( 3 ) . Th i s h y p o th e s i s i s c e n t r a l to th e p r e s e n t ly d e s c r ib e d s tu d ie s in wh ic h th ep r e s e n c e o f p i l l i s f o u n d to b e a s s o c ia t e d wi th e n h a n c e d a t t a c h m e n t o f g o n o c o c c it o h u m a n a m n i o n c e ll s i n v i tr o . L i g h t a n d e l e ct r o n m i c r o s c op e o b s e r v a t i o n ss u p p o r t t h i s fi n d i n g a n d t h e l a t t e r p r o v i d e p a r t i a l v i s u a l i z a t i o n o f t h e a t t a c h -m e n t o f g o n o co c c i m e d i a t e d b y t h e i r p i l l.

Malerials and MethodsGonococci.--The strains used were freshlv isolated from patient material at the MountSinai Hospital , New York. Iso lation, identification, and pro pagat ion have been describedprevio usly in de tail (3 ). Gonococci used in experiments were grown for 18 h on GC aga r basesupplemented with IsoVitaleX (Baltimore Biological Laboratories, Baltimore, Md.) and weresuspended in Earle's basic salt solution (Grand Island Biological Co., Grand Island, N. Y.)for determination of optical opacity and suitable dilution before their addition to tissue* This work was supported by contract D ADA 17 72 C 2146 of the U.S. Army MedicalResearch and Development Command, and by research grant AI-11187 from the U.S. PublicHealth Service.

THE JOURNAL OF EXPERIMENTAL MEDICINE VOLUME 137, 1973 571

Published March 1, 1973

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572 STUDIES ON GONOCOCCUS INI?ECTION. IVculture cells. Dilution and plat ing of organisms for enumeration of colony-forming units werecarried out in Medium 199 (Grand Island Biological Co.) and on Mueller-Hinton agar (DifcoLaboratories, Inc., Detroit, Mich.) plates, respectively. The inoculated plates were incubatedovernight in either candle extinction jars or in an incubat or with 5% CO2 atmosphere.

Tissue Culture Cdls.--Human amnion cells were obtained from Dr. Joseph Sonnabend(Mount Sinai School of Medicine) and were maintained in either minimal essential mediumor Medium 199 (M-199) (Grand Isla nd Biological Co.) Mth Ear le' s salt s, L-glutamine, and10% heat-inactivated (at 56C for 30 rain) fetal calf serum (Grand Island Biological Co.).Penicillin and streptomycin were included in the medium for propagation of the tissue cul-tures but were removed by repeated washing of the monolayers with antibiotic-free mediumbefore use of the cells for atta chme nt experiments.

Incubation of Gonococci with Amnion C el ls .-With monolayers: Monolayers of human amnion cells, propagated as described above,

were utilized when they were about 75-80% confluent on 9-cm diameter tissue culture dishes.The monolayers were repeatedly washed with antibiotic-free medium to remove penicillinand streptomycin and then were overlaid with 2 ml of a suspension of gonococci (approx.1 )< l 0 s colony-forming units/ ml) in M-199 containing 2% bovine serum al bumin (BSA) 1(Schwarz-Mann, Orangeburg, N. Y.). Incuba tion in a 5% CO2 atmosphere at 37C proceededfor 1-2 h a fter which the monolayers were vigorously washed with f resh M-199, fixed with 2%glutaraldehyde in 0.1 M sodium cacodylate, and stained with 1% Giemsa stain for 30 rain.These incubations were carried out with both colony types 2 and 4 (T-2 and T-4) in parallelstudies. Evaluation of comparative results on attachment of gonococci to cells of the mono-layers was carried out with a Zeiss photomicroscope (Carl Zeiss, Inc., New York).

With cells in suspension: Amnion cells that had been removed from monolayer cultureswith 0.004% ethylenedi aminetet raaceti c acid disodium and 0.1% t rypsin were centrifugedand washed with M-199. After enumeration the cells were diluted in M-199 with 2% BSAto a final concentra tion of 4-6 X 106/ml. 2 ml of this amnion cell suspension was mixed withan equa l volume of the gonococcal suspension contai ning approx. 1 X 104-1 X 105 colony -forming units/ml in M-199 Mth 2% BSA. The total volume of the incubation mixture was 4ml and this was rotated at 30 rpm in a polypropylene tube (12 x 75 ram) at 37C. 1-ml ali-quots were removed immediately after mixing bacteria and amnion cells and after 50- and100-rain rotation-inc ubations. These were centrifuged (500 rpm for 5 min) and the super-nat ant was removed for dilution and enumera tion of colony-forming units present. T he nu m-ber of organisms enumerated by dilution and plating of this supernatant represents the or-ganisms not associated with amnion cells. The sediment containing amnion cells and cell-assoc iated gonococci was washed with i ml of M-199, resed imen ted (500 rpm for 5 rain),diluted in 1 ml of fresh M-199, placed in a sonicating bath for 5 rain, diluted, and plated fornumber of gonococci prese nt. Similar condi tions were used for suspensions of gonococci th atwere rotate d-incuba ted in the absence of amnion cells to determine the organisms that appearin e ach fraction in the absence of cells.

