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Molecular basis for sortase-catalyzed pilus tip assembly.

Aadil H Bhat1, Chungyu Chang1, Asis Das2

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Summary
This summary is machine-generated.

The cell wall sorting signal (CWSS) of the CafA pilin is essential for determining its position at the tip of Gram-positive bacterial pili. This mechanism, involving the FLIAG motif, is crucial for pilus assembly and has potential bioengineering applications.

Keywords:
Actinomyces oriscell wall anchoringcoaggregationpilus assemblysecretionsortasetip pilin

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Biochemistry

Background:

  • Gram-positive bacteria assemble pili using sortase enzymes that crosslink pilin monomers via cell wall sorting signals (CWSS).
  • The molecular mechanisms governing pilus tip assembly, specifically the selection of tip pilins, remain largely unknown.

Purpose of the Study:

  • To elucidate the molecular determinants responsible for pilus tip assembly in the Gram-positive bacterium *Actinomyces oris*.
  • To investigate the role of the cell wall sorting signal (CWSS) and its conserved motifs in directing tip pilin localization.

Main Methods:

  • Genetic manipulation of pilin CWSS in *Actinomyces oris* to create hybrid proteins.
  • Analysis of pilus assembly and localization of engineered proteins using microscopy and functional assays.
  • Investigating the role of the conserved FLIAG motif within the CWSS.

Main Results:

  • Swapping the CWSS of the tip pilin CafA with another pilin's CWSS resulted in a functional hybrid localizing at the pilus tip.
  • The conserved FLIAG motif within the CafA CWSS is critical for proper pilus tip localization and function.
  • Heterologous proteins (GspA, SpaA) engineered with the CafA CWSS were successfully targeted to the pilus tip, demonstrating the sufficiency of the CWSS.

Conclusions:

  • The CWSS of the tip pilin CafA, particularly the FLIAG motif, is both necessary and sufficient for its specific localization at the pilus tip during assembly.
  • This mechanism provides a molecular basis for sortase-catalyzed pilus tip assembly in Gram-positive bacteria.
  • Findings suggest potential bioengineering applications for controlling the display of proteins on bacterial surfaces.