Identification of fibronectin-binding proteins in Mycoplasma gallisepticum strain R

Abstract

We have determined that virulent Mycoplasma gallisepticum strain Rlow is capable of binding the extracellular matrix protein fibronectin. Fibronectin was found to be present in M. gallisepticum Rlow protein extracts by Western blotting and peptide sequencing. Mycoplasma gallisepticum Rhigh, the attenuated, high-passage derivative of Rlow, is deficient in this ability. MGA_1199, the M. gallisepticum homologue of the cytadherence-associated protein P65 from Mycoplasma pneumoniae, and MGA_0928, the M. gallisepticum homologue of the M. pneumoniae cytoskeletal protein HMW3, were identified as fibronectin-binding proteins. Peptides from the regions of MGA_1199 and MGA_0928 exhibiting the highest degree of homology with known fibronectin-binding proteins were shown to bind the gelatin/heparin-binding domain of fibronectin. MGA_1199 and MGA_0928 were shown to be absent and aberrant, respectively, in Rhigh, explaining its lack of fibronectin-binding capability. Consistent with its M. pneumoniae counterpart, MGA_1199 (renamed PlpA) was demonstrated to be surface exposed, despite a lack of classical membrane-spanning domains. Due to its demonstrated topology and the strength of interaction between its binding peptide and fibronectin, we propose that PlpA functions as a fibronectin-binding protein in vivo and may possess atypical transmembrane domains.

FIG. 1.

Protein profiles of R_low and R_high analyzed by SDS-PAGE and Western blotting. SDS-PAGE (A, B, and C) and Western blotting (D, E, and F) demonstrate that three proteins were clearly absent (A, C, D, and F) or aberrant (D and E) in R_high. Arrows indicate the presence or absence of the implicated proteins, and pertinent molecular masses are noted. Western blot analysis was performed with anti-fibronectin (D), anti-HMW3 (E), and anti-PlpA (F).

FIG. 2.

Sample peptide alignments. (A) Peptides generated from MALDI-MS analysis of the 158-kDa band aligned with PlpA. (B and C) Peptides generated from the 200-kDa band aligned with Hlp3 or with fibronectin.

FIG. 3.

Nucleotide sequencing of hlp3 and plpA. (A) A 54-bp deletion occurs in hlp3 in R_high. (B) A duplication of 17 bp occurs in plpA in R_high.

FIG. 4.

Suspected fibronectin-binding peptides. (A) AlignX Blocks was used to define homologous regions between PlpA, Hlp3, and known fibronectin-binding proteins. (B) Peptides derived from the most homologous regions of each block are shown. Peptides shown to bind fibronectin are displayed in red. (C) Peptides 1 (Hlp3) and 7 (PlpA) are aligned. Identical amino acids are shown in red, similar amino acids are shown in purple, and dissimilar amino acids are shown in blue. These peptides, each shown to bind fibronectin, are 60% identical and 80% similar.

FIG. 5.

Peptide-binding assays. Biotinylated peptides from the homologous blocks were shown to bind fibronectin immobilized on nitrocellulose, as indicated by arrows. Peptide 1 from Hlp3 (A) and peptide 7 from PlpA (B) were both shown to bind. These peptides were 60% identical and 80% similar to each other. (C) Peptide 7 was exposed to immobilized subunits of fibronectin and shown to interact with the gelatin/heparin-binding domain. Lane 1, whole fibronectin; lane 2, fragment IIIC; lane 3, adhesion-promoting peptide; lane 4, residues 1377 to 1398; lane 5, gelatin/heparin-binding domain.

FIG. 6.

Binding inhibition assay to demonstrate surface exposure of PlpA. Decreasing titers of R_low were incubated with preimmune rabbit serum IgG (stippled bars) and with anti-PlpA IgG (striped bars) and then exposed to immobilized fibronectin in microtiter plates. Bound organisms were detected by chicken anti-R_high, followed by horseradish peroxidase (HRP)-conjugated goat anti-chicken. At each titer, a significant difference (*) (P < 0.02) in the level of colorimetric signal (o-phenylenediamine) generated was observed.

FIG. 7.

Growth inhibition by complement-mediated lysis. R_low cells were incubated in the presence of hyperimmune rabbit serum raised against the MID and CTD of PlpA and guinea pig serum or in guinea pig serum alone. Following incubation at 37°C, Hayflick's medium was added. Growth of R_low was assessed by optical density after 24 h. Incubation in the presence of complement components and anti-PlpA antiserum significantly (P < 0.004) impeded the subsequent growth of R_low (spotted bars) compared to the growth of R_high (striped bars) incubated in the presence of anti-PlpA antiserum and complement components or R_low (stippled bars) incubated in the presence of complement components alone.

FIG. 8.

Investigation of putative transmembrane regions of PlpA. GxxxG (GG₄), IxxxI (II₄), and GxxxGxxxG (GG₄E) motifs from PlpA were examined for their abilities to interact with membranes. Liposomes were generated in the presence of biotinylated peptides representing these motifs. Excess peptide was removed by washing. Liposomes were immobilized on paraffin wax, after which incorporated peptides were detected with streptavidin-HRP. (A) Empty liposomes, the hydrophilic peptide (PlpA⁻), the GG₄ peptide, and the II₄ peptide did not show any reactivity, indicating that no peptide was incorporated during synthesis. The ponticulin peptide and the expanded motif of GG₄ did show reactivity (arrows), indicating that they were incorporated into the liposomes. In addition, biotinylated peptides were introduced into the membrane of M. gallisepticum by electroporation. Resulting cells were spotted onto nitrocellulose for the detection of embedded peptide by streptavidin-HRP. (B) Panels are labeled with the name of each peptide; those marked with a plus sign represent electroporated cells used to detect embedding, and those marked with a minus sign represent nonpulsed cells mixed with peptides to detect nonspecific binding. The hydrophilic peptide (PlpA⁻), the GG₄ peptide, and the II₄ peptide did not embed in the membrane or bind to it nonspecifically. The ponticulin peptide embedded in the membrane but also bound the cell surface nonspecifically. The expanded repeat of GG₄ embedded in the cell membrane and did not bind nonspecifically.