SDS Interferes with SaeS Signaling of Staphylococcus aureus Independently of SaePQ

et al. (2013) SDS Interferes with SaeS Signaling of Staphylococcus aureus Independently of SaePQ. PLoS ONE 8(8): e71644. doi:10.1371/journal.pone.0071644 SDS Interferes with SaeS Signaling of Staphylococcus aureus Independently of SaePQ Phuti E. Makgotlho 0 Gabriella Marincola 0 Daniel Scha fer 0 Qian Liu 0 Taeok Bae 0 Tobias Geiger 0 Elizabeth Wasserman 0 Christiane Wolz 0 Wilma Ziebuhr 0 Bhanu Sinha 0 Tarek Msadek, Institut Pasteur, France 0 1 Institute for Hygiene and Microbiology, University of Wu rzburg , Wu rzburg, Germany , 2 Institute for Medical Microbiology and Hygiene, University of Tu bingen , Tu bingen, Germany , 3 Department of Microbiology and Immunology, Indiana University School of Medicine-Northwest, Gary, Indiana, United States of America, 4 Department of Pathology, Stellenbosch University , Cape Town , South Africa , 5 Institute for Molecular Infection Biology, University of Wu rzburg , Wu rzburg, Germany , 6 Department of Medical Microbiology and Infection Prevention, University Medical Center Groningen , Groningen , The Netherlands 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 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. - Funding: This study was supported by the International Research Training Group IRTG 1522, the BMBF-funded MedVetStaph Research Network (FKZ 01KI1014E) and the Deutsche Forschungsgemeinschaft Wo578/7-1 and TransRegio34 Projects B1, B4, and C6/11. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Staphylococcus aureus is part of the commensal flora, colonizing predominantly the anterior nares of approximately 2050% of the human population [1]. However, it is also a facultative pathogen able to cause a wide spectrum of infections, ranging from skin and soft tissue infections and abscess formation to complicated systemic diseases such as osteomyelitis, endocarditis, sepsis and toxic shock syndrome [2,3]. S. aureus has the ability to rapidly adapt to different environmental conditions, including heat, pH, and a range of chemical components. There is now growing evidence that S. aureus can also invade and persist within different cell types. The invasion potential is due to the production of various proteins such as fibronectin binding proteins (FnBPs) and extracellular adhesive proteins (Eap) [4] which are controlled by the regulatory SaePQRS system [5]. Strain Newman uses Eap rather than FnBPs as invasin since both FnBPs are secreted due to a point mutation resulting in a truncation of these proteins [6]. SaeR and SaeS are part of a bacterial two-component system coding for a response regulator and a histidine kinase, respectively [7]. They are encoded in the saePQRS operon together with other two ORFs, which are predicted to encode a lipoprotein (SaeP) and a membrane protein (SaeQ). Recently it was suggested that these two proteins play a role in the deactivation of the the sae system by inducing the phosphatase activity of SaeS [8,9]. A total of four overlapping transcripts (T1T4) are expressed in the sae operon from two promoters (P1 and P3) (Fig. 1A) [10]. The T1 transcript is transcribed from the strongly auto-activated P1 promoter [10]. The most abundant and stable T2 transcript is generated by endoribonucleolytic cleavage of T1 by RNase Y [10,11]. T3 is transcribed from the weak constitutive P3 promoter [10] and, finally, T4 is a monocistronic transcript coding just for saeP [12,13]. The sae system can be activated by environmental stimuli such as, H2O2, low pH, and sub-inhibitory concentrations of adefensins and antibiotics [10,1416]. We could show previously that sub-inhibitory concentrations of sodium dodecyl sulfate (SDS) lead to a decrease of sae target gene expression (e.g. eap) in some S. aureus strains, but causes an increase in strain Newman [17]. This opposing effect was mirrored by a decrease and increase of the invasion capacity of the strains upon SDS treatment, respectively. Strain Newman is characterized by a high, constitutive expression of the sae operon due to an amino acid substitution (Proline for Leucine, L18P) within the putative N-terminal transmembrane domain of the sensor histidine kinase SaeS (SaeSP). Several lines of evidence led to the conclusion that the SaeSP allele renders the kinase constitutively active [5,9,10,12]. Thus the Sae system of strain Newman is thought to be nonresponsive to environmental signals. Of note, SDS is the only signal described so far which seems to activate the SaeS of strain Newman. Here we analyze the possible involvement of the upstream part of the sae operon (i.e. saePQ) in SDS-mediated sae activation. We could show that the upstream region does not interfere with SDS signaling in any of the strains analyzed (i.e. carrying either SaeSP or SaeSL). Moreover, we could show that the auto-regulated promoter P1 is also dispensable in SDS-mediated sae activation. Materials and Methods Bacterial Strains and Growth Conditions Strains and plasmids used in this study are listed in Table 1. S. aureus strains were grown in TSB medium. For strains carrying resistance genes, antibiotics were used only in overnight cultures at the following concentrations: 10 mg ml21 erythromycin and 5 mg ml21 tetracycline. Bacteria from overnight cultures were diluted in fresh TSB to an initial optical density at 600 nm (OD600) of 0.05 with and without SDS 0.004% (w/v), and grown with shaking at 200 rpm at 37uC to the desired growth phase. SDS MIC of the strains used in this study was determined by microdilution (not shown). The concentration of SDS was chosen at 30% of the MIC. Construction of the saeP Mutant The saeP locus was replaced by a kanamycin resistant cassette. Briefly, two fragments fla (...truncated)