Critical Point Drying.--Amnion cells were propagated on glass microscope slides cut to fitthe critical point drying (CPD) appa ratus specimen chamber, cleaned, autoclaved, and placedin a 9 cm P etri dish. After washing to remove antibiotics the tissue culture cells were exposedto a suspension of T-2 gonococci and were incubated for 1 h at 37C in a CO2 atmosphere.The slides were then thoroughly washed to remove bacteria not attached to amnion cells,immersed in 2% glutaraldehyde in 0.1 M sodium cacodylate for 5 rain, washed in 0.1 M caco-dylate, and imme rsed for 5 min in 1% osmium tetroxide in 0.9% sodium chloride. The speci-men slide was passed through graded ethanol and amyl ac etate solutions and sub.iected tocritical point drying with CO2 in a CPD-1 apparatus (Denton Vacuum, Inc., Cherry Hill,

1 Abbreviations used in this paper: BSA, bovine serum albumin; CPD, critical point drying.


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jo~IN SWANSON 573N. J . ) as descr ibed in the opera t ing brochure supplied with the equipment (5) . The onlymodif ica t ion ut i l ized was tha t f ina l venting t ime was 2 h af ter wh ich specimens were removedand placed in a Balzers 360 M appara tus (Balzers High Vacuum Corp. , Santa Ana, Calif . )for p la t inum -c a rbon e va pora t ion . The p la t inum -sha dowe d c a rbon-s ta b i l i z e d spe c im e n wa sdipped in 0.2% Formvar in e thylene dichlor ide , br ief ly dr ied, and immersed in Clorox.Fragments of the replica re leased from the s l ide af ter 12-36 h immersion in Clorox werewashed severa l hours in dis t i l led water and were placed on naked copper gr ids for e lec tronmicroscope examination. All photographs were pr inted without reversa l .

Electron Microscopy. --Amnion cells for thi n sec t ioning were e i ther f ixed in s i tu a s m ono-layers overla id with gonococci or were sedimented from rota t ion-incubation with gonococciand f ixed as a suspension of ce lls . Fixa t ion with 2% glutara ld ehyde in 0.1 M sodium cacodyla tepH 7.0 was followed by osmicat ion, en bloc s ta in ing w i th 1% ura nyl a c e ta te , de hydra t ion , a ndembedding as previously descr ibed (3) . Elec tron micrographs were taken e i ther with an AEIEM8 01 microscope (AEI Scientific App ara tus , Inc . , E lmsford, N. Y.) or with a SiemensElmiskop IA (Siemens Corp. , Ise l in, N. J . ) .

RESULTS

Exposure o f Am nio n Cell Monolayers to Gonococci (Light Mic rosco py).--In cu-bat io n of T 4 gonococci with am nion cel l mon olaye rs resul ts in l i t t le associa t ionbetween bacteria and amnion cel ls (F ig. 1) . Occas ional gonococci appear toadhere to the plas t ic subs tra te of the t issue cul ture dish between cel ls of them onolaye r , bu t few gonococc i a re ac tua l ly in con tac t wi th the t i s sue cu l tu rece l l s . Th i s i s in m arked con t ra s t to the p ic tu re ob ta inab le a f t e r incuba t ion o fam nion ce ll m onolaye rs wi th T -2 gonococci equ iva len t in num ber to those usedin the T -4 expe r im ents . T -2 gonococc i exh ib i t m ark ed adhe rence to the am nioncel ls (F ig. 2) . Not only are the organisms dumped in their associa t ion with theeuka ryo t ic ce ll s, bu t a l so the num b ers o f c lum ps a re abundan t . Num erousgonococci appear to adhere in the perinuclear region of the amnion cel ls . Thism ay be re la ted to the re la t ive ly f l a t con tour o f the pe r iphe ra l cy top la sm , theraised convex contour of the nucleus , and the concave profi le of the perinuclearzone. This variable thickness of different port ions of the t issue cul ture cel lmak es different ia t ion of extracel lular from the intracel lular gonococci imposs iblebecause gonococci may be seen a t a level , through fine focus ing of the l ightmicroscope, below that a t which the top of the nucleus is in focus.Rotation-Incubation of Amnion Cells with Gonococci.--To de te rm ine whe the rdifferences in a t tachment of gonococci to amnion cel ls could be corre la ted withcolony type, s tudies were carried out ut i l iz ing T-2 and T-4 gonococci exposed tosuspens ions of amnion cel ls in paral le l experiments . Associa t ion with or a t tach-ment to amnion cel ls is defined as appearance of gonococci in the sediment asopposed to the supe rna tan t , which con ta ined gonococc i tha t d id no t cen t r i fu -gal ly separate with amnion cel ls and, therefore , are not cel l associa ted. Controlstha t d id no t con ta in anm ion ce ll s were inc luded to de te rm ine the num ber o fgonococc i tha t appea red in the s ed im ent bu t d id no t represen t ce l l -a s soc ia tedorgan i sm s . The da ta a re t abu la ted e i the r a s num ber o f co lony- fo rm ing un i t s(Tab le I ) o r a s the pe rcen tage o f gonococc i recove rab le a t a g iven t im e po in t in


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574 STUDIES ON GONOCOCCUS INFECTION. IV

FIGS. 1 and 2. Amnion cells in monolayer culture have been exposed to gonococci, washed,fixed, and stained for light microscopy with Giemsa stain. In Fig. 1 only a few nonpilated,T-4 gonococci (arrows) are found in association with the amnion cells. This is in contrast toamnion cells that have been exposed to pilated, T-2 gonococci (Fig. 2) and that have numer-ous, clumped organisms on their surfaces (arrows), particularly in the perinuclear regions.X 1,400.

TABLE IDistribution of Gonococci after Rotatlon-Incubation with A mnlon Cells

Rotation incubationCells added 0 time 50 m[n 100 rain

(total) Supernate Sed ime nt Sup er na te SedimentT- 2a lo ne 2 X 10 6 1.3 X 10 6 7.5 X 10 ~ 1 X 10 6 3 X 101T- 2q -a m~ qo n 2 X 10 6 5.3 X 10 5 2.8 X 10 6 7.7 X 10 4 1.8 X 10 ~

cellsT-4 alone 3 X l0 G 9 X 10 ~ 2.3 X 10 5 4. 5 X fOR 2.3 X 10 4T-4 -?a m:~ ion 3 X 10 6 1.5 X 10 6 5.6 X 10 5 1.4 X 10 5 2.8 X 10 4

cellsT-2 and T-4 gonococci were incubated either alone or with amnion cells as described inMateria ls and Met hods. The nu mbe r of gonococci noted (0 time [total]) were rotat ed with

6 X 10 a amni on cells where designated. After variable times of incu bation at 37C allquo tswere removed, centrifuged, and the n umber of gonococcal colony-forming units determined inthe sup etnate a nd sediment flactions. T-2 alone and T-4 alone are specimens not co ntainingamnion cells that were included to determme the distribution of gonococci that is independentof the presence of amnion cells.


