Mycoplasma pneumoniae initiates infection in the human host by attachment to respiratory epithelium. The organism attaches by a specialized terminal structure. Monoclonal antibodies to an organism surface protein (P1) inhibited attachment to respiratory epithelium and were localized to the tip structure by a ferritin antibody label. The P1 protein was degraded by trypsin treatment to smaller polypeptides that possessed the same antigenic determinants as the larger P1 protein when reacted with the specific monoclonal antibody, and evidence has been provided for the existence of multiple antigenic determinants on the attachment protein

Clyde, W. A.

Collier, A. M.

Hu, P. C.

English

PubMed

Mycoplasma pneumoniae initiates infection in the human host by attachment to respiratory epithelium. The organism attaches by a specialized terminal structure. Monoclonal antibodies to an organism surface protein (P1) inhibited attachment to respiratory epithelium and were localized to the tip structure by a ferritin antibody label. The P1 protein was degraded by trypsin treatment to smaller polypeptides that possessed the same antigenic determinants as the larger P1 protein when reacted with the specific monoclonal antibody, and evidence has been provided for the existence of multiple antigenic determinants on the attachment protein.ImagesFIG. 1FIG. 

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THE YALE JOURNAL OF BIOLOGY AND MEDICINE 56 (1983), 671-677Location of Attachment Moiety on MycoplasmapneumoniaeA.M. COLLIER, M.D., P.C. HU, Ph.D., AND W.A. CLYDE, Jr., M.D.Department of Pediatrics, Center for Environmental Health and Medical Sciencesand Frank Porter Graham Child Development Center, University of NorthCarolina, Chapel Hill, North CarolinaReceived January 4, 1983Mycoplasma pneumoniae initiates infection in the human host by attachment to respiratoryepithelium. The organism attaches by a specialized terminal structure. Monoclonal antibodiesto an organism surface protein (P1) inhibited attachment to respiratory epithelium and werelocalized to the tip structure by a ferritin antibody label. The P1 protein was degraded by tryp-sin treatment to smaller polypeptides that possessed the same antigenic determinants as thelarger P1 protein when reacted with the specific monoclonal antibody, and evidence has beenprovided for the existence of multiple antigenic determinants on the attachment protein.INTRODUCTIONMycoplasma pneumoniae is an extracellular pathogen of the human respiratorytract and is the most common cause of pneumonia in teenagers and young adults.The pathogenic form of M. pneumoniae is filamentous in morphology and attachesto the respiratory epithelium by a specialized terminal structure which consists of adense central core with a bulbar tip surrounded by an electron-lucent spaceenveloped by the cell membrane of the organism [1]. In vitro tracheal organ culturestudies have provided evidence that attachment of viable, virulent M. pneumoniaeorganisms to the respiratory epithelium is a prerequisite for host cell injury [2,3,4].The attachment moiety on the mycoplasma membrane has been demonstrated to bea trypsin-sensitive protein, designated P1 5].Monoclonal antibody has been developed that reacts specifically with the P1 pro-tein. This antibody interferes with attachment of M. pneumoniae to respiratoryepithelium in hamster tracheal organ culture. Electron microscopic studies utilizingthe mouse monoclonal antibody and ferritin-conjugated rabbit antimouse IgG havepermitted the localization of the P1 protein on the filamentous M. pneumoniaeorganism [6].Studies on the fine analysis of antigenic determinants of the P1 protein may leadto a better understanding of the structure-function relationship of this molecule.This paper presents preliminary evidence for the existence of multiple antigenicdeterminants of P1 molecules by means of limited protease digestion andmonoclonal antibodies.671Address reprint requests to: Dr. A.M. Collier, Dept. of Pediatrics, 535 Burnett-Womack Building229H, University of North Carolina, Chapel Hill, NC 27514Copyright t 1983 by The Yale Journal of Biology and Medicine, Inc.All rights of reproduction in any form reserved.MATERIALS AND METHODSMycoplasma CulturesMycoplasmapneumoniae strain M-129 (ATCC No. 29342) was grown in Hayflickmedium supplemented with 20 percent agamma horse serum and 10 percent yeastdialysate. The cultures were prepared as described previously [5] and in some ex-periments the monolayers were treated with trypsin [5].Organ CulturesTracheal organ cultures were prepared from adult male, syrian hamsters andmaintained in Hayflick medium [7]. The tracheal rings were infected with M.pneumoniae as previously described [5].ElectrophoresisSodium dodecyl sulfate (SDS)-polyacrylamide slab gels were employed to separatethe M. pneumoniae proteins. The separated proteins were transferred tonitrocellulose paper for radioimmunobinding studies as fully described before [8].Monoclonal AntibodiesHybridoma cell lines that produced antibodies to the antigens of M. pneumoniaewere produced by hybridization of