Interaction between the P1 protein of Mycoplasma pneumoniae and receptors on HEp-2 cells

Abstract

The human pathogen Mycoplasma pneumoniae can cause atypical pneumonia through adherence to epithelial cells in the respiratory tract. The major immunogenic protein, P1, participates in the attachment of the bacteria to the host cells. To investigate the adhesion properties of P1, a recombinant protein (rP1-II) covering amino acids 1107-1518 of the P1 protein was produced. This protein inhibited the adhesion of M. pneumoniae to human HEp-2 cells, as visualized in a competitive-binding assay using immunofluorescence microscopy. Previous studies have shown that mAbs that recognize two epitopes in this region of P1 also reduce M. pneumoniae adhesion. Therefore, peptides covering these epitopes, of 8 and 13 aa, respectively, were synthesized to further investigate the adhesion region. None of these synthetic peptides reduced the binding of M. pneumoniae to the receptors on the host cells. Instead, 10 overlapping synthetic peptides covering the whole of rP1-II were evaluated in the competitivebinding assay using immunofluorescence microscopy. A reduction in the number of M. pneumoniae microcolonies was seen, which was confirmed for five peptides using a POLARstar OPTIMA reader to measure fluorescence intensity. The number of M. pneumoniae microcolonies adhering to the host cells was significantly reduced by these five peptides. Further investigations showed that inhibiting peptide 7 (amino acids 1347-1396) of the major adhesin protein P1 bound directly to host receptors, suggesting that the observed M. pneumoniae-inhibiting peptides occupied HEp-2 receptors, which are otherwise available for P1-mediated M. pneumoniae adhesion.

Strategy To Create Chimeric Proteins Derived from Functional Adhesin Regions of Mycoplasma pneumoniae for Vaccine Development

“…In fact, antibodies against RP10 were able to decrease cytadherence of M. pneumoniae cells to MRC-5 lung fibroblasts by 42% but had no measurable effect on the interaction of mycoplasmas with HeLa or HBEC. The divergent findings of the studies mentioned in comparison with results of the present report might be explained by the use of different methods and/or different host cells such as Hep-2 cells or erythrocytes (8,27,29,46), which might differ in the receptor configuration (37,38,50). Despite the fact that M. pneumoniae has a high binding capacity to HeLa cells, MRC-5 lung fibroblasts, and primary bronchial epithelial cells, it is still unclear if attachment of M. pneumoniae to different cell types is mediated by the same adherence mechanisms.…”

“…The use of flow cytometry to quantify the mycoplasmas attached to eukaryotic cells turned out to be a rapid method for obtaining quantitative, reproducible, and objective results. Thus, this approach could be an excellent alternative to replace time-consuming, more subjective or laborious techniques that are currently used to enumerate at- (8,16,46). To summarize, only antibodies against recombinant protein RP14 of the P1 adhesin and against the recombinant protein derived from protein P30 decreased M. pneumoniae adherence to all three human cell lines tested.…”

“…Despite the immunogenicity of P116 (9), it was suggested that this protein might serve as an additional adhesin for attachment to Hep-2 cells. Further investigations are necessary to identify other protein regions that are useful [26]; G [2]; and J [3]), nonimmunoreactive epitopes (a [3] and b to g [23]), adherence-mediating sites (I, III, V to VII, IX, and X [13]; II and IV [25]; VIII [6]; XI [46]; and XII [8]), and a noncytadherent region (i [46] for vaccination experiments. In the search for such candidate antigens, the results of the present study led to a chimeric protein combining the immunogenic and adherence-mediating properties of both the RP14 and RP30 regions.…”

“…To circumvent the problem, different strategies have been applied (14). The use of recombinant proteins covering defined adhesin regions turned out to be a suitable tool to identify protein regions with antigenic and adherence-mediating properties (3,8,46). However, these previous investigations were applied to only a few protein regions of P1.…”

Infection of the Circulifer haematoceps cell line Ciha-1 by Spiroplasma citri: the non-insect-transmissible strain 44 is impaired in invasion

“…These cellular models are currently used to study the mechanisms of cell infection by a wide variety of animal pathogens, including mycoplasmas (Burnett et al, 2006;Drasbek et al, 2007;Fleury et al, 2002;Giron et al, 1996;Svenstrup et al, 2002;Winner et al, 2000). However, leafhopper cell lines that have been established to study the interactions between plant pathogenic mollicutes and their specific insect vectors are still very few (Omura & Kimura, 1994;Steiner et al, 1984;.…”