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J O I I N S W A N S O N 5 7 5

t he se d ime n t f r a c t ion a s c ompa re d w i th t he t o t a l num be r o f gonoc oc ci r e c ove re din the spec imen (sediment p lus superna te ) (Fig . 4) .Li t t le d i f ference is found in the to ta l re cover y of T-2 an d T-4 g onococc ifrom incub a t ion w i th amnion cel ls . Both co lony types exhibi t a decrease inthe to ta l c o lony-fo rming uni ts recovered a f te r 50- or 90-min incuba t io ns wi thamnio n ce l ls (Fig . 3) . No chan ge in colonial morp holo gy was foun d by com par-

C9

8O80Z0r..O:-1

0

107 -

106 -

105 -

6n Ce l l s

! !50 MIN 100 MIN

I N C U B A T I O N T I M E

FiG. 3. The to tal recovery of gonococci from mixtures of T-2 or T-4 organisms ~it h amnioncells appears to b e independent of colony type. Am nion cells and gonococci wer e mix ed asdescribed in Materials and M ethods and the to tal number of colony-forming units (CFU)recovered in both the sediment and supernate fractions determined by dilution and plating ofaliquots after 5Q- and lee-rain incubations.

ing input organisms wi th those recovered a f te r exposure to amnion ce l ls , i .e . ,there d id not seem to be a se lec t ive process for one or another colony typeduring the short incuba t ions of these exper iments .

Segrega t ion of gonococc i in to ce l l -assoc ia ted (sediment) and non-ce l l -asso-c ia ted (superna te ) f rac t ions is s t r ik ingly dependen t on the colony type ofgonococc i used . T-2 gonococc i consistent ly d ist r ib ute pr edom inan t ly in thece l l -assoc ia ted f rac t ion , whereas T- 4 gonococc i show prefe rent ia l d is t r ibu t ionin the non-ce l l -assoc ia ted or superna te f rac t ion . A typica l exper iment demon-st ra t ing th is d i f fe rence is shown in Table I in which the number of colony-forming uni ts recovered is g iven from each frac t ion . In the amnion ce l l - f reecontro ls (T-2 a lone and T-4 a lone , Tabl e I ) as wel l as in the mix ture of T-4gonococc i and a mnion ce lls (T-4 -I - amnio n ce l ls) , the superna te conta ins tw oto fou r fold m ore c o lony - fo rming un i ts t ha n a re found in the se d ime n t . Bycontras t , a f te r e i ther 50- or 100-min incuba t ions wi th amn ion ce l ls (T-2 - t -amnion ce lls), the n um ber of T-2 gonococc i in the ce l l -assoc ia ted f rac t ion is a t


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576 S TU D I ES O N G O N OC O C C U S I NF EC TI O N . I V

1 0 0

80 .2;

z 6a

rjUO 40(.)O

20 "+ +

! 150 MIN 100 MIN

INCUBATION INCUBATIONFIG. 4. The distribution of gonococci in the sediment, cell-associated fraction is quite

different depending on whether nonpilated T-4 or pilated T-2 gonococci are incubated withamnion cells. Controls which do no t contain amnion cells (T-4 and T-2) were utilized to de-termine the number of gonococci that appear in the sediment fraction but which are not asso-ciated with amnion cells. The se probably represent organisms that are clumped in suspensionto the extent that they sediment under the centrifugation conditions noted in Materials andMethods. In these experiments the number of colony-forming units (CFU) present in thesediment fraction was compared with th e total CFU after 50- and 100-rain incubations alone(T-4 and T-2) and with amnion cells (T-4 + Am C and T-2 + Am C).

l e a s t t w i c e t h a t f o u n d i n t h e s u p e r n a t e . T h i s p r e f e r e n t i a l d i s t r i b u t i o n i s s e e np e r h a p s m o r e c l e a r l y i n t h e c o m p o s i t e d a t a d e r i v e d f r o m f o u r e x p e r i m e n t scarr ied out on d i f ferent days (F ig . 4 ) . In th is f igure the amnion ce l l - free contro lv a l u e s ( T - 2 a l o n e a n d T - 4 a l o n e ) a r e s i m i la r . T h e p e r c e n t a g e o f c o l o n y - f o r m i n gu n i t s f o u n d i n t h e c e l l - a s s o c i a t e d f r a c t i o n ( s e d i m e n t ) i s m a r k e d l y i n c r e a s e dw i t h T - 2 g o n o c o c c i a s c o m p a r e d w i t h T - 4 o r g a n i s m s ( T - 2 + a m n i o n c e ll s v s .T - 4 + a m n i o n c e l ls ) .

Electron M icroscopy of Gonococci-Amnion Cell M ix tur es.--A dequate e v a l u a -t i o n o f t h e a s s o c i a t i o n b e t w e e n g o n o c o c c i a n d a m n i o n c e l l s re q u i re s e l e c t r o nm i c r o s c o p e e x a m i n a t i o n o f b o t h t h i c k a n d u l t r a t h i n " t h i n " s e c t i o n s a s w e l l a sc r i ti c a l p o i n t d r i e d s p e c i m e n s . T h e m e r e d i f f e re n c e i n s e c t i o n t h i c k n e s s i s i n s t r u -m e n t a l i n v i s u a l i z a t i o n o f t w o a p p a r e n t l y d i s t i n c t m o r p h o l o g i c a l t y p e s o f a s s o -a c i t i o n b e t w e e n t h e p r o k a r y o t i c a n d t h e e u k a r y o t i c c e l l i n t h i s s y s t e m . I n


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JOHN SWANSON 577

thicker sections (dark-to-medium gold interference colors) pili are visualizedfor considerable distances along their lengths even if they do not course pre-cisely parallel to the p lane of section. In ultra thin sections (silver to gra y inter-ference colors) only short segments of the pili usually are visible due to theimprobability of extensive portions of pili lying in a plane parallel to the verythin section. Thus, pili are seen in the thick sections as electron-opaque 80-fitthick fibrils extending radially from the gonococcus' surface (Figs. 5-7). Innumerous instances multiple pili from a single bacterium appear to contactdifferent points of an amnion cell (Figs. 5 and 6). The actual point of contactfor each pilus with the plasma membrane is not visible; therefore, one can notdetermine whether the tip or a lateral surface of the pilus contacts the amnioncell's membrane. Pili are not seen in similar thick sections of T 4 gonococciand amnio n cells as expected from the absence of pili on these organism s demon-strat ed b y negative stainin g (3) and freeze-etching (6).