spleen cells from BALB/c mice immunized withM. pneumoniae and myeloma cells Sp2/0 Agl4 (ATCC No. CRL-1581). Hybridsthat produced antibodies against M. pneumoniae were screened by radioim-munoassay. Ascitic fluids were produced in BALB/c mice by injecting the mice in-traperitoneally with hybridoma cells. Monoclonal antibodies against specific M.pneumoniae antigens were identified by protein blot and radioimmunobindingtechniques [8,6].Trypsin Cleavage of Attachment ProteinA band of P1 protein was cut off from fixed or unfixed 7 percent SDS-gel anddigested with trypsin in the sample wells of a second SDS gel (12 percent). Details ofthis method have been described previously [9]. The best results were obtained using1 mM EDTA in the gel solution on 1.5 mm thick gels with 3 cm stacking gel and 8cm separation gel. The current was turned off for 30 minutes when the dye frontreached the bottom of the stacking gel to assure adequate digestion.Electron MicroscopyTracheal rings and M. pneumoniae organisms were fixed in 2 percentglutaraldehyde-2 percent paraformaldehyde in 0.1 M phosphate buffer. Secondaryfixation was accomplished in 1 percent OsO4-Veronal acetate buffer followed bydehydration and embedding in Epon. In ferritin experiments M. pneumoniae wereincubated with mouse monoclonal antibody M-218 specific for the P1 protein,washed, and then incubated with ferritin-conjugated rabbit antibody to mouse IgGprior to fixation. Thin sections were cut on a LKB ultramicrotome and examined ina Zeiss EM-10 electron microscope at an accelerating voltage of 60 kV.RESULTSElectron MicroscopyExamination of hamster tracheal organ cultures infected with M. pneumoniaeshowed the filamentous organisms to be located in an extracellular location at the672 COLLIER ET AL.ATTACHMENT MOIETY OF MYCOPLASMA PNEUMONIAE 673lumenal border of the respiratory epithelium. The organisms were oriented with thespecialized terminal structure proximal to the host cell membrane. Some organismshad the membrane lining the sides of the specialized tip structure in close approx-imation to the host cell membrane as well as the terminal portion of the tip (Fig.1A).When M. pneumoniae was incubated with the monoclonal antibody M-218 andsubsequently with ferritin-conjugated rabbit antibody to mouse IgG, the ferritinlocalized the M-128 antibody to be specifically concentrated on the surfaces of theunit membrane surrounding the organism's terminal structure (Fig. 1B). In thatmonoclonal antibodies secreted by hybridoma cell line M-218 have been shown to bespecific for the P1 protein by SDS-gel/protein blot radioimmunobinding [8], theresults obtained by indirect ferritin staining localized the P1 protein to the cell mem-brane surrounding the terminal organelle of the filamentous M. pneumoniaeorganism.Evidence of Multiple Antigenic Determinants on P1 ProteinSince P1 is a large protein molecule (molecular weight, 190 Kd), it is conceivablethat a number of antigenic determinants may exist on this molecule. In an effort toinvestigate this possibility, bands of P1 protein were cut out of the first SDS gel andsubjected to a limited trypsin digestion followed by SDS gel electrophoresis in com-bination with protein blot and radioimmunobinding. As shown in Fig. 2, when theP1 protein band, cut off from a 7 percent gel, was re-electrophoresed on a 12 percentSDS gel, it migrated as a single band, as revealed by Coomassie blue staining (laneA). Its immunoreactivity also was not distorted by the re-electrophoresis, since there-electrophoresed P1 protein was capable of reacting with both hyperimmunizedrabbit antiserum and monoclonal antibody M-218 (lanes B and D), respectively,following the transfer to nitrocellulose filters. Lanes C and E served as the negativecontrols which were incubated with '25I-labeled goat anti-rabbit IgG or rabbit anti-SS,' ........................~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.... ..FIG. 1. A Electron micrograph of M. pneumoniae attached to theciliated respiratory epithelium of hamster tracheal organ culture. Note theclose association of the side of the specialized tip with the cilia (arrow).B Electron micrograph ofM. pneumoniae organism incubated withmonoclonal antibodies specific to P1 protein, and indirectly stained withferritin-conjugated rabbit anti-mouse IgG. Ferritin grains indicate thelocation of P1 protein on the organism cell membrane surrounding theterminal organelle of M. pneumoniae. Bar equals 0.1 tM.COLLIER ET AL.FIG. 2. Immunoradiobinding studies of the cleaved products of P1protein following limited trypsin digestion. A A PI protein bandcut off from a 7 percent gel and re-electrophoresed on a 12 percent gel andstained with Coomassie blue. B through E P1 protein bands pre-pared the same way as A but transferred to nitrocellulose paper by Westernblot technique. The nitrocellulose blots were incubated with: B serumfrom a rabbit hyperimmunized with M. pneumoniae followed by'2ll-labeled goat anti-rabbit IgG; C '2ll-labeled goat anti-rabbit IgGonly; D monoclonal antibody M-218, followed by '2ll-labeled rabbitanti-mouse IgG; and E '251-labeled rabbit