ILNON AND ImmUNITY, June 1977, p. 766-772Copyright C 1977 American Society for Microbiology

Vol. 16, No. 3Printed in U.S.A.

Differences in the Attachment of Mycoplasma pneumoniaeCells and Membranes to Tracheal Epithelium

MICHAEL G. GABRIDGE,* Y. DEE BARDEN-STAHL, ROBERT B. POLISKY,1 AND JEFFERY A.ENGELHARDT2

Department ofMicrobiology, School ofBasic Medical Sciences, University ofIllinois, Urbana, Illinois 61801

Received for publication 14 January 1977

Hamster trachea organ cultures were exposed to isolated membranes ofMycoplasma pneumoniae, PI 1428. Attachment, monitored by the uptake oftritiated membranes, was relatively insensitive to neuraminidase pretreatment,unlike the attachment of viable cells. Membrane attachment was optimal whenexplants were incubated with 50 to 100 ug of membrane protein per 'ml inminimal essential medium broth while gently being rotated (1 rpm) in a rollerapparatus for 90 to 120 min at 3700. Saturation of the receptor sites with viablecells failed to inhibit subsequent membrane attachment. Induction of squamousmetaplasia by extended cultivation of tracheal explants in a vitamin A-freemedium reduced the content of ciliated cells without significantly affecting totalcell viability, but did not alter the attachment of M. pneumoniae membranes.Collectively, the data indicate that the mechanism of attachment ofM. pneumo-niae membranes to respiratory epithelium is distinct from the receptor site-mediated attachment of M. pneumoniae cells.

Mycoplasma pneumoniae, the only myco-plasma conclusively proven to cause respira-tory disease in humans, is known to attach tosialic acid-containing receptor sites on ciliatedepithelial cells (24, 31). However, the mannerin which this microbe elicits necrosis subse-quent to specific attachment is not currentlyunderstood. In the course of examining the cy-totoxic potential of cellular components, wenoted (10) that high doses of M. pneumoniaemembranes could induce necrosis in trachealexplants. This phenomenon was in accord withan earlier suggestion by Piercy (27) that cellmembranes might be responsible for the celldamage associated with M. mycoides infection.It was also consistent with our reports on thecytotoxic potential of M. fermentans mem-branes for laboratory mice (11) and cell cultures(13) and with the recent observation that nonvi-able Ureaplasma preparations can induce cil-iostasis and necrosis in bovine oviduct explants(32).The ability of purified M. pneumoniae mem-

branes to induce cell damage has been evalu-ated with several different assay systems andmembrane preparation methods (10, 12, 14; M.G. Gabridge and R. B. Polisky, submitted for

I Present address: Department of Microbiology, Univer-sity of British Columbia, Vancouver, British Columbia,Canada.

2 Present address: Department of Microbiology, Univer-sity of Notre Dame, Notre Dame, IN 46556.

publication). In view of the wide variety of en-zymatic activities associated with mycoplasmamembrane proteins (28, 30), as well as the cyto-toxic activity of lipids (23, 25), some form ofmembrane-associated cytopathic activity wasnot surprising. Still to be resolved, however,was the manner in which mycoplasma mem-branes physically interacted with epithelialcells. The data obtained in the current studydemonstrate that the attachment of M. pneu-moniae membranes occurs without any signifi-cant involvement by the receptor sites that me-diate the specific attachment of intact myco-plasma cells.

MATERIALS AND METHODSMycoplasmas. The source of M. pneumoniae, PI

1428, and the composition of G-199 medium havebeen described (12, 15). Cells (passages 3 to 12 sincereceipt) were grown on the surface of plastic tissueculture flasks (75 cm2). When the sheet was nearlyconfluent with the pH c 6.75 (normally after 5 dayswith a 10 to 20% log culture inoculum and two mediachanges), the medium was decanted and the cellsheet was gently rinsed two times with phosphate-buffered saline (PBS, pH 7.4). Cells were removedby shaking vigorously with 5 to 10 ml of organculture medium and 1 to 2 g of sterile glass beads (3-mm diameter).Membranes were prepared after freeze-thaw cell

lysis and isolation on a sucrose step gradient aspreviously described (14). Membranes were washedfour times in 0.05 M NaCl or distilled water and

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were suspended in organ culture medium. Clumpswere dispersed before use of the membrane suspen-sion by homogenization with a sterile, ground-glasstissue grinder.