Par tic ipation of pil i radiating from the gonococcal surface in a tta chm ent oforganisms to amnion cells can be assessed also through critical point drying.This relatively little-used technique (7, 8), although somewhat limiting resolu-tion, has a dist inct advan tage over other metho ds of specimen preparation forobserving thL type of association. Negative sta ining delineates structure ofradiating pil l but does not a l low adequate v isualization of bacter ia l or am nioncell surface topology. Freeze-fracture, freeze-etching ade qua tely exhibits eitheramnio n cell or gonococcal surface structure, bu t definition of the former necessi-tates use of glycerol, which precludes visualization of the freeze-etch exposedexterior surfaces of bacteria. Further, pili radiating from gonococci are poorlyseen by freeze-fracture, freeze-etch techniques. Thi n sections offer little appre-ciation of the third dimension in spatial relationships between gonococci andamnion cells . Simple a ir drying and subsequent h eavy m etal shadowing yieldserious artifacts of shrinkage with poor visualization of cell surface topology.Crit ical point drying (CPD) has the advan tage tha t volume and configurationartifacts are minimized such that relationships between gonococcal and amnioncell surfaces can be seen, as in Figs. 8-12.

Gonococci prepared by CPD have th e appearance shown in Figs. 8-12 an dseen best in Fig. 8. The co nvoluted gonococcal exterior has adher ent pili (analo-gous to the freeze-fracture, freeze-etch appearance) as well as pili radiatingfrom its exterior . Radiating pil i lying on the substra te hav e diameters similarto those obtained by other methods (3, 6), but if pili are elevated above thesubst rate level, their diameters are increased due to the accumu lated thicknessesof carbon, pla tinum, and F ormv ar of the replica . This enhanced thickness, aswell as the position of the shad ow derived from each pilus, is helpful in iden tify-ing those surface appendages oriented in space above the plane of the substrateon which the cells are supported.After CPD preparation , am nion cells of mono layers hav e gonococci atta che dvia their pili as shown in Figs. 9-12. The majority of gonococcal pilus-amnion


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F IG S. 5 - 7 . P i l a t ed T - 2 gonococc i a r e numer ous in a s s oc ia t ion w i th amn io n ce l ls bo th a f t e rexpos u r e to th e ce l l s in s u s pens ions ( F ig . 5 ) o r a s mono lave r s ( F ig s . 6 and 7 ). P i l i can bes een ex tend ing f r om the gonococc i ( a r r ow s) tow ar d th e p la s ma m em br an es o f amn ion ce l l sin thes e th i ck s ec t ions . I n s ome in s t ance s ( F ig s . 5 and 6 ) s eve r a l p i l i cou r s e f r om each o r gan -ism to the amn ion cel l surface. X 80,000 (F igs . 5 and 6) an d X 60,000 (F ig . 7) .


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JOHN SWANSON 579

cell connect ions appear to invo lve f ingerl ike surface project ions of the amnioncel ls . These project ions are found on most parts of amnion cells af ter C PD andare especia l ly numero us cen trad from the peripheral , ap ronl ike leading edge ofcytoplasm. Str iking examples of pi lus-cytoplasmic project ion a t tachmentsinvolve bacter ia l and amnion cel l s t ructures ra ised above the plane of the sup-port ing subs tra te as ascerta ined by heavy metal shadows derived from theses t ructures (Figs . 10-12). As noted in thick sections of gonococci and amnioncells , several pili from a single bacterium may communicate with a singleamnion cell (Fig. 9).

Not a l l gonococci associa ted with amnion cel ls have vis ible pi lus-mediatedconnections to the cells . In many instances (Fig. 11) gonococci appear to reston the amnion cell exterior in contact with fingerlike cell projections, but pilir ad ia t ing f rom the bac te r ium a re no t appa ren t . Thi s p robably represen t s asecond type of associa t ion found between gonococci and amnion cel ls as de-scribed below.The second type of associa t ion, between plasma membrane of amnion cel lsand the cell wall of gonococci, is seen clearly only in ultrathin sections. Over-lapping segments of gonococcal cel l wal l and amnion cel l cytoplasm precludeclear visual izat ion of this type of interact ion in thick sect ions . In the u l t ra thinsections, however, segments of the gonococcal cell wall outer membrane lie inclose appos i t ion to areas of the amnion cel l plasma membrane that have con-tours similar to those of the gonoeoccal exteriors (Figs. 13-16). An electron-lucent space 70-90 A wide is cons is tent ly seen between the external-most aspectof the gonococcal cel l wal l and th e exter ior l imi t of the amn ion cell plasma mem -brane. An analogous space is not present in the region of appos i t ion betweenT-4 gonococci and amnion cel ls . The cel l wal ls of T-4 gonococci appear toimpinge direct ly on the plasma m embranes of amnion cells (Figs. 17-20) wi thoutthe intervening space described with T-2 gonococci (Figs . 13-16). Only a fewT-4 organisms have been found closely appl ied to amnion cel ls in numeroussect ions examined, whereas numerous T-2 gonococci are avai lable for morpho-logical s tudy because of the di ffer ing avidi ty wi th which these colony typesadhere to the eukary ot ic cells. Nevertheless , th e di ffer ing morphologies ofassocia t ions between the am nion cells and the two colony types of gonococci:seem con sistent.

