Adhesion and inhibition assay of Mycoplasma genitalium and M. pneumoniae by immunofluorescence microscopy

Abstract

Adhesion of Mycoplasma pneumoniae and the closely related M. genitalium to HEp-2 cells was investigated. The main surface proteins known to be involved in adhesion are P1 of M. pneumoniae and its homologue, MgPa, of M. genitalium. Both proteins are also immunodominant proteins. Protein P116 is another immunodominant protein of M. pneumoniae. These immunogenic proteins were investigated for their surface exposure and involvement in adhesion to host epithelial cells. Immunofluorescence microscopy (IFM) was used to detect M. pneumoniae and M. genitalium adhering to HEp-2 cells. Monospecific antibodies were produced against fragments of the surface proteins lacking tryptophan stop codons and were used for adhesion detection, surface exposure and adhesion inhibition IFM assays. Three monospecific antibodies were made against MgPa covering regions in the N-terminal, the middle and the C-terminal part; two monospecific antibodies were produced against P1 covering regions of the N-and the C-terminal part and one monospecific antibody was made against most of P116. Only the C-terminal parts of P1 and MgPa were surface exposed and blocking of these regions with the monospecific antibody resulted in inhibition of cytadsorption. Protein P116 was shown to be surface exposed and an essential protein involved in adhesion because the anti-P116 antibody prevented attachment of M. pneumoniae to the HEp-2 cells independently of P1. This study adds to the understanding of the molecular biology of M. genitalium and M. pneumoniae and presents a method to study the proteins involved in adhesion of these mycoplasmas.

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

“…The recombinant protein M. pneumoniae rP1-II aa 1107-1518 and the recombinant M. genitalium protein rMgPa aa 628-1350 were produced as described previously (Svenstrup et al, 2002). To reduce the concentration of urea in the purification buffer, the proteins were first diluted to 0.2 mg ml 21 in growth medium and then dialysed against the same medium with 10 mg gentamicin ml 21 for 2 h at 4 uC.…”

“…The HEp-2 cells were washed twice in PBS and fixed in 100 % methanol at 4 uC for 1 min. To detect the adhering mycoplasmas, the infected HEp-2 cells were incubated with primary antibodies (PabFH or PabG37) diluted 1 : 500 for 30 min at 37 uC (Svenstrup et al, 2002), washed with PBS and incubated with 100 ml per well of secondary FITC-conjugated 'Affinipure' goat anti-rabbit (GaR) IgG (H+L) (Jackson Immuno Research Laboratories) diluted 1 : 100 in PBS with 0.002 % Evans blue, for 30 min at 37 uC. The wells were washed twice with PBS before attached M. pneumoniae were visualized using a fluorescence microscope.…”

“…The recombinant rP1-II protein contained a 66His tag, which was used for nickel-affinity purification under denaturing conditions in a urea-containing buffer (Svenstrup et al, 2002). However, urea is toxic to HEp-2 cells, so rP1-II was diluted to 0.2 mg ml 21 and subjected to dialysis against growth medium for 2 h prior to use.…”

“…In addition, the surface protein P65 contains an Arg-Gly-Asp (RGD) domain, raising the possibility that M. pneumoniae attaches to binding sites for fibronectin on the host cell (Krause, 1996). Recently, it has been shown that antibodies against the C-terminal part of the M. pneumoniae P1 protein and the homologous Mycoplasma genitalium MgPa protein inhibit host-cell adhesion of M. pneumoniae and M. genitalium, respectively (Svenstrup et al, 2002). mAbs that recognize epitopes in this region (Dallo et al, 1988;Jacobs et al, 1990) also inhibit the binding of M. pneumoniae to host cells.…”

“…Adhering mycoplasmas contain several adhesins, and M. pneumoniae expresses at least three different adhesins, termed P1, P30 and P116, suggesting that there are multiple receptors for M. pneumoniae on the host cells (Razin & Jacobs, 1992;Svenstrup et al, 2002). Monospecific antibodies raised against P116 block the adhesion of M. pneumoniae to HEp-2 cells, as do antibodies against the P30 protein (Baseman et al, 1987).…”