For labeling studies, cells were initially culti-vated on plastic surfaces for 24 h in normal G-199medium. This was then replaced with "G-199 label-ing medium," which did not contain the lactalbuminhydrolysate, but did have 10 mCi of 3H-labeledamino acids (sterile, aqueous, protein hydrolysate;Schwarz/Mann, Div. of Becton, Dickinson & Co.,Orangeburg, N.Y.) per liter. After an additional 48h of cultivation (with pH adjustments as necessary),the radioactive medium was decanted and the cellsheet was rinsed with five separate PBS washesbefore cells were detached as described above. Incor-poration efficiency averaged approximately 104 cpm/ml containing 106 colony-forming units (CFU). Oneseries of experiments (see Table 1) employed a[14C]oleic acid label (400 uCi per liter of completeG-199).Organ cultures. Tracheal explants ("rings" or

transverse slices) were prepared from adult ham-sters as previously described (8). These organ cul-tures were maintained in a shallow layer (approxi-mately 0.7 ml/35-mm plastic petri dish) of minimalessential medium (MEM; with Spinner salts andcomplete with glutamine and bicarbonate), supple-mented with 10% horse or fetal calfserum and 500 Uof penicillin G per ml. Relative activity (10) meas-urements, i.e., the percentage ofthe epithelium thatremained intact (0 to 100%) multiplied by the rela-tive vigor of ciliary beating (0 to 3+), were madedaily during inspection with phase optics (x225).Culture medium was replaced every 3 to 4 days.Methods for determining the adenosine 5'-triphos-phate (ATP) content (Gabridge and Polisky, submit-ted for publication) or dehydrogenase activity (12)have been described, as has the induction of squa-mous metaplasia and the extended maintenance ofnormal explants in retinol-supplemented MEM (7).

Attachment. Suspensions of cells (approximately107 CFU/ml) or membranes (50 to 100 ,.g of proteinper ml) were added to sterile tubes containing 3 to 12explants. Tubes were placed in a roller apparatusand rotated at 1 rpm for 90 to 120 min at 37C.Explants were then rinsed three to five times inMEM and cultured in dishes or dried in vacuo oversilica gel for at least 24 h. Dried rings were weighedand placed in scintillation vials (three per vial). Theepithelium was dissolved with 0.1 ml of BioSolv(Beckman Instruments, Inc., Fullerton, Calif.) orProtosol (New England Nuclear Corp., Boston,Mass.). A 5-ml portion of Aquasol 2, Econofluor(New England Nuclear Corp.), or equivalent scintil-lation cocktail (2) was then added, and radioactivitywas measured in an Isocap 300 liquid scintillationsystem (Nuclear-Chicago Corp., Des Plaines, Ill.).When internal standards were available (tritiatedtoluene; New England Nuclear Corp.), counts perminute were converted to disintegrations per min-ute to increase reliability and accuracy.

Biochemicals. Neuraminidase (Clostridium per-fringens, purified type V; Sigma Chemical Co., St.Louis, Mo.) was used at a level of 5 U/ml in PBS.

Waymouth MAB 87/3 medium was obtained fromGrand Island Biological Co. (Grand Island, N.Y.)and used without supplements. Protein determina-tions were made with the Lowry assay (10), incorpo-rating a crystalline bovine serum albumin standard.

RESULTS

Attachment methodology. In view ofthe factthat mycoplasma cells (and membranes) cansettle out of solution at 1 x g, it appeared thatthe usual static incubation may not provideconditions appropriate for maximum attach-ment to explants. Accordingly, series of tra-cheal explants were established and were incu-bated with viable, radioactive M. pneumoniaecells in either petri dishes (static; 0.7 ml/35-mmdish) or vials (in roller apparatus, 1 rpm; 0.7ml/vial). Incubation, or contact with the taggedcells, proceeded for 60 min. In addition, eachgroup had been initially divided into two subca-tegories for pretreatments; one was incubatedin PBS for 30 min, whereas the other was pre--treated with neuraminidase (5 U/ml of PBS) for30 min, prior to being placed in contact with themycoplasmas.