Occasional amnion cells , either after exposure to T-2 or to T-4 gonococci,contain int racel lular gonococci (Figs . 21-23). T he bacter ia are enclosed withinmemb rane-l imited ves ic les , appear morpho logical ly intact , and are often seenin the perinuc lear region where membran e-enclosed gonococci appear to impingeclosely on the perinuc lear c is tern (Fig. 23) .

DISCUSSIONGonococci capable of becoming es tabl ished in the male u rethra and producing

class ical s igns and symp toms of acute gonorrheal venereal disease have d is t inc-


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58 0 S T U D I E S O N G O N O C O C C U S I N F E C T I O N . I Vt ive charac te r is t ics . This was shown by Kel logg e t a l. (1 , 2) who not o nly def inedthe colony forms ( ty pes 1 and 2) tha t resu l t f rom growth of v i ru lent gonococc ibut a lso showed tha t o ther organisms (colony types 3 and 4) , which a re Neisseriagonorrhoeae by the usua l b iochemica l and l ight microscope c r i te r ia , a re notassoc ia ted wi th produc t ion of gonorrhea in exper imenta l subjec ts . These f indingsprovided a model system tha t could be used to s tudy v i ru lence fac tors throughcomparison of the pa thogenic and the nonp a thogenic forms of gonococc i . Pil la re demons trable appendages of the ce ll wal ls of v i ru lent co lony type gonococc ibut a re absent f rom the gonococca l surfaces of the avi ru lent co lony types (3 , 4) .

The present s tudy provides evidence for a mechanism through which p i l imay p lay a ro le in de te rmining v i ruJence of Neisseria gonorrhoeae. Th e p re se n c eof p i l l i s assoc ia ted wi th enhanced a t tachment of gonococc i to anmion ce l ls invi t ro . Whether p i l i have a s imi la r func t ion in v ivo in promot ing a t tachment ofgonococc i to cel ls of the urogeni ta l t rac t , an orec ta l mucos a , or nasopha ryngea lt r a c t a w a i t s fu r th e r s tu d y . Su c h a n a t t a c h me n t h y p o th e s i s w i th r e g a rd togonococca l p i l i has been suggested previously (3) and is a t t rac t ive because ofthe "flushing" by secretions, excretions, etc., of the cell-l ined surfaces sus-cept ib le to gonorrhea l infec t ions. Enhancement in gonococca l a t tachment tothese surfaces could conce ivably account for v i ru lence of p i lus-bear ing or-ganisms in contrast to the avi ru lence of gonococc i lacking p i l i .

At tachment of gonococc i to amnion ce l ls seems to have two morphologica l lyd i f fe ren t c o mp o n e n t s . Th e f i r s t c o mp o n e n t o r t y p e o f a t t a c h m e n t i s me d ia t e dthrough p i l l tha t rad ia te f rom the gonococcus ' surface and a re seen in sec t ionsor in c r i t ica l poin t dr ied spec imens. The d iameter of gonococcus bear ing radia l lyor iented p i l l is much grea te r t han tha t of a gonococcus devoid of pi ll . Fur ther ,bac te r ia l p i l l o f ten d isp lay a "st icky" na ture re la t ive to mammalian ce l ls (9) .These a t t r ibutes of p i li a re possib ly influent ia l in de te rmining a t tac hme nt ofgonococci to cells of host targ et t issues.

Th e second morphologica l typ e of a t tac hme nt of gonococc i to amnion ce l lsappears to be more in t imate and involves segments of the organism's ce l l wal lwi th corresponding port ions of the amnion ce l l' s p lasm alemm a. One quest ionconcerning th is type of a t ta chm ent a r ises because of the demonst rable d i f fe rencebe tween T-2 and T-4 gonococc i in the i r apposi t ion to amnion ce l ls . In theformer , T-2 , p i lus-bear ing organisms there i s a consis ten t space separa t ing the

FIG. 8. Gonococci prepared by critical point drying exhibit detail on their surfaces in-cluding pili adherent to the exterior of the organism (arrows). Pill that lie on the substrate(P~) have adherent globular material but have diameters similar to those obtained by nega-tive staining. Pili that are elevated above the substrate surface (Pc) have thicker profiles byvirtue of accumulation of metal and For mvar . X 45,000.Fins. 9 and 10. Pilated gonococci appe ar attach ed to projections of amnion cells by pillwhose profiles (Pc) and shadows (s) demonstrate their position as being above the plane ofthe substrate. The double shadows (s) of the pilus in Fig. 10 reflect platinum and carbonevapor ation b oth of which produced shadows beneath the pilus. X 18,00 0 (Fig. 9)and )< 27,000 (Fig. 10).


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58 2 STUDIES ON GONOCOCCUS INFECTION. IVbac te r ium from the amnion ce l l . With the la t te r , T-4 , nonpi la ted organisms thece l l wal l appears to abut d i rec t ly on the amnion ce l l surface . The d i f fe rencema y be expl icable by v i r tue of the adherent , surface-coa t ing or ien ta t ion of somegonococca l p i l i , demonstra ted recent ly by f reeze-frac ture , f reeze-e tching (6) .Thus, the space present be tween T-2 gonococc i and amnion ce l l membranescould represent p i l i d i f f icu l t to reso lve by e lec t ron microscopy in th is par t icu la ror ien ta t ion and loca t ion. I t i s unc lear whether the two morphologica l types ofgonococcus-am nion ce l l assoc ia t ion r epresent a sequence of events . I t i s possib letha t the in i t ia l a t t achm ent of gonococc i to the eukaryot ic ce lls i s medi a tedthrough the organisms' e longa ted , rad ia l ly ex tended, s t icky p i l l . This mayp ro v id e t e mp o ra ry a t t a c h me n t fo r b a c t e r iu m to a mn io n c el l; e v e n tu a l ly t h etwo ce l ls may come toge ther in the more in t imate contac t media ted by the i rrespec t ive exte rna l membranes. The d i f fe r ing proximity of ce l l wal ls of T-2and T-4 to the amnion ce l l p lasma membrane seems somewhat paradoxica l .T-2 gonococc i adhere more avid ly to the t i ssue cul ture ce l ls ; ye t the i r ce l l wal lsa re separa ted f rom the p lasmalemma of the amnion ce l ls by spaces not presentwi th T-4 gonococc i . I t i s possib le tha t th is c lose or in t imate assoc ia t ion of bac-te r ia wi th eukaryot ic ce l ls i s re la ted to phagocyt ic uptake of the bac te r ia a l -though the smal l number of in t race l lu la r organism s seen in our exper imentsmakes th is unl ike ly . I t i s a lso possib le tha t surv iva l and mul t ip l ica t ion ofgonococc i a re , in some manner , inhib i ted when they a re in such c lose contac twi th eukaryot ic ce l ls .

