The effect of calcium binding on adhesion and pilus biogenesis in the PilC family of proteins Public Deposited

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  • March 21, 2019
Creator
  • Johnson, Michael D. L.
    • Affiliation: School of Medicine, Department of Biochemistry and Biophysics
Abstract
  • Pseudomonas aeruginosa is an opportunistic pathogen prevalent in people on immunosuppressants, recent open wounds, or cystic fibrosis patients. P. aeruginosa attaches to the host cell via polar type IV pili (tfp). These tfp are composed of many proteins including the major pilus subunit PilA, retraction protein PilT, and one focus of this dissertation, 1163 residue protein PilY1. PilY1 shares homology to other bacterial adhesion and pilus biogenesis proteins such as the PilC family of proteins in Neisseria gonorrhoeae, Neisseria meningitidis, and Kingella kingae thus, it is often characterized as a pilus biogenesis protein and the P. aeruginosa adhesin. However, no known mechanism for either situation had been elucidated. In this study, we have identified two calcium binding sites which affect both functional pilus biogenesis and adhesion to host cells. In addition, we have also located an RGD (integrin binding motif) that mediates PilY1 binding to integrin. The C-terminal most PilY1 calcium binding site (CBS) was found from residues 851-859 in PAK and is conserved in other strains of P. aeruginosa. This CBS is also conserved in the PilC proteins over 100 different bacteria. We found, through mutating the bidentate aspartic acid in vitro and in vivo, that this CBS controlled functional pilus biogenesis with the calcium bound and unbound states corresponding to pilus extension and retraction respectfully. We also characterized the homologous CBSs in N. gonorrhoeae and K. kingae finding similar results. Upon further examination, we also located separate CBS from residues 600-608. This site was in close proximity to an RGD. This CBS was conserved in other strains of P. aeruginosa, but not in other bacteria. We found that purified PilY1 bound integrin in an RGD dependent manner. Furthermore, we found that this interaction was mediated by the calcium bound states of both CBSs. Here, we demonstrate that P. aeruginosa PilY1 uses calcium to mediate both functional pilus biogenesis factor and adhesion to host cells. Future studies will hopefully include exploiting both calcium binding domains to prevent pilus formation and binding to host cells and characterizing the in vivo effects of these three sites.
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  • In Copyright
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  • "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biochemistry and Biophysics."
Advisor
  • Redinbo, Matthew R.
Degree granting institution
  • University of North Carolina at Chapel Hill
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Place of publication
  • Chapel Hill, NC
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  • Open access
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