Table 1 shows that almost twice as much cellattachment occurs when the tracheal explantsare constantly agitated and repeatedly mixed(via the gentle rolling), as compared with theusual static method in which mycoplasmas aresimply added to the explants and both are incu-bated in a static fashion. Approximately one-half of the mycoplasma attachment was me-diated by specific receptor sites, since pretreat-ment with neuraminidase reduced the counts-per-minute incorporation by 42 to 44% for bothmethods. Thus, the ratio of specific to nonspe-cific attachment remained nearly constant,whereas the overall attachment increased withroller incubation. Inasmuch as this dynamictype of exposure led to an increased attachmentefficiency and probably more closely reflected

TABLE 1. Relative effectiveness of static and rollerincubation methods for promoting specific and

nonspecific attachment of[14C]oleic acid-labeled M.pneumoniae cells to hamster tracheal explants

Mycoplasma attach-ment"

Mode Pretreatment% of

cpm control

Static PBS 276 (33) 100Neuraminidase 161 (26) 58

Roller PBS 495 (40) 100Neuraminidase 278 (63) 56

a Mean value (+ standard error of the mean) from 12explants, with assay of 3 explants per vial; results ex-pressed as counts per minute per vial or as percentage ofcontrol value attained after neuraminidase pretreatment.

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768 GABRIDGE ET AL.

the natural exposure experienced by epithelialtissue, remaining experiments employed thismethodology.Suspending media and attachment. To de-

termine the optimal suspending medium formycoplasmas in attachment and infection stud-ies, roller incubation techniques were repeatedwith the tritiated mycoplasma cells suspendedin either PBS or MEM (complete with 10%serum). Samples were withdrawn at varioustime intervals so that the overall attachmentand the attachment kinetics could be comparedfor both protein (MEM) and salts (PBS) sus-pending media. Preliminary studies indicatedthat neither solution would actually supportthe growth of mycoplasmas in the absence oftracheal tissue.

Figure 1 shows that the mycoplasma cellsattach to the respiratory epithelial explants ata somewhat faster rate in the presence ofMEM,but that the final levels of attachment werequite similar. It was noted that with MEM themajority of cells had attached by 60 1nin, butwith PBS it was necessary to incuba e for an-other 30 min to maximize attachment (al-though efficiency was lower after the 90-minperiod). The optimal approach for attachmentstudies thus involved a dynamic (roller) incuba-tion in the presence of a nutritive explant me-dium and mycoplasmas for 60 to 120 min at370C.Attachment of M. pneumoniae membranes

to explants. When purified mycoplasma mem-branes were prepared and then incubated withtracheal explants, definite attachment wasnoted (Table 2). Initial dosage levels were 100

2500PBS

2000/ MEM

1500

z

I

a1000 /

500

0 30 60 90 120

TIME (min)

FIG. 1. Attachment of tritiated (3H-labeled aminoacids) M. pneumoniae cells to tracheal explants whilesuspended in PBS orMEM containing 10% fetal calfserum. Mean values from duplicate assays, threeexplants per point, from a total of 60 explants.

TABLE 2. Attachment of 3H-labeled M. pneumoniaemembranes to tracheal organ cultures

Membrane attachment (cpm)Y Neuramin-

Expt Pretreatment ~~~~idase effectExpt Pretreatment Pretreatment (% of con-with PBS aminidase trol)

1 777 (114) 646 (+78) 832 1,779 (16) 1,345 (+71) 76

Experiments 1 and 2 are from different lots ofmembranes (100 ,ug of protein per ml). Mean value( standard error of the mean) from 12 explants,with assay of 3 explants per vial; results expressedas counts per minute per vial or as percentage ofcontrol value after neuraminidase pretreatment.

,ug of protein per ml of MEM, equal to approxi-mately 1.3 x 105 cpm/ml. Thus, individual ex-plants were associated with 259 (777/3) to 393(1,179/3) cpm, equivalent to approximately 0.3,tg of protein. This is less than 1% uptake andhence somewhat less than that noted previ-ously with intact cells (Fig. 1).When the effect of neuraminidase pretreat-

ment on this attachment was evaluated (Table2), it appeared that removal of sialic acid-con-taining receptor sites did not significantly altermembrane attachment. Neuraminidase did notreduce attachment of membranes to near the50% control level noted when viable cell attach-ment was measured. More than three-fourthsof the membrane attachment was apparentlyunrelated to the presence of sialic acid com-pounds, and an analysis of the combined datain Table 2 showed that the neuraminidase re-duction of membrane attachment was not sta-tistically significant in the t test (P = 0.26).

