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Phosphorylation of cytadherence-accessory proteins in Mycoplasma pneumoniae
L B Dirksen1, K A Krebes, D C Krause
1Department of Microbiology, University of Georgia, Athens 30602.
Abstract:
Attachment to host cells of the respiratory epithelium by Mycoplasma pneumoniae is a complex, multicomponent process, requiring a number of accessory proteins in addition to adhesins directly involved in receptor binding. In this study, protein phosphorylation of the cytadherence-accessory proteins HMW1, HMW2, and HMW4 of M. pneumoniae was examined using biochemical and immunological techniques. The initial indication of protein modification came from Western immunoblot analysis of the two-dimensional polyacrylamide gel electrophoresis (PAGE) profile of M. pneumoniae proteins, revealing multiple spots for both HMW1 and HMW4 that varied in pI but not in size. M. pneumoniae cultured in the presence of H3(32)PO4 exhibited numerous phosphorylated proteins as detected by sodium dodecyl sulfate-PAGE and autoradiography. These included proteins corresponding to HMW1, HMW2, and HMW4 in electrophoretic mobility. The Triton X-100 partitioning characteristics of these phosphorylated proteins was identical to that described previously for HMW1, -2, and -4. Furthermore, these protein bands were absent when a noncytadhering variant deficient in HMW1-5 was examined in the same manner. Finally, the availability of antiserum to HMW1 and -4 enabled us to confirm by radioimmunoprecipitation that HMW1 and HMW4 are phosphoproteins. Phosphoamino acid analysis of acid-hydrolyzed HMW1 and HMW2 identified primarily phosphothreonine and, to a lesser extent, phosphoserine in HMW1 and predominantly phosphoserine, with a trace of phosphothreonine, in HMW2. Neither protein contained phosphotyrosine. HMW1-HMW5 are components of a cytoskeleton-like structure in M. pneumoniae that is thought to function in cell division, changes in cell morphology, gliding motility, and the localization of adhesins in the mycoplasma membrane. Phosphorylation may regulate cytoskeleton dynamics involving these cytadherence-accessory proteins.
Insights
Mycoplasma pneumoniae cytadherence proteins HMW1, HMW2, and HMW4 are phosphorylated, primarily on threonine and serine residues. This phosphorylation may regulate the dynamics of the M. pneumoniae cytoskeleton, impacting cell functions.
Area of Science:
- Microbiology
- Molecular Biology
- Cell Biology
Background:
- Mycoplasma pneumoniae attachment to respiratory epithelium involves adhesins and accessory proteins.
- Cytadherence-accessory proteins, including HMW1, HMW2, and HMW4, play a crucial role in M. pneumoniae host cell attachment.
- The HMW1-HMW5 protein complex forms a cytoskeleton-like structure involved in various cellular processes.
Purpose of the Study:
- To investigate protein phosphorylation of cytadherence-accessory proteins HMW1, HMW2, and HMW4 in Mycoplasma pneumoniae.
- To determine the specific amino acid residues phosphorylated in HMW1 and HMW2.
- To explore the potential role of phosphorylation in regulating M. pneumoniae cytoskeleton dynamics.
Main Methods:
- Two-dimensional polyacrylamide gel electrophoresis (PAGE) and Western immunoblot analysis.
- Sodium dodecyl sulfate-PAGE and autoradiography following culturing with H3(32)PO4.
- Triton X-100 partitioning and radioimmunoprecipitation.
- Phosphoamino acid analysis of acid-hydrolyzed proteins.
Main Results:
- HMW1 and HMW4 exhibited multiple spots in 2D PAGE, indicating post-translational modification.
- Phosphorylation was detected in proteins with electrophoretic mobility corresponding to HMW1, HMW2, and HMW4.
- HMW1 and HMW4 were confirmed as phosphoproteins; HMW1 contained phosphothreonine and phosphoserine, while HMW2 contained predominantly phosphoserine.
- Phosphotyrosine was not detected in HMW1 or HMW2.
Conclusions:
- HMW1, HMW2, and HMW4 are phosphoproteins in Mycoplasma pneumoniae.
- Phosphorylation of these cytadherence-accessory proteins, particularly on threonine and serine residues, is confirmed.
- Protein phosphorylation may be a regulatory mechanism for the M. pneumoniae cytoskeleton and associated functions like cytadherence.