The ro le of p i l i in promot ing a t ta chm ent of gonococc i to eukaryot ic cel ls i snot nove l . Duguid and Gi l l ies (10) demonstra ted tha t Shigella tha t bear p i l iadhere to in test ina l ep i the lia l ce l ls in v i t ro . These authors corre la ted thepresence of pi l i and a t ta chm ent of these organisms to ep i the l ia l ce lls to v i ru lenceof these Shigella sp. More recent ly Si lverb la t t has shown a corre la t ion be tweenviru lence and p i la t ion of Proteus sp. and h is micrographs of exper imenta l infec-t ions s t rongly suggest tha t a t tachment of the organisms to ep i the l ium of theu r in a ry t r a c t i s me d ia t e d b y p i l l3 Th e se p i lu s -a t t a c h me n t c o r rel a t io n s m a y h a v egenera l impl ica t ions regarding bac te r ia l pa thogenic i ty . Brin ton (9) demon-st ra ted the presence of p i l i on bac te r ia l i so la tes of var ious genera f rom c l in ica lur inar y t r ac t infect ions. I t i s conce ivable tha t p i la t ion is a requisi te for v i ru lence

2 Silverblatt, F. Pe rsonal communication.FI ts . 11 and 12. Pilus-mediated attac hme nt of gonococci can be visualized also onamnion cell projections tha t are abov e the level of the su bstra te (compare position of theshadow of projection to which organisms are attached with those of projections lying to theright o f that projection in Fig. 11) . Gonococci sometimes appear to be completely above the

plane of the substrate , have pili extending to amnlon cell projections (arrows), and r adiat eother pill that are partially adherent to the substrate (P, and Pc). Note also the absence ofdiscernible pill radiat ing fr om gonococci (gc) that lie on the surface of the amnion cell (Fig. 12is an enlarged portion of Fig. 11.) ;< 12,000 (Fig. 11) and X 75,000 (Fig. 12).


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F ins . 13 - 16 . I n u l t r a th in s ec t ions p i li a r e no t v i s ua l i zed on the s u r f aces o f thes e p i l a t ed ,T- 2 gonococc i . The ce l l w a l l s o f the gonococc i a r e f oca l ly appos ed to the p la s ma membr aneso f amn ion ce l l s, bu t an e l ec t r on - lucen t s pace ( a r r ow s ) i s s een be tw een the l imi t ing me mbr anesof bacte r ia a nd t is sue cul ture cel ls. >( 60,0G0.

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FlOs. 17 20 . Occasional nonpi la ted , T -2 gonococci a re seen adhere n t to am nion ce l l s . Th ecel l wal l s o f these organ isms appear to impinge d i rec t ly on foc i o f the amnion ce l l membranes(arrow s). X 75,000 (Figs. 17 and 20) and X 60,000 (Figs. 18 and 19).

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FIGS. 21-23. Gonococci are somet imes observed w ithin the conf ines of amnion cel ls . Thebacter ia are conta ined in membran e- l imited vesicles and may l ie at a distan ce f rom the nucleus(n) as in Fig. 21 or may be located immediately ad jacen t to the per inuc]ear cistern (Figs.22 and 23). (Fig. 23 is an enlarged portion of the cell in Fig. 22.) X 60,000 (Figs. 21 and 23)and X 22,000 (Fig. 22).

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J O H N S WA N S O N 587

o f s e v e ra l k i n d s o f g r a m - n e g a t i v e b a c t e r i a . I t is d o u b t f u l t h a t p i li a l w a y s e n d o wo r g a n i s m s w i t h p a t h o g e n i c a c t i v i t y . N u m e r o u s "nonpathogen ic" Neisseria sp.hav e p i l i (11) bu t a r e no t a s soc i a t ed wi t h c l i n i ca l in f ec t i ons o f hum an hos t s i nw h o m t h e b a c t e r i a r e s i d e . O n t h e o t h e r h a n d , Neisseria sp. a r e c o m m o n l y e n-c o u n t e r e d a s p a r t o f t h e " n o r m a l f l o r a " a n d i t i s p o s s ib l e t h a t t h e i r m a i n t a i n i n ga r e l a t i onsh i p wi t h hos t ce l l s and r ema i n i ng a pa r t o f t h i s f l o r a depend on t he i rpossess ing pi l i .