Effect of CFU pretreatment. To verify thesuggestion that cell-specific receptor sites didnot account for membrane attachment, tra-cheal explants were treated with intact cells soas to block the receptors before exposure tomembranes. In each experiment, pools of ap-proximately 24 explants were divided into twogroups. The first was incubated in MEM,whereas the second was incubated in MEMcontaining approximately 107 CFU of viable M.pneumoniae cells without label (roller; 90-minincubation) per ml. After being rinsed twotimes in sterile PBS, both explant groups werethen incubated for 120 min with tritiated mem-branes. Table 3 shows that loading the specificreceptor sites (at near-saturation levels basedon the data shown in Fig. 1) with M. pneumo-niae cells failed to significantly alter the subse-quent attachment of M. pneumoniae mem-branes. Differences in membrane attachmentwith and without previous exposure to viablecells were not statistically significant (P = 0.2

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and 0.4 in experiments 1 and 2, respectively),and, in some instances, attachment actuallyincreased slightly. Thus, removal or saturationof the sialic acid-containing, cell receptor sitesdid not affect the basic attachment of myco-plasma membranes.Attachment of mycoplasma membranes to

nonciliated epithelium. If the attachment ofM. pneumoniae membranes to tracheal ex-plants was not mediated by receptor sites lo-cated on ciliated epithelial cells, as the previousdata suggested, then removal or reduction ofciliated cells should not affect membrane at-tachment. Significant decreases in ciliation ofrespiratory epithelium were induced in vitro byextended cultivation of tracheal explants in avitamin A-free medium, such as WaymouthMAB 87/3. When hamster tracheal organ cul-tures were maintained for 30 days in eitherMEM or MAB medium, the ciliary layer waspartially reduced in the former, whereas it wasalmost completely replaced by sheets of squa-mous cells in the latter. Table 4 shows thatMEM medium allowed maintenance of relativeciliary activity near the 200 level (maximumpossible was 300), whereas squamous metapla-sia was associated with a relative ciliary activ-ity of less than 25. This was not due to loss ofviability, as evidenced by the fact that values ofmetabolic activity (as reflected in dehydrogen-ase levels and ATP content) were normal. Ex-

TABLE 3. Effects of blocking receptor sites withmycoplasma cells on the subsequent attachment of

mycoplasma membranes

Membrane attachment (cpm)a ChangeExpt Pretreatment Pretreatment (% of

with MEM CFU control)

1 855 (22) 913 (55) 1062 2,553 (338) 1,879 (344) 74a Mean value ( standard error ofthe mean) from

12 explants, with assay of 3 explants per vial; resultsexpressed as counts per minute per vial or as per-centage of control after pretreatment with viableorganisms (CFU).

plants cultivated for 30 or more days in MABmedium thus represented viable respiratory ep-ithelial explants with markedly reduced num-bers of ciliated cells and contained squamousepithelial cells (verified by histopathologicaland scanning electron microscopic examination[6]).Paired sets of tracheal explants that had

either a ciliated or a squamous epithelium wereincubated for 120 min with tritiated M. pneu-moniae membranes after pretreatment for 60min with either PBS or neuraminidase. Beforethis treatment, relative ciliary activity, after 35days of incubation, was 154 (n = 15) for ciliatedexplants and 48 (n = 21) for explants with themajority of ciliated cells replaced by squamouscells. Table 5 shows that the induction of squa-mous metaplasia (i.e., significant replacementof ciliated cells) had little effect on membraneattachment. Specific attachment in terms ofdisintegrations per minute per milligram (de-signed to minimize or eliminate any artifactualdifferences due to gain or loss of cell mass dur-ing the extended incubation period) was essen-tially the same for both epithelial types (438versus 289; not significant at the P = 0.01 levelwith a t test). Pretreatment of both types oforgan cultures with neuraminidase reducedmembrane attachment in only a very limitedfashion (approximately 15% reduction), againindicating the minimal participation by specificreceptor sites. Thus, the data suggest that dim-

TABLE 5. Attachment of 3H-labeled M. pneumoniaemembranes to hamster tracheal explants cultivated

for 35 days in standard MEM (ciliated) orWaymouth MAB 87/3 medium (squamous)

AttachmentaPretreatment

Ciliated SquamousPBS (control) 434 (+139) 289 (+55)Neuraminidase 370 (75) 243 (42)a Disintegrations per minute per milligram of dry

weight; mean values (t standard deviation) fromfive to six replicates of three explants per vial.

