Mycoplasma genitalium adhesin P110 binds sialic-acid human receptors

Abstract

Adhesion of pathogenic bacteria to target cells is a prerequisite for colonization and further infection. The main adhesins of the emerging sexually transmitted pathogen Mycoplasma genitalium, P140 and P110, interact to form a Nap complex anchored to the cell membrane. Herein, we present the crystal structures of the extracellular region of the virulence factor P110 (916 residues) unliganded and in complex with sialic acid oligosaccharides. P110 interacts only with the neuraminic acid moiety of the oligosaccharides and experiments with human cells demonstrate that these interactions are essential for mycoplasma cytadherence. Additionally, structural information provides a deep insight of the P110 antigenic regions undergoing programmed variation to evade the host immune response. These results enlighten the interplay of M. genitalium with human target cells, offering new strategies to control mycoplasma infections.

Fig. 1

Overall structure of P110. a Two views, 90° apart from each other, of the extracellular region of P110 that is formed by a large N-domain, with a seven blade β-propeller (green), the crown (brown), and the C-domain (orange). In the right side panel the view is along the central axis of the β-propeller. The situation of the seven blades in the propeller is explicitly indicated showing that the two terminal blades I and VII are close to the C-terminal domain and opposite to the crown

Fig. 2

Binding of sialic acid oligosaccharides to P110. a Schematic representation of the disposition of P110 with respect to the mycoplasma membrane (same color code as in Fig. 1). The predicted transmembrane helix follows in sequence to the C-domain, which accordingly is expected to be close to the cell membrane. The overall structure of P110 can be sketched as a capital letter P with the sialic binding site (indicated in the figure with a rectangle) located in the crown, away from the cell membrane. Detail of the binding to P110 of sialic acid oligosaccharides b 3SL and c 6SL. Left panels show the electron density corresponding to oligosaccharides in a sigma weighted (Fo–Fc) omit map at two sigma. The binding site and the binding interactions with P110 for both oligosaccharides are very similar, but not identical

Fig. 3

Comparison of sialic binding sites in bacterial adhesins. Binding of the oligosaccharides neuraminic acid moiety to a adhesin P110 from M. genitalium and to the serine-rich repeat (SRR) adhesins from b SrpA (PDB accession code 5IJ1), and c GspB (PDB accession code 5IUC). The extended tripeptide X-Tyr/Phe–Ser/Thr (residues 456–458 in P110) appears to be a common binding motif for sialic acid. Carbon atoms for the oligosaccharides are represented in light blue, while for amino acid residues are represented in brown. Nitrogen and oxygen atoms are depicted in dark blue and red, respectively

Fig. 4

Affinity of oligosaccharides by SPR. Characterization of M. genitalium P110 variants. a SPR sensorgrams showing the binding of the erP110 β-propeller N-domain to immobilized 3SL and 6SL (upper and lower left panels, respectively). Data fitting of duplicates assuming a Langmuir 1:1 affinity model depicting the K_D calculated in steady state for 3SL and 6SL (upper and lower right panels, respectively). b SDS–PAGE of whole cell lysates from WT and different P110 mutant strains. c HA assays determined by FACS analysis. Graphic depicts inverse Langmuir plots obtained with a fixed amount of mycoplasma cells and increasing amounts of hRBCs. Plots were generated using data from, at least two biological repeats for each strain. d HA assays of the WT strain using neuraminidase-treated hRBCs

Fig. 5

Binding of Potassium to P110. a Overall views of the potassium binding site (indicated by a rectangle) in the C-terminal domain of erP110. Detail of the potassium binding site in the presence (b) and in the absence (c) of potassium. Electron density corresponding to the potassium is shown with a sigma weighted (Fo–Fc) omit map at four sigma

Fig. 6

MgPar regions of P110. a Sequence alignment of the KL variable region of P110 and different MgPars encompassing the sialic binding site. KL region of MgPar-3 carries several STOP codons within the aligned segment and it was not included in the analysis. Similarly, the MgPar-6 region was omitted because it does not contain variable sequences homologous to P110. Residues shown to be critical for cell adhesion in this work (black star) appear to be fully conserved in the KL variable region of all the MgPar repeats. These residues are also conserved in the M. pneumonia protein MPN_142, orthologous to P110 (lower part of the panel). b Two views, 90° apart from each other, of the P110 surface. The KL (residues 42–294 in red) and LM (residues 462–517 in blue) variable regions and the poly-Serine tract (409–419 in yellow) of the P110 protein are depicted. Variable regions surround the receptor binding site where a bound oligosaccharide is shown (green)