A l t h o u g h b a c t e r i a l p il i a re l o n g - re c o g n i z e d c o m p o n e n t s o f m a n y g r a m - n e g a -t i v e b a c t e r i a l s p e c i e s , p r i m a r y a t t e n t i o n h a s c e n t e r e d o n t h e s e s t r u c t u r e s a sa t t a c h m e n t s i te s f o r b a c te r i o p h a g e s a n d a s a p p e n d a g e s p o s s i b l y r e la t e d t o s e x u a lm a t i n g o f b a c t e ri a ( 9 ). P i li h a v e a ls o b e e n s t u d i e d a s a n t i g e n s, b u t o n l y r e c e n t l yh a s t h i s c h a r a c t e r i s t i c b e e n e x p l o i t e d f o r d i a g n o s t i c p u r p o s e s . T h e s t u d y o fB u c h a n a n e t a l. ( 1 2) h a s s h o w n t h a t a n t i b o d i es f o r m e d i n r e s p o n s e to g o n o c o c c a lp i li c a n b e d e t e c t e d b y a r a d i o i m m u n o a s s a y w h i c h m a y p r o v e u s e f ul f o r d ia g n o s -i ng gonor rhe a l d isease e spec i a ll y i n unsuspec t i ng , a s sym pt om at i c i nd i v i dua l s .I t i s n o t c l e a r a t p r e s e n t w h e t h e r a n t i p i l u s a n t i b o d i e s f u n c t i o n i n m o d i f y i n ga t t a c h m e n t o f g o n o co c c i t o e u k a r y o t i c c e ll s P r e l i m i n a r y s t u d ie s sh o w t h a t t h e s ea n t i b o d i e s m o d i f y d a m a g e o f t is s u e c u l t u r e c e lls p r o d u c e d b y T - 2 g o n o c o c c i .3W h e t h e r t h i s r e p r e s e n t s a c t i v i t y o f t h e a n t i b o d i e s i n m o d i f y i n g a t t a c h m e n t o ft he p i l a t ed T -2 o rgan i sms o r r e f l ec t s agg l u t i na t i on o f t he o rgan i sms i s no tk n o w n .

T h e p r esen ce o f gonococc i w i t h i n amni on ce ll s i s me re l y an i nc i den t a l f i nd i nga t t h i s p o i n t . A r e c e n t s t u d y r e p o r t i n g a n e x p e r i m e n t a l a n i m a l m o d e l f o r g o n o r -rhea (13) has shown i n t r ace l l uJa r o rgan i sms i n t he g r anu l a t i on t i s sue used fo rg r o w t h o f g o n o c o c c i i n v i v o . F u r t h e r , t h e m o s t c o m p l e t e h i s to p a t h o l o g i c a ldesc r i p t i on known t o me (14) no t es , i n pas s i ng , t he p r esence o f i n t r ace l l u l a rg o n o c o c c i i n s o m e b i o p s y s p e c i m e n s f r o m p a t i e n t s w i t h g o n o r r h e a . T h e s e o b -s e r v a t io n s d o n o t r e l a t e t o i n t r a l e u k o c y t i c go n o c o c c i b u t t o b a c t e r i a w i t h inep i t he l i a l o r con nec t i ve t i s sue ce ll s. T h e i n t r ace l l u l a r l oca t i on o f gonococc i hasi m p o r t a n t p o s s i bl e im p l i c a ti o n s t o c h r o n i c i t y a n d t o t h e r a p e u t i c r e f r a c t i v e n e s sof gonococca l i n f ec t i ons . E xper i ment s on seve ra l a spec t s o f i n t r ace l l u l a r gono-cocca l i n f ec t i ons a r e unde r i nves t i ga t i on . However , t he r e l a t i on o f i n t r ace l l u l a rgonococc i t o genes i s o r con t i nua t i on o f gonococca i i n f ec t i ons i s cu r r en t l y un-c l e ar . O n e g r o u p o f in v e s t i g a t o r s h a s r e c e n t l y s u g g e s t e d t h a t g o n o c o c c i s u r v i v eon l y i f t hey r ema i n on t he mucosa l su r f ace o f ep i t he l i um- l i ned t i s sues (15) .E i t h e r t h i s l oca t i on o f gonococc i o r i n t r ace l l u l a r s i t e s mi g h t d epend , i n i t i a ll y ,o n a n c h o r i n g o f o rg a n i s m s t o t h e e p i t h e li u m .

SUMMARYA t t a c h m e n t o f Neisseria gouorrhoeae t o amn i on ce l ls i n t i s sue cu l t u r e i s fac i l i-

t a t e d i f t h e g o n o c o c c i b e a r p i li . T h i s h a s b e e n d e t e r m i n e d b y s t u d y i n g t h e3 Swanson, J., and M. A . Siam. Unpu blished observation.


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5 8 8 STUDIES ON GONOCOCCUS INFECTION. IVn u m b e r o f p i l a t e d , c o l o n y t y p e 2 g o n oc o c c i a s s o c i a t e d w i t h a m n i o n c e ll s a f t e ri n c u b a t i o n i n v i t ro a s c o m p a r e d w i t h t h e n u m b e r o f n o n p i l a t e d , c o l o ny t y p e 4g o n o c o c c i p r e s e n t w i t h a m n i o n c e l l s u n d e r t h e s a m e c o n d i t i o n s . T h e s e d a t a a r es u p p o r t e d b y l i g h t m i c r o s c o p e f i n d i n g s . E l e c t r o n m i c r o s c o p e s t u d i e s p r o v i d ev i s u a l i z a t i o n of f i ne s t r u c t u r e o f g o n o c o c c a l a t t a c h m e n t G o n o c o c c i a r e a l s of o u n d w i t h i n a m n i o n c e ll s i n t h i s i n v i t r o s y s t e m .