anti-mouse IgG only. Fand G P1 protein bands treated with trypsin (250 ,g/ml) in the samplewells of a 12 percent gel and electrophoresis continued; the digestedfragments separated on the gel were then transferred to nitrocellulosepaper. F was incubated with monoclonal antibody M-218, followedby '2ll-labeled rabbit anti-mouse IgG; and G was incubated withhyperimmunized rabbit serum, followed by I2'l-labeled goat anti-rabbitIgG. Molecular weight standards were ovalbumin (43,000), c-chymotry-sinogen (25,700), ,3-lactoglobulin (18,400), and Cytochome C (12,300).mouse IgG, respectively, without prior incubation with specific antibodies. Theseresults indicate that the binding of labeled second antibodies to the nitrocelluloseblots was mediated specifically through the bound P1 protein-specific antibodiespresent in the immunized rabbit antiserum or the monoclonal antibody M-218 whichhas been shown to be specific to P1 protein [6].When the P1 protein bands were subjected to a limited trypsin treatment followedby SDS-gel electrophoresis, a number of very faint bands were observed in theCoomassie blue-stained gels (data not shown). However, when the nitrocelluloseblots were prepared from these gels, intact antigenic determinants were preserved inthe cleaved fragments. As demonstrated in lanes F and G, two bands were detectedby monoclonal antibody M-218, and six bands were detected with the immunizedrabbit antiserum. The two bands which appeared on the nitrocellulose strip in-cubated with monoclonal antibodies are anticipated to be two polypeptides possess-ing the same antigenic determinants but with different molecular weights. The fouradditional bands detected by incubation with the immunized rabbit antiserum mustrepresent antigenic determinants other than the ones detected by monoclonal an-674ATTACHMENT MOIETY OF MYCOPLASMA PNEUMONIAEtibody M-218. This preliminary result confirmed that P1 protein possesses morethan one antigenic determinant.DISCUSSIONThe only natural host of M. pneumoniae is man. This pathogen is the causativeagent of atypical pneumonia [10]. The morphology of this organism in culture wasreported by Biberfeld and Biberfeld [11]. Use of both transmission and scanningelectron microscopy showed the organism to be filamentous and to possess a dif-ferentiated terminal knob-like structure. Collier and Clyde [12] studied trachealorgan cultures infected with M. pneumoniae and observed the organism to be in anextracellular location with the differentiated terminal structure in apposition to hostcell membranes. This suggested that the terminal structure was important for the at-tachment of M. pneumoniae. This observation was supported by later studies ofradioactively labeled mycoplasmas in tracheal organ culture [5] and of sputumsamples from patients [1]. Earlier studies of the interaction of M. pneumoniae withhost cells in tracheal organ culture showed that host cell injury could be inducedonly by metabolically active mycoplasmas attached to respiratory epithelium.Transfer of infected tracheal organ cultures to medium non-permissive to M.pneumoniae 24 hours following infection prevented the alterations of grossmacromolecular biosynthesis in host cells [4]. Based upon these data, it was pro-posed that M. pneumoniae infection was a two-step process, consisting of specificattachment of M. pneumoniae to the sialic acid-containing receptor sites on hostcells followed by a yet-to-be-defined mechanism of cell injury [5].Studies have been done in our laboratory to identify and characterize the surfacemoiety of M. pneumoniae responsible for the specific attachment of M. pneumoniaeorganisms to respiratory epithelium. This adhesion is thought to be a prerequisite forthe colonization of M. pneumoniae in the respiratory tract and subsequent develop-ment of disease [5,13]. The proteinaceous nature of the adhesion was demonstratedby mild treatment of this organism with trypsin followed by SDS-gel elec-trophoresis. We found that the trypsin treatment of M. pneumoniae nullifies itsability to adhere to tracheal epithelium. The ability of M. pneumoniae to attach,removed by trypsin treatment, could be restored by incubating the treated organismsin growth medium. SDS-gel electrophoresis of M. pneumoniae proteins preparedfrom trypsin-treated organisms showed the disappearance of a major band on thecell surface. We proposed this protein, designated P1, to be the attachment factor,as the failure of the mycoplasmas to attach correlated with its absence. Moreover,regeneration of P1 by incubation in fresh growth medium for six hours (approx-imately one generation time of M. pneumoniae) renewed the attachment capacity,and P1 also reappeared on the polyacrylamide gel profile [5]. Results shown in Fig.1B and one previous publication [6] demonstrated evidence that P1 protein isphysically located on the tip structure of M. pneumoniae organisms.Another important characteristic of virulent M. pneumoniae is its ability to ad-sorb erythrocytes [14]. Trypsin-treated colonies of virulent M. pneumoniae areunable to adsorb either erythrocytes or tracheal epithelial cells [15]. This suggeststhat M. pneumoniae might utilize the same mechanisms for attachment to both celltypes. The concomitant loss of the ability to attach and the hemadsorption activity(HA) in an avirulent strain [16] also implies the possibility that the catalytic sites ofthese two activities could be in close proximity, if not identical. On the other hand,there are reports [17,18,19] suggesting that HA is probably mediated by a differentmoiety in that protein bands other than P1 were found missing in certain HA-675676 COLLIER ET AL.mutants as revealed by gel electrophoresis. Unfortunately, authors of these studiesfailed to provide evidence that the bands were surface-located, which is a basic re-quirement to qualify their role in the hemadsorption, a surface contactphenomenon.The fine analysis of antigenic determinants of P1 using monoclonal antibodiesmay lead to answers as to whether P1 is responsible for hemadsorption as well as at-tachment of M. pneumoniae organisms. Similar approaches have been used todelineate the antigenic composition of hemagglutinin molecules of influenza virusand for analysis of the mechanism of antigenic drift, which has been suggested to beresponsible for the recurrence of influenza infection [20]. The preliminary resultspresented in this paper indicate the feasibility of this approach for studying thestructure/function relationship of the P1 protein. These studies should lead to a bet-ter understanding of the biological activities of this organism and provide informa-tion that would be useful in altering the interaction of M. pneumoniae with man tofavor the human host.ACKNOWLEDGEMENTThe authors wish to thank Chien-Hui Huang and Nancy Hu for their technical assistance and SarahHenderson for her secretarial assistance.The studies were supported in part by NIH grant HL-19171 and by Contract 023404 and CooperativeAgreement CR 807392 from the Environmental Protection Agency.REFERENCES1. Collier AM, Clyde WA: Appearance of Mycoplasmapneumoniae in lungs of experimentally infectedhamsters and sputum from patients with natural disease. Am Rev Resp Dis 110:765-773, 19742. Powell DA, Hu PC, Wilson MH, et al: Attachment of Mycoplasma pneumoniae to respiratoryepithelium. Infect Immun 13:959-966, 19763. Hu PC, Collier AM, Baseman JB: Alterations in the metabolism of hamster tracheas in organ cultureafter infection by virulent Mycoplasma pneumoniae. Infect Immun 11:704-710, 19754. Hu PC, Collier AM, Baseman JB: Further studies on the interaction of virulent Mycoplasmapneumoniae with hamster tracheal organ culture. Infect Immun 14:217-224, 19765. Hu PC, Collier AM, Baseman JB: Surface parasitism by Mycoplasma pneumoniae of respiratoryepithelium. J Exp Med 145:1328-1343, 19776. Hu PC, Cole RM, Huang YS, et al: Mycoplasma pneumoniae infection: Role of a surface protein inthe attachment organelle. Science 216:313-315, 19827. Collier AM, Clyde WA, Denny FW: Biologic effects of Mycoplasma pneumoniae and othermycoplasmas from man on hamster tracheal organ culture. Proc Soc Exp Biol Med 132:1153-1158,19698. Hu PC, Huang YS, Graham JA, et al: Identification of immunogens of Mycoplasmapneumoniae byprotein blotting. Biochem Biophys Res Commun 103:1363-1370, 19819. Cleveland DW, Fisher JG, Nirschner MW, et al: Peptide mapping by limited proteolysis in sodiumdodecyl sulfate and analysis by gel electrophoresis. J Biol Chem 252:1102-1106, 197710. Denny FW, Clyde WA, Glezen WP: Mycoplasma pneumoniae disease: Clinical spectrum,pathophysiology, epidemiology, and control. J Infect Dis 123:74-92, 197111. Biberfeld G, Biberfeld P: Ultrastructral features of Mycoplasma pneumoniae. J Bact 102:855-861,197012. Collier AM, Clyde WA: Relationships between Mycoplasma pneumoniae and human respiratoryepithelium. Infect Immun 3:694-701, 197113. Collier AM: Attachment of Mycoplasma pneumoniae to respiratory epithelium. In BacterialAdherence. Edited by EH Beachey. London, Chapman and Hall, 1980, pp 159-18314. Manchee RJ, Taylor-Robinson D: Haemadsorption and haemagglutination by mycoplasmas. J GenMicrobiol 50:465-478, 196815. Sobeslavsky 0, Prescott B, Chanock RM: Adsorption of Mycoplasma pneumoniae to neuraminicacid receptors of various cells and possible role in virulence. J Bact 96:695-705, 1968ATTACHMENT MOIETY OF MYCOPLASMA PNEUMONIAE 67716. Lipman RP, Clyde WA: The interrelationship of virulence, cytadsorption, and peroxide formationin Mycoplasma pneumoniae. Proc Soc Exp Biol Med 131:1163-1167, 196917. Hanson EJ, Wilson RM, Bereman JB: Isolation of mutants of Mycoplasma pneumoniae defective inhemadsorption. Infect Immun 23:903-906, 197918. Hanson EJ, Wilson RM, Clyde WA Jr: Characterization of hemadsorption-negative mutants ofMycoplasma pneumoniae. Infect Immun 32:127-136, 198119. Krause DC, Leith DK, Wilson RM, et al: Identification of Mycoplasma pneumoniae proteinsassociated with hemadsorption and virulence. Infect Immun 35:809-817, 198220. Gerhard W, Webster RG: Antigenic drift in influenza A viruses. I. Selection and characterization ofantigenic variants of A/PR/8/34 [HONIJ influenza virus with monoclonal antibodies. J Exp Med148:383-392, 1978

