Epitope Mapping of the Variable Repetitive Region within the MB Antigen ofUreaplasma urealyticum

One of the major surface structures of Ureaplasma urealyticumrecognized by antibodies of patients during infection is the MB antigen. Previously, we showed by Western blot (immunoblot) analysis that any one of the anti-MBmonoclonalantibodies(MAbs)3B1.5,5B1.1,and10C6.6couldblockthebindingofpatientantibodies to MB. Subsequent DNA sequencing revealed that a unique six-amino-acid direct tandem repeat region composedthecarboxytwo-thirdsofthisantigen.Inthepresentstudy,usingantibody-reactivepeptidescanning of this repeat region, we demonstrated that the amino acids defining the epitopes for MAbs 3B1.5, 5B1.1, and 10C6.6 are EQP, GK, and KEQPA, respectively. Peptide scanning analysis of an infected patient's serum antibody response showed that the dominant epitope was defined by the sequence PAGK. Mapping of these continuous epitopes revealed overlap between all MAb and patient polyclonal antibody binding sites, thus explaining the ability of a single MAb to apparently block all polyclonal antibody binding sites. We also show that a single amino acid difference in the sequence of the repeats of serovars 3 and 14 accounts for the lack of reactivity with serovar 14 of two of the serovar 3-specific MAbs. Finally, the data demonstrate the need to obtain the sequences of the mba genes of all serovars before an effective serovar-specific antibody detection method can be developed. Ureaplasma urealyticum has been implicated in perinatal morbidity and mortality, including chorioamnionitis, prema- ture birth, and respiratory disease in newborns (1, 2). Addi- tional studies have also shown thatU. urealyticumis the single most common microorganism isolated from the central ner- voussystemofnewborninfants(13,14).Sincediseaseoccursin only a subpopulation of infected individuals, it has been pos- tulated that only some of the 14 established serovars may be responsible for causing diseases. Earlier studies suggested that a serovar-specific antibody may be required for protection against invasive diseases caused by this organism. Our prelim- inary studies (18) suggested that invasiveness was not likely to be limited to one or a few particular serovars and may be mediated by an adaptive property inherent in all serovars, i.e., the ability to accommodate a new environment via easily al- terable surface antigens. This ability to provide a highly plastic interface between organism and host has been found in many mycoplasmas(16).ThechangeoftopographyofU.urealyticum consequent to changes of its MB antigen may reflect such a property of this organism, thus providing any serovar with a possible immune system avoidance mechanism and the poten- tial ability to alternate between a commensal and a pathogenic state. We have shown that this size-variable antigen, which presents a multiple-banding pattern on an immunoblot, is one ofthemajorantigensrecognizedduringinfectionbyantibodies of patients and that antibodies against this antigen have me- tabolism inhibition activity in vitro (11, 15). Recently, we cloned and sequenced thembagene of the serovar 3 reference strain and found that the associated antigen contains a region atthecarboxyendofthemoleculewhichconsistsexclusivelyof directtandemrepeatunits(17).Eachrepeatunitconsistsofsix