The effects of 10 mM sodium metaperiodate or 6 U of proteinase K on the preformed biofilms developed by the wild type (WT) strain HC474 and the derivative <i>lrgB</i> and <i>yycI</i> knockout and complemented strains.

<p>(A) HC474 (WT), M1HC474 (<i>lrgB</i>::pLGEM), and C1HC474 (P_cad-<i>lrgB</i>). (B) HC474 (WT), M2HC474 (<i>yycI</i>::pYGEM), and C2HC474 (P_cad-<i>yycI</i>). For graphic presentation purposes, the amount of biofilm was transformed into a percentage taken as reference (defined as 100%) the mean value obtained for M1HC474 (upper panel) or M2HC474 (bottom panel). ***<i>p</i> < 0.0001.</p

The effects of 56 U of DNase I on preformed biofilms developed by the wild-type (WT) HC474 strain and its derivative <i>lrgB</i> and <i>yycI</i> knockout and complemented strains.

<p>Confocal laser scanning microscopy of biofilms before (left panels) and after (right panels) DNase I treatment using a microtiter-based method. (A) HC474 (WT); (B) M1HC474 (<i>lrgB</i>::pLGEM); (C) C1HC474 (P_cad-<i>lrgB</i>); (D) HC474; (E) M2HC474 (<i>yycI</i>::pYGEM); and (F) C2HC474 (P_cad-<i>yycI</i>). For graphic presentation purposes, the amount of biofilm was transformed into a percentage taken as reference (defined as 100%) the mean value obtained for M1HC474 or M2HC474. ***p < 0.0001.</p

Inactivation of the Autolysis-Related Genes <i>lrgB</i> and <i>yycI</i> in <i>Staphylococcus aureus</i> Increases Cell Lysis-Dependent eDNA Release and Enhances Biofilm Development <i>In Vitro</i> and <i>In Vivo</i>

<div><p><i>Staphylococcus aureus ica</i>-independent biofilms are multifactorial in nature, and various bacterial proteins have been associated with biofilm development, including fibronectin-binding proteins A and B, protein A, surface protein SasG, proteases, and some autolysins. The role of extracellular DNA (eDNA) has also been demonstrated in some <i>S</i>. <i>aureus</i> biofilms. Here, we constructed a Tn<i>551</i> library, and the screening identified two genes that affected biofilm formation, <i>lrgB</i> and <i>yycI</i>. The repressive effect of both genes on the development of biofilm was also confirmed in knockout strains constructed by allelic recombination. In contrast, the superexpression of either <i>lrgB</i> or <i>yycI</i> by a cadmium-inducible promoter led to a decrease in biofilm accumulation. Indeed, a significant increase in the cell-lysis dependent eDNA release was detected when <i>lrgB</i> or <i>yycI</i> were inactivated, explaining the enhanced biofilm formed by these mutants. In fact, <i>lrgB</i> and <i>yycI</i> genes belong to distinct operons that repress bacterial autolysis through very different mechanisms. LrgB is associated with the synthesis of phage holin/anti-holin analogues, while YycI participates in the activation/repression of the two-component system YycGF (WalKR). Our <i>in vivo</i> data suggest that autolysins activation lead to increased bacterial virulence in the foreign body animal model since a higher number of attached cells was recovered from the implanted catheters inoculated with <i>lrgB</i> or <i>yycI</i> knockout mutants.</p></div

Transcriptional levels of <i>lrgB</i> and <i>yycI</i>.

<p>(A) and (B) Total RNA was isolated from free-living or sessile cells of the HC474 strain. (C) and (D) Total RNA was obtained from sessile cells of BMB9393 or GV69 isolates. Tests were performed using real-time qRT-PCR. The results were considered significant when the difference in gene expression varied by two-fold or more. RQ, Relative Quantity. Right bottom panel: Biofilm accumulated by the isolates BMB9393 (strong biofilm producer) and GV69 (moderate producer) on inert polystyrene microtiter plates.</p

Most relevant strains used in this study.

<p>Most relevant strains used in this study.</p

Virulence comparisons between the <i>S</i>. <i>aureus</i> wild type (WT) strain and the <i>lrgB</i> or <i>yycI</i> isogenic knockouts in a foreign-body infection model.

<p>Difference in the number of bacterial cells recovered from the catheters implanted in mice inoculated with WT (HC474) or (A) M1HC474 (<i>lrgB</i>::pLGEM) or (B) M2HC474 (<i>yycI</i>::pYGEM). Columns represent the mean number of sessile cells recovered from the catheters implanted in the animals. For graphic presentation purposes, CFU/mL was transformed into a percentage taken as reference (defined as 100%) the average CFU/mL value recovered for (A) M1HC474 or (B) M2HC474. *<i>p</i> < 0.01.</p

eDNA assay using wild type (WT) strain HC474 and the isogenic (A) <i>lrgB</i> and (B) <i>yycI</i> knockouts.

<p>The concentration of eDNA was determined in the biofilm supernatants (*<i>p</i> < 0.01).</p

Biofilm development by the wild type (WT) strain HC474, knockout mutants for <i>lrgB</i> (M1HC474) and <i>yycI</i> (M2HC474) and respective complemented mutants (C1HC474 and C2HC474).

<p>(A) Percentage of biofilm accumulation on inert polystyrene surfaces by HC474 (WT), M1HC474 (<i>lrgB</i>::pLGEM), and C1HC474 (P_cad-<i>lrgB</i>). (B) Images obtained by confocal laser scanning microscopy (CLSM) of the biofilms accumulated by the WT HC474 (14.10 μm thick), M1HC474 (18.13 μm thick), and C1HC474 (6.04 μm thick). (C) Percentage of biofilm accumulated on inert polystyrene surfaces by HC474 (WT), M2HC474 (<i>yycI</i>::pYGEM), and C2HC474 (P_cad-<i>yycI</i>). (D) Images obtained by CLSM of the biofilms accumulated by HC474 (14.10 μm thick), M2HC474 (24.19 μm thick), and C2HC474 (8.06 μm thick). For graphic presentation purposes, biofilm data were transformed into a percentage taken as reference (defined as 100%) the mean value obtained for M1HC474 (A) or M2HC474 (C). ***p < 0.0001.</p

Autolytic activity of wild type (WT) strain HC474 and the isogenic (A) <i>lrgB</i> and (B) <i>yycI</i> knockouts in the presence of 0.1% Triton X-100.

<p>Cell lysis was measure at OD_600nm every 15 min until 4 h. The increase in the autolytic activity detected for both <i>lrgB</i> and <i>yycI</i> mutants were extremely significant ((p<0.0001).</p

Plasmids used in this study.

<p>Cm, chloramphenicol; Amp, ampicillin; Ery, erythromycin.</p><p>Plasmids used in this study.</p