TABLE 4. Comparison of ciliary motion, dehydrogenase activity, and ATP levels in tracheal explantscultivated for 30 days in different media to retard (MEM) or induce (Waymouth MAB 87/3) squamous

metaplasiaa

Relative ciliary activity Tetrazolium reduction ATP contentMedium Epithelial type (ODJMg)b (ng/mg)

Day 0 Day30(04,m)(nmgMEM Ciliated 271 (1) 202 (12) 1.02 (+0.12) 89 (+9)MAB 87/3 Squamous 270 (+1) 14 (+5)c 0.76 (+0.15) 105 (+29)a Mean values (t standard error of the mean) from 5 to 13 replicate analyses.b OD4,o, Optical density at 490 nm.c Statistically significant with t test (P < 0.001); all other paired groups (MEM versus MAB) were not

statistically different.

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inution of the quantity of ciliated cells by theinduction of squamous metaplasia does not sub-stantially inhibit the attachment of myco-plasma membranes to tracheal epithelium.The kinetics of attachment of membranes to

both types of surfaces were quite similar (Fig.2). It was noted that membranes attached at afairly regular rate for both squamous and cil-iated epithelium for approximately 90 min. Atthat time, normal (i.e., ciliated) epithelium hadslower attachment, and continued incubationadded only small amounts of attached mem-brane protein. It was noted, however, that con-tinued contact of squamous epithelium and my-coplasma membranes resulted in significant in-creases in incorporation. For example, extend-ing the time period from 120 to 150 min showedan attachment increment of more than 50%(namely, 1,831 cpm at 120 min to 3,089 cpm at150 min). This suggests that the presence ofciliation affects the final incorporation or actualload of membranes, but not the basic rate atwhich host and parasite membranes interactwith each other.

DISCUSSIONThe attachment of numerous viruses, toxins,

and hormones to the surface of eucaryotic cellsis known to involve specific receptor sites. Theinteraction of mycoplasmas with epithelial cellsand erythrocytes is similarly receptor site me-diated. Gesner and Thomas (16) were amongthe first to note the critical role played by sialicacid-containing receptors when they observed

100 SQUAMOUSCILIATED

E 80 /

CI 60-

2- _zuJ2 40-IC-)'2

< 20~-

TIME (min.)

FIG. 2. Attachment of tritiated (3H-labeled aminoacids) M. pneumoniae membranes to tracheal ex-plants that had been cultivated for 30 days in eitherMEM (ciliated explants) or Waymounth MAB 37/3medium (squamous explants). Mean data from twoseparate experiments, each run in duplicate for atotal of 12 explants per time sample.

that hemagglutination by M. gallisepticumcould be inhibited by mucoproteins, neuramini-dase, or sialic acid itself. They also reportedthat neuraminidase pretreatment of the muco-proteins (e.g., egg white, gastric mucin, ovo-mucoid) with neuraminidase reduced their ef-fectiveness as inhibitors of subsequent myco-plasma attachment.

Sobeslavsky et al. (31) and Manchee and Tay-lor-Robinson (24) later demonstrated that M.pneumoniae attachment to erythrocytes, cellcultures, and isolated epithelial cells is alsomediated by a sialic acid receptor. The bindingsite on the M. pneumoniae cells may be lipid orlipoprotein in nature, since glycerophospholi-pid haptens can block hemadsorption-inhibit-ing antibodies. The cellular site is apparentlyheat stable (560C for 30 min) and trypsin resist-ant (0.025% for 30 min at 37C). These and other(19) reports also demonstrated that the sialicacid-containing receptor is not common to allmycoplasma attachment processes, since M.hominis, M. salivarium, and M. dispar attachto an unidentified receptor that is not affectedby neuraminidase.The data presented here indicate that the

attachment of purified membranes of M. pneu-moniae to respiratory epithelial surfaces is ap-parently not mediated by the same mechanismthat accounts for the attachment ofM. pneumo-niae cells. Attachment of membranes, unlikethat of viable cells, was not markedly affectedby neuraminidase pretreatment of the trachealexplants. That fraction of the attached mem-brane counts (17 to 24%) that appears to beneuraminidase sensitive may represent attach-ment of those membrane fragments that retainthe attachment tip. Further experiments on thefate of the attachment tip in the membraneisolation process would be desirable. Similarly,it would be of basic interest to better define thebiochemical and biophysical nature of both theM. pneumoniae attachment tip and the epithe-lial cell receptor site.Attachment of membranes was not signifi-

cantly affected by the loss of ciliated cells (9)that accompanied squamous metaplasia. Thisis in contrast to the results previously noted (7)with intact cells. In that instance, the metapla-sia-induced reduction in the number of ciliatedcells markedly diminished the attachment ofM. pneumoniae cells. Thus, the current studysuggests that the attachment of membranes isnot specific for ciliated respiratory epithelialcells.We also observed that pretreatment of ex-

plants with viable mycoplasmas in an attemptto saturate the specific cell receptors had noeffect on the attachment of membranes. Thus,

