# Functional Analysis of the Major Pilin Proteins of Type IV Pili in Streptococcus sanguinis CGMH010

## TL;DR

This study explores how different pilin proteins affect the function of Type IV pili in Streptococcus sanguinis, revealing their roles in motility and host cell invasion.

## Contribution

The study identifies PilA1 as critical for twitching motility and structured biofilm formation in S. sanguinis.

## Key findings

- PilA1 is essential for the assembly of twitching-active Type IV pili in S. sanguinis CGMH010.
- Recombinant strains expressing motility form more structured biofilms under shearing forces.
- Tfp retraction, not twitching, is essential for host cell invasion in S. sanguinis.

## Abstract

The pil gene cluster for Type IV pilus (Tfp) biosynthesis is commonly present and highly conserved in Streptococcus sanguinis. Nevertheless, Tfp-mediated twitching motility is less common among strains, and the factors determining twitching activity are not fully understood. Here, we analyzed the functions of three major pilin proteins (PilA1, PilA2, and PilA3) in the assembly and activity of Tfp in motile S. sanguinis CGMH010. Using various recombinant pilA deletion strains, we found that Tfp composed of different PilA proteins varied morphologically and functionally. Among the three PilA proteins, PilA1 was most critical in the assembly of twitching-active Tfp, and recombinant strains expressing motility generated more structured biofilms under constant shearing forces compared to the non-motile recombinant strains. Although PilA1 and PilA3 shared 94% identity, PilA3 could not compensate for the loss of PilA1, suggesting that the nature of PilA proteins plays an essential role in twitching activity. The single deletion of individual pilA genes had little effect on the invasion of host endothelia by S. sanguinis CGMH010. In contrast, the deletion of all three pilA genes or pilT, encoding the retraction ATPase, abolished Tfp-mediated invasion. Tfp- and PilT-dependent invasion were also detected in the non-motile S. sanguinis SK36, and thus, the retraction of Tfp, but not active twitching, was found to be essential for invasion.