SDS interferes with SaeS signalling of Staphylococcus aureus independently of SaePQ

CITATION: Makgotlho, P. E. et al. 2013. SDS interferes with SaeS signalling of Staphylococcus aureus independently of SaePQ. PLoS ONE, 8(8): e71644, doi:10.1371/journal.pone.0071644.The original publication is available at http://journals.plos.org/plosoneThe Staphylococcus aureus regulatory saePQRS system controls the expression of numerous virulence factors, including extracellular adherence protein (Eap), which amongst others facilitates invasion of host cells. The saePQRS operon codes for 4 proteins: the histidine kinase SaeS, the response regulator SaeR, the lipoprotein SaeP and the transmembrane protein SaeQ. S. aureus strain Newman has a single amino acid substitution in the transmembrane domain of SaeS (L18P) which results in constitutive kinase activity. SDS was shown to be one of the signals interfering with SaeS activity leading to inhibition of the sae target gene eap in strains with SaeSL but causing activation in strains containing SaeSP. Here, we analyzed the possible involvement of the SaeP protein and saePQ region in SDS-mediated sae/eap expression. We found that SaePQ is not needed for SDS-mediated SaeS signaling. Furthermore, we could show that SaeS activity is closely linked to the expression of Eap and the capacity to invade host cells in a number of clinical isolates. This suggests that SaeS activity might be directly modulated by structurally non-complex environmental signals, as SDS, which possibly altering its kinase/phosphatase activity.http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0071644Publisher's versio

<i>saeP</i> deletion does not affect SDS-mediated <i>sae</i> activity.

<p>(<b>A</b>) Schematic representation of the <i>sae</i> locus with its four ORFs. Two promoters, P1 and P3 generate three primary transcripts (T1, T3, and T4). T1 processing by an endonucleolytic enzyme, RNase Y, results in T2. (<b>B, C, D, E</b>) Wild type and <i>saeP</i>-deleted strain in Newman and ISP479C backgrounds were grown in TSB without (-) or with (+) SDS (30% MIC) until late exponential growth phase. (<b>B</b>) Relative expression of <i>saeR</i> in relation to <i>gyrB</i> was assessed by qRT–PCR. The results represent means ± SEM of at least three independent experiments performed in triplicates. (<b>C</b>) (<b>Lower panel</b>) Expression of Eap was monitored by SDS PAGE and silver staining. (<b>Upper panel</b>) Expression of SaeR was monitored by Western blot analyses with specific antibody against SaeR. (<b>D</b>) Relative expression of <i>eap</i> in relation to <i>gyrB</i> was assessed by qRT–PCR. The results represent means ± SEM of at least three independent experiments performed in triplicates (<b>E</b>) Cellular invasion of 293 cells was measured and expressed as relative invasiveness compared to <i>S. aureus</i> strain Cowan I. Results represent means ± SEM of at least three independent experiments performed in duplicates. (<b>B,D,E</b>) Asterisks indicate the significance of comparisons (***P<0.001; **P = 0.001–0.01; *P = 0.01–0.05; ns P>0.05).</p

<i>saePQ</i> is not needed for <i>sae</i> mediated response to SDS.

<p>Wild type (Newman), <i>sae</i>-deleted, and <i>sae</i>-deleted strains complemented with <i>saePQRS^P</i>, <i>saeRS^P</i>, <i>saePQRS^L</i>, <i>and saeRS^L</i> were grown in TSB with or without SDS (30% MIC) until late exponential phase. (<b>A</b>) RNA was hybridized with a digoxigenin-labelled <i>saeR</i>-specific probe. 16S rRNA detected in ethidium bromide-stained gels is shown as a loading control. (<b>B</b>) Relative expression of <i>saeR</i> in relation to <i>gyrB</i> assessed by qRT–PCR. Results represent means ± SEM of at least three independent experiments performed in triplicates. (<b>C</b>) RNA was hybridized with a digoxigenin-labeled <i>eap</i>-specific probe. (<b>D</b>) Cellular invasiveness was measured in 293 cells and expressed as relative invasiveness compared to <i>S. aureus</i> strain Cowan I. Results represent means ± SEM of at least three independent experiments performed in duplicates. (<b>B,D</b>) Asterisks indicate the significance of comparisons (**P = 0.001–0.01; *P = 0.01–0.05).</p

Oligonucleotides.

<p>Oligonucleotides.</p

SDS Interferes with SaeS Signaling of Staphylococcus aureus Independently of SaePQ

<p>The Staphylococcus aureus regulatory saePQRS system controls the expression of numerous virulence factors, including extracellular adherence protein (Eap), which amongst others facilitates invasion of host cells. The saePQRS operon codes for 4 proteins: the histidine kinase SaeS, the response regulator SaeR, the lipoprotein SaeP and the transmembrane protein SaeQ. S. aureus strain Newman has a single amino acid substitution in the transmembrane domain of SaeS (L18P) which results in constitutive kinase activity. SDS was shown to be one of the signals interfering with SaeS activity leading to inhibition of the sae target gene eap in strains with SaeS(L) but causing activation in strains containing SaeS(P). Here, we analyzed the possible involvement of the SaeP protein and saePQ region in SDS-mediated sae/eap expression. We found that SaePQ is not needed for SDS-mediated SaeS signaling. Furthermore, we could show that SaeS activity is closely linked to the expression of Eap and the capacity to invade host cells in a number of clinical isolates. This suggests that SaeS activity might be directly modulated by structurally non-complex environmental signals, as SDS, which possibly altering its kinase/phosphatase activity.</p>