Relationship between Expression of the Family of M Proteins and Lipoteichoic Acid to Hydrophobicity and Biofilm Formation in Streptococcus pyogenes

Harry S Courtney,Bernd Kreikemeyer,David L Hasty,Morgan A Pence,Andreas Podbielbski,Victor Nizet,James B Dale,Thomas Penfound,Itzhak Ofek,Matthew E Falagas

doi:10.1371/journal.pone.0004166

Abstract

BackgroundHydrophobicity is an important attribute of bacteria that contributes to adhesion and biofilm formation. Hydrophobicity of Streptococcus pyogenes is primarily due to lipoteichoic acid (LTA) on the streptococcal surface but the mechanism(s) whereby LTA is retained on the surface is poorly understood. In this study, we sought to determine whether members of the M protein family consisting of Emm (M protein), Mrp (M-related protein), Enn (an M-like protein), and the streptococcal protective antigen (Spa) are involved in anchoring LTA in a manner that contributes to hydrophobicity of the streptococci and its ability to form biofilms.Methodology/Principal FindingsIsogenic mutants defective in expression of emm, mrp, enn, and/or spa genes of eight different serotypes and their parental strains were tested for differences in LTA bound to surface proteins, LTA released into the culture media, and membrane-bound LTA. The effect of these mutations on the ability of streptococci to form a hydrophobic surface and to generate biofilms was also investigated. A recombinant strain overexpressing Emm1 was also engineered and similarly tested. The serotypes tested ranged from those that express only a single M protein gene to those that express two or three members of the M protein family. Overexpression of Emm1 led to enhanced hydrophobicity and biofilm formation. Inactivation of emm in those serotypes expressing only a single emm gene reduced biofilm formation, and protein-bound LTA on the surface, but did not alter the levels of membrane-bound LTA. The results were more varied in those serotypes that express two to three members of the M protein family.Conclusions/SignificanceOur findings suggest that the formation of complexes with members of the M protein family is a common mechanism for anchoring LTA on the surface in a manner that contributes to hydrophobicity and to biofilm formation in S. pyogenes, but these activities in some serotypes are dependent on a trypsin-sensitive protein(s) that remains to be identified. The need for interactions between LTA and M proteins may impose functional constraints that limit variations in the sequence of the M proteins, major virulence factors of S. pyogenes.

Highlights

The hydrophobic properties of bacterial surfaces are a major determinant in the adhesion of bacteria and in the formation of biofilms by bacteria on animate and inanimate surfaces [1]
Conclusions/Significance: Our findings suggest that the formation of complexes with members of the M protein family is a common mechanism for anchoring lipoteichoic acid (LTA) on the surface in a manner that contributes to hydrophobicity and to biofilm formation in S. pyogenes, but these activities in some serotypes are dependent on a trypsin-sensitive protein(s) that remains to be identified
Decreased expression of Emm1 led to a decrease in the amount of LTA released by trypsin and to a decrease in hydrophobicity and biofilms. These findings suggest that variations in the amount of M proteins expressed on the surface of streptococci can have a direct impact on the amount of protein-bound LTA, hydrophobicity and biofilm formation

Summary

Introduction

The hydrophobic properties of bacterial surfaces are a major determinant in the adhesion of bacteria and in the formation of biofilms by bacteria on animate and inanimate surfaces [1]. Lipoteichoic acid (LTA) is a major hydrophobin that contributes to the hydrophobicity of a variety of Gram-positive bacteria [1,2,3,4]. Hydrophobicity is an important attribute of bacteria that contributes to adhesion and biofilm formation. We sought to determine whether members of the M protein family consisting of Emm (M protein), Mrp (M-related protein), Enn (an M-like protein), and the streptococcal protective antigen (Spa) are involved in anchoring LTA in a manner that contributes to hydrophobicity of the streptococci and its ability to form biofilms

Methods

Results

Conclusion