This w ork was carried out with the excellent technical assistance of Carol Par rott , Bar baraZeligs, and Dr. M onir A. Siam.Note Added in Proof.--Since s u b m i s s i o n o f t h i s p a p e r , a n a r t i c l e d e a l i n g w i t h a d -

h e r e n c e o f g o n o c o c c i t o u r e t h r a l m u c o s a l c e l ls fr o m i n d i v i d u a l s w i t h g o n o r r h e a h a sb e e n p u b l i s h e d ( W a r d , M . E . , a n d P . J . W a t t . 1 9 72 . A d h e r e n c e o f Neisseria gonor-rhoeae t o u r e t h r a [ m u c o s a l c e ll s . A n e l e c t r o n - m i c r o s c o p i c s t u d y o f h u m a n g o n o r r h e a .J. Infect. Dis. 1 2 6 : 6 01 ) . I n t h a t s t u d y g o n o c o c c i a r e s h o w n t o a d h e r e t o e p i t h e l i a lc e l l s v i a e x t e n s i v e z o n e s of i n t i m a t e c o n t a c t . I t i s n o t c l e a r w h e t h e r t h o s e z o n e s o fc o n t a c t c o r r e s p o n d t o t h e a t t a c h m e n t r e g i o n s d es c r i b ed i n t h e p r e s e n t p a p e r . F u r t h e r ,p i l l a r e n o t i d e n t i f ie d e it h e r b y t h e a u t h o r s o f t h a t p a p e r o r i n t h e i r a c c o m p a n y i n ge l e c t r o n m i c r o g r a p h s . H o w e v e r , t h e e x t e n t o f g o n o c o c c a l a t t a c h m e n t , a s w e l l a s a p -p a r e n t p a r t i a l p e n e t r a t i o n o f t h e g o n o c o c c i i n t o t h e e p i t h e l i a l c e l l s i n t h o s e s p e c i m e n sf r o m h u m a n s w i t h g o n o r r h e a , i s o f c o n s i d e r a b l e i n t e r e s t i n r e l a t i o n s h i p t o t h e f i n d i n g sh e r e i n r e p o r t e d f o r a n i n v i t r o s y s t e m

R E F E R E N C E S1 . K e l l o g g , D . S . , W . L . P e a c o c k , J r . , W . E . D e a c o n , L . B r o w n , a n d C . L . P i r k l e .

1963. Neisseria gonorrhoeae. I . V i r u l e n c e g e n e t i c a l l y l i n k e d t o c l o n a t v a r i a t i o n .J. Bacteriol. 85 :1274 .

2 . K e l l o g g , D . S . , I . R . C o h e n , L . C . N o r i n s , A . L . S c h r o e t e r , a n d G . R e i s i n g . 1 96 8 .Neisseria gonorrhoeae. I I . C l o n a l v a r i a t i o n a n d p a t h o g e n i c i t y d u r i n g 3 5 m o n t h sin vitro. J. Bacteriol. 96 :596 .

3 . S w a n s o n , J . , S . K r a u s , a n d E . C . G o t s c h l i c h . 1 9 7 1 . S t u d i e s o n g o n o c o c c u s i n fe c -t i o n . I . P i l l a n d z o n e s o f a d h e s i o n : t h e i r r e l a t i o n t o g o n o c o c c a l g r o w t h p a t t e r n s .J. Exp. Med. 134 :886 .

4 . J e p h c o t t , A . E . , A . R e y n , a n d A . B i r c h - A n d e r s e n . 1 9 7 1 . B r i e f r e p o r t : Neisseriagonorrhoeae. I I I . D e m o n s t r a t i o n o f p re s u m e d a p p e n d a g e s t o c e l l s f r o m d i f f e r e ntc o l o n y t y p e s . Acta Pathol. Microbiol. Scan& Sect. B: Microbiol. [mmunol.79 :437 .

5 . O p e r a t i n g i n s tr u c t i o n s fo r D e n t o n V a c u u m C P D - 1 C r i t i c a l P o i n t D r y e r , f o r m n o .2 1 6. 1 9 7 2. D e n t o n V a c u u m , I n c . , C h e r r y H i l l , N . J .

6 . S w a n s o n , J . 1 9 7 2 . S t u d i e s o n g o n o c o c c u s i n f e c t io n . I I . F r e e z e - f r a c t u r e , f r e e z e -e t c h s t u d i e s o n g o n o c o c c i. J . Exp. Med. 136:1258.

7 . A n d e r s o n , T . F . 1 9 5 2 . A m e t h o d f o r e l i m i n a t i n g g r o s s a r t i f a c t s i n d r y i n g s p e c i -m e n s . In E x t r a i t d u C o n g r e s d e M i c r o s c o p i e E l e c t ro n i q u e . E d i t i o n s d e l aR e v u e d ' O p t i q u e , P a r i s .

8 . A n d e r s o n , T . F . 1 96 6 . E l e c t r o n m i c r o s c o p y o f m i c r o - o r g a n i s m s . In P h y s i c a lT e c h n i q u e s i n B i o l o g i c a l R e s e a r c h . A . A . W . P o l l i s t e r , e d i t o r . A c a d e m i c P r e s s ,I n c . , N e w Y o r k . 3 : 3 1 9 .


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J'OHN SWANSON 5899. Brinto n, C. C. 1967. Contr ibutions of pill to the specificity of the bacterial surface

and a unitary hypothesis of conjugal infectious heredity. I n The Specificityof Cell Surfaces. B. D. Davis and L. Warren, editors. Prentice-Hall, Inc.,Englewood Cliffs, N.J. 37.

10. Dugu id, J. P., and R, R. Gillies. 1957 . Fimbr iae and adhes ive properties indyse ntery bacilli. J. Pathol. Bacteriol. 74:397.

11. Wistreich, G. A., and R. F. Baker. 197l. The presence of fimbriae (pili) in threespecies of Neisseria. J. Gen. Microbiol. 65:167.

12. Buchanan, T. M., J. Swanson, and E. C. Gotschlich. 1972. Antibody responseto gonococcal pili in patient s with gono rrhea. Clin. Invest. 51:17a. (Abstr.)

13. Arko, R. J. 1972. Neisseria gonorrhoeae: experimental infection of experimentalanimals. Science (Wash. D.C.) . 177:1200.14. Harkness, A. H. 1948. The pathology of gonorrhoeae. Br. J . Vener. Dis. 9.4:137.

15. Ward, M. E., P. Watt, and A. A. Glynn. 1972. Quoted in Theory of mechanismsof gonococ ci explains difficulties of era dicatio n. Infect. Dis. 9.:1.


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J Exp Med 1973 Swanson 571 89