The surface-exposed carboxyl region of Mycoplasma pneumoniae elongation factor Tu interacts with fibronectin.
Mycoplasma pneumoniae is a bacterial pathogen of the human respiratory tract that causes a wide range of airway diseases as well as extrapulmonary symptoms. It possesses a distinct, differentiated terminal structure, termed the attachment organelle, that mediates adherence to the host respiratory epithelium. Previously, we reported that surface-associated M. pneumoniae elongation factor Tu (EF-Tu, also called MPN665) serves as a fibronectin (Fn)-binding protein, facilitating interactions between mycoplasmas and extracellular matrix. In the present study, we determined that binding of M. pneumoniae EF-Tu to Fn is primarily mediated by the EF-Tu carboxyl region. A 179-amino-acid region spanning the carboxyl terminus (designated EC; amino acids 192 to 394) binds Fn in a dose-dependent manner. Further analysis of carboxyl constructs (ED3 and ED4) and their deletion truncations (ED3.1, ED3.2, and ED4.1) revealed that the carboxyl region possessed two distinct sites with different Fn-binding efficiencies. Immunogold electron microscopy using antibodies raised against recombinant ED3 and ED4 demonstrated the surface accessibility of the EF-Tu carboxyl region. Competitive binding assays using intact radiolabeled mycoplasmas and purified recombinant ED3 and ED4 proteins, along with antibody blocking assays, reinforced the role of the surface-exposed EF-Tu carboxyl region in Fn binding. Alkali and high-salt treatment of mycoplasma membranes and Triton X-114-partitioned mycoplasma fractions confirmed the stable association of EF-Tu within the mycoplasma membrane. These observations highlight the unique, multifaceted, and unpredictable role of the classically defined cytoplasmic protein EF-Tu relative to cellular function, compartmentalization, and topography.
Balasubramanian
Sowmya
S
Department of Microbiology and Immunology, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA.
Kannan
T R
TR
Baseman
Joel B
JB
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002245:Carbon Dioxide / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D008854:Microscopy, Electron; D009177:Mycoplasma pneumoniae / Q000235:genetics / Q000378:metabolism*; D010444:Peptide Elongation Factor Tu / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D005353:Fibronectins; D011994:Recombinant Proteins; D002245:Carbon Dioxide; C041069:carboxyl radical; D010444:Peptide Elongation Factor Tu