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

Background: Hydrophobicity 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 Findings: Isogenic 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 an

Effect of inactivation of <i>emm</i>, <i>mrp</i>, <i>enn</i> or <i>spa</i> in patterns C, D, and E serotypes on biofilm formation, hydrophobicity, and LTA expression.

<p>The values for wild type parental strain was used as the control and set at 100% and the data for mutants are provided as percent of control (data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004166#pone-0004166-t001" target="_blank">table 1</a>). The horizontal line at the 100% mark represents the values for parental controls, which are not individually shown.</p

Effect of variable expression of Emm1 on hydrophobicity, biofilm formation, and protein-bound LTA.

<p>A. Microtiter wells were coated with the indicated streptococci and then reacted with rabbit anti-SM1(1-26) serum followed by peroxidase-conjugated, goat anti-rabbit Ig. B. The hydrophobicity of the streptococci was determined by adhesion to hexadecane as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004166#s2" target="_blank">Materials and Methods</a>. C. The ability of the streptococci to form biofilms was determined by the microtiter assays as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004166#s2" target="_blank">Materials and Methods</a>. D. The amount of protein-bound LTA (trypsin extractable) for the streptococci was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004166#s2" target="_blank">Materials and Methods</a>. All assays were done in triplicate and the S.D. is shown.</p

Effect of growth medium on biofilm formation.

<p>A. The growth of M type 4 <i>S. pyogenes</i> in the indicated media was determined by measuring the A₅₃₀ after a 48 hour incubation at 37°C. B. The formation of biofilms by M type 4 <i>S. pyogenes</i> grown for 48 hours at 37°C in the indicated media. These experiments were done in quadruplicate and the mean±SD is shown.</p

Biofilm formation and hydrophobicity of <i>S. pyogenes</i>.

<p>A. Biofilm formation by the various serotypes was determined by the microtiter assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004166#s2" target="_blank">Materials&Methods</a>. Black bars indicate biofilm formation by various serotypes and white bars indicate formation of biofilms in the presence of trypsin. B. Hydrophobicity of <i>S. pyogenes</i>. Black bars indicate the degree of hydrophobicity of the various serotypes as measured by their ability to bind to hexadecane. White bars indicate the percent hydrophobicity after trypsin treatment. All experiments were done in triplicate and performed at least twice. The mean±SD is shown.</p

Biofilms, hydrophobicity, and surface, membrane-bound, and culture released LTA of <i>Streptococcus pyogenes</i>

a<p>biofilm formation was determined in microtiter wells as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004166#s2" target="_blank">Materials&Methods</a>, biofilm formation by parental strains (wt) was the positive control and set at 100%.</p>b<p>hydrophobicity was determined by adhesion of streptococci to hexadecane as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004166#s2" target="_blank">Materials&Methods</a>, adhesion by parental strains was the control and set at 100%.</p>c<p>µg of LTA complexed to surface proteins was obtained by trypsin extraction of 1 ml of streptococci (OD₅₃₀ = 1.0) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004166#s2" target="_blank">Materials&Methods</a>.</p>d<p>µg of LTA obtained by phenol extraction of 1 ml of trypsinized streptococci (OD₅₃₀ = 1.0) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004166#s2" target="_blank">Materials&Methods</a>.</p>e<p>Lauth, X., M. von Kockritz-Blickwede, C.W. McNamara, S. Myskowski, A.S. Zinkernagel, B. Beall, P. Ghosh, R. L. Gallo, and V. Nizet. 2008. M1 protein allows group A streptococcal survival in phagocyte extracellular traps by cathelicidin inhibition. (submitted).</p>*<p>indicates that the mutant is significantly different from the parent, p≤0.05 as determined with students t-test.</p><p>All experiments were performed in triplicate. The mean±SD is shown.</p

Effect of inactivation of <i>emm</i> in pattern A serotypes on biofilm formation, hydrophobicity, and LTA expression.

<p>The values for the wild type parental strain was used as the control and set at 100% in each case and data for the mutants are provided as percent of control (data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004166#pone-0004166-t001" target="_blank">table 1</a>). The horizontal line at the 100% mark represents the values for parental controls, which are not individually shown.</p

Correlation of membrane-bound LTA with trypsin extracted LTA and with LTA released into the culture media.

<p>The amount of LTA bound to membranes was compared to the amount of LTA released into the culture media (A) and to the amount of LTA extracted with trypsin (B). There was a significant degree of correlation in each case, r = 0.730.</p