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the purified M. pneumoniae membranes attachto ciliated and nonciliated epithelial cells by amechanism that apparently does not involvethe N-acetylneuraminic (sialic) acid receptor toany significant extent. If it did, then pretreat-ment with mycoplasma cells would haveimpeded maximal membrane attachment.Since no difference was found after cellular in-fection, it is conceivable that the small mem-brane fragments simply attached directly to theepithelial surface in between the M. pneumo-niae cells. These kinds ofdata therefore suggestthat isolated membranes of M. pneumoniae at-tach to tracheal epithelium in a manner dis-tinct from the receptor site mediation commonto intact cells.

There are reports in the literature that indi-cate that nonviable M. pneumoniae cells havelittle cytotoxic potential (5) or do not attachefficiently to tracheal epithelium (29). Thismay indicate that mycoplasmas must be meta-bolically active in order to infect, or it couldmean that the inactivation processes (heat andperiodate) destroyed some critical, labile mem-brane component and prevented any effectivehost-parasite interaction. The possible viabilityrequirement and the membrane interactiveprocess suggested here may, in fact, be compat-ible if membrane-to-membrane attachment re-quires small sections with relatively large, ex-posed surface areas in order to maximize physi-cal contact. The small membrane particles maybe able to lodge between the cilia and cells andattach to many portions of the epithelial mem-brane, which would present a spatial hindranceto intact cells. One must also consider the possi-bility that inner surfaces of the mycoplasmamembrane could be especially efficient at at-tachment. The relative attachment efficiency ofinner and outer membrane surfaces remains tobe assessed.The isotope labeling method developed for

this study yielded relatively high activity inmycoplasma cells and membranes so that at-tachment to epithelial surfaces could be moni-tored. Difficulty in the labeling of mycoplasmamembranes may have been one of the majorcauses of failure when another laboratory (21)attempted to observe membrane attachment totracheal explants. The lack of success in thatstudy may also have been due, in part, to nu-merous technique differences (for example, ra-dioactive label, mycoplasma broth, explant me-dium, membrane isolation, parameter used tomeasure cytotoxicity, maximum membraneconcentration, length of incubation/observationperiod, and use of membrane aggregates) be-tween their study and ours.The precise manner in which mycoplasma

membranes attach to respiratory epithelium isnot currently understood. One possible expla-nation may be actual membrane fusion. Grantand McConnell (17) and Teuber and Bader (33)both found that Acholeplasma laidlawii cellscan fuse with isolated lipid vesicles. In addi-tion, Apostolov and Windsor (3) recently usedelectron microscopy to demonstrate membranefusion between M. gallisepticum and erythro-cytes. This is consistent with previous reports(1, 6, 22, 26, 35) that noted a-reiaably inti-mate association between the menib-`- i:fvarious mycoplasmas and the-cells to wichthey attached. If such fusion occurs upon infec-tion with M. pneumoniae cells, it might alsofunction with isolated membranes as well andcould serve to explain the membrane attach-ment that we observed.Some degree of fusion between cells (and/or

membranes) could conceivably be associatedwith the pathogenesis of mycoplasma disease.Receptors could provide the mechanismwhereby mycoplasma cells (covered with a typi-cal biological membrane) are held in close prox-imity to the epithelial membrane. Cell fusioncould then ensue at or between the cilia, whichare membrane bound (34). This would serve tointroduce a wide variety ofpotentially cytotoxicproteins (enzymes) and lipids into the cell with-out containment in the typical, protective vesi-cle associated with normal phagocytosis andparticle uptake. The new "patch" on the epithe-lial cell membrane may also provide a rela-tively weak area that could leak ions or essen-tial metabolites or that could eventually yieldto stress and result in cytoplasmic leakage. Inaddition, a fusion process would be compatiblewith the fact that M. pneumoniae can appar-ently infect certain nonciliated cells and alsocause autoimmune reactions (4, 18, 19). Therole of fusion in the interaction of M. pneu-moniae cells and membranes with mammaliantissue is currently under investigation.

ACKNOWLEDGMENT

This project was supported by Public Health Service grantAI 12559 from the National Institute of Allergy and Infec-tious Diseases.

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