Location of attachment moiety on Mycoplasma pneumoniae.

al: Identification ofimmunogens ofMycoplasmapneumoniae by protein blotting.

al: Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis.

Appearance ofMycoplasmapneumoniae in lungs ofexperimentally infected hamsters and sputum from patients with natural disease. Am Rev Resp Dis 110:765-773,

Attachment of Mycoplasma pneumoniae to respiratory epithelium. In Bacterial Adherence.

Bereman JB: Isolation of mutants ofMycoplasmapneumoniae defective in hemadsorption.

Biberfeld P: Ultrastructral features of Mycoplasma pneumoniae.

Chanock RM: Adsorption of Mycoplasma pneumoniae to neuraminic acid receptors of various cells and possible role in virulence.

Clyde WA: Relationships between Mycoplasma pneumoniae and human respiratory epithelium. Infect Immun 3:694-701,

Clyde WA: The interrelationship of virulence, cytadsorption, and peroxide formation in Mycoplasma pneumoniae. Proc Soc Exp Biol Med 131:1163-1167,

et al: Attachment of Mycoplasma pneumoniae to respiratory epithelium. Infect Immun 13:959-966,

et al: Identification of Mycoplasma pneumoniae proteins associated with hemadsorption and virulence. Infect Immun 35:809-817,

Further studies on the interaction of virulent Mycoplasma pneumoniae with hamster tracheal organ culture. Infect Immun 14:217-224,

FW: Biologic effects of Mycoplasma pneumoniae and other mycoplasmas from man on hamster tracheal organ culture. Proc Soc Exp Biol Med 132:1153-1158,

Glezen WP: Mycoplasma pneumoniae disease: Clinical spectrum, pathophysiology, epidemiology, and control.

JB: Alterations in the metabolism ofhamster tracheas in organ culture after infection by virulent Mycoplasma pneumoniae. Infect Immun 11:704-710,

Jr: Characterization of hemadsorption-negative mutants of Mycoplasmapneumoniae. Infect Immun 32:127-136,

Mycoplasmapneumoniae infection: Role of a surface protein in the attachment organelle.

RG: Antigenic drift in influenza A viruses. I. Selection and characterization of antigenic variants of A/PR/8/34 [HONIJ influenza virus with monoclonal antibodies.

Surface parasitism by Mycoplasma pneumoniae of respiratory epithelium.

Taylor-Robinson D: Haemadsorption and haemagglutination by mycoplasmas.

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Location of attachment moiety on Mycoplasma pneumoniae.

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