The success of Staphylococcus aureus, as both a human and animal pathogen, stems from its ability to rapidly adapt to a wide spectrum of environmental conditions. Two-component systems (TCSs) play a crucial role in this process. Here, we describe a novel staphylococcal virulence factor, SpdC, an Abi-domain protein, involved in signal sensing and/or transduction. We have uncovered a functional link between the WalKR essential TCS and the SpdC Abi membrane protein. Expression of spdC is positively regulated by the WalKR system and, in turn, SpdC negatively controls WalKR regulon genes, effectively constituting a negative feedback loop. The WalKR system is mainly involved in controlling cell wall metabolism through regulation of autolysin production. We have shown that SpdC inhibits the WalKR-dependent synthesis of four peptidoglycan hydrolases, SceD, SsaA, LytM and AtlA, as well as impacting S. aureus resistance towards lysostaphin and cell wall antibiotics such as oxacillin and tunicamycin. We have also shown that SpdC is required for S. aureus biofilm formation and virulence in a murine septicemia model. Using protein-protein interactions in E. coli as well as subcellular localization in S. aureus, we showed that SpdC and the WalK kinase are both localized at the division septum and that the two proteins interact. In addition to WalK, our results indicate that SpdC also interacts with nine other S. aureus histidine kinases, suggesting that this membrane protein may act as a global regulator of TCS activity. Indeed, using RNA-Seq analysis, we showed that SpdC controls the expression of approximately one hundred genes in S. aureus, many of which belong to TCS regulons.

Staphylococcus aureus is a major human pathogen, and has become a significant worldwide health concern due to the rapid emergence of antibiotic resistant strains. Like most bacteria, S. aureus adapts to its environment by adjusting its genetic expression through sensing and regulatory systems. We show here that the SpdC membrane protein is a novel virulence factor of S. aureus, controlling biofilm formation and pathogenesis. We show that SpdC interacts with the WalK histidine kinase to inhibit activity of the WalKR two-component system. SpdC also interacts with nine other histidine kinases of S. aureus, suggesting it acts as a pleiotropic global regulator.

In an effort to identify additional factors controlling the WalKR system, we showed that the S. aureus SpdC Abi-domain protein negatively affects WalK activity and expression of WalKR-regulated genes. We showed that SpdC, previously identified as playing a role in the display of surface proteins [ 31 ], forms a complex with the WalK histidine kinase and that the two membrane proteins preferentially localize at the division septum, suggesting that this interaction regulates WalK histidine kinase activity. The ΔspdC mutant displays a pleiotropic phenotype, including altered resistance towards compounds targeting the cell wall, as well as strongly diminished biofilm formation and virulence. Using RNA-Seq analysis, we showed that SpdC controls the expression of approximately one hundred genes in S. aureus. Indeed, SpdC activity appears to extend well beyond the WalKR system, since we have shown it also interacts with several other S. aureus histidine kinases suggesting it could be involved in controlling multiple regulatory/adaptive pathways.

In Bacillus subtilis, the WalK histidine kinase is thought to coordinate cell wall plasticity with cell division, with two membrane-bound accessory proteins, WalH and WalI, inhibiting WalK kinase activity [ 11 , 27 – 29 ]. However, the WalH and WalI proteins of S. aureus share no significant sequence similarities with those of B. subtilis and their role is not as clear-cut. Indeed, although they are also membrane proteins and interact with the WalK kinase, WalH and WalI do not seem to play a major role in negatively controlling WalKR activity, suggesting that their functions have evolved [ 30 ].

The WalKR system is the only S. aureus TCS shown to be essential for cell viability, suggesting that its activity may respond not only to environmental conditions but could also be controlled by intrinsic bacterial factors [ 9 – 11 ]. Indeed, it is becoming increasingly apparent that so-called two-component systems frequently involve additional proteins regulating the phosphorylation levels of the response regulator [ 12 , 13 ]. These include accessory phosphatases such as CheZ, Spo0E or RapA, respectively dephosphorylating the E. coli CheY [ 14 ], B. subtilis Spo0A [ 15 ] and Spo0F [ 16 ] response regulators, or antikinases such as KipI and Sda that inhibit B. subtilis KinA [ 17 , 18 ] and FixT which inhibits the Sinorhizobium meliloti FixJ kinase [ 19 ]. A sub-class of histidine kinases, known as intra-membrane sensing kinases [ 20 ], require the permease component of an associated ABC transporter for signal sensing, such as the S. aureus BraS [ 21 ] and GraS [ 22 ] kinases. Many TCS histidine kinases act as so-called bifunctional sensors, acting on their cognate response regulators both as kinases and phosphoprotein phosphatases [ 23 ]. Accordingly, several accessory proteins act by binding to the histidine kinase to inhibit its kinase activity or stimulate its phosphatase activity towards the response regulator. These include the PII protein acting on the NtrB kinase to control nitrogen assimilation by dephosphorylating NtrC [ 24 ], the Streptococcus agalactiae Abx1 Abi-domain membrane protein which interacts with the CovS histidine kinase to inhibit activity of the CovR response regulator [ 25 ], and the SaePQ protein complex, which stimulates the phosphatase activity of the SaeS histidine kinase in S. aureus [ 26 ].

Two-component systems (TCSs) are composed of a histidine kinase, usually membrane-bound and acting as an environmental sensor, which phosphorylates a coupled response regulator, often controlling gene transcription. Although these systems have been extensively studied and play an essential role in bacterial adaptation to the environment, the signal(s) to which they respond and additional factors positively or negatively controlling their activities remain mostly unknown. Staphylococcus aureus, a major human pathogen, causes diseases ranging from superficial cutaneous abscesses to life-threatening infections affecting all major organs [ 1 ]. S. aureus is also a commensal bacterium, colonizing approximately half the human population asymptomatically, essentially within the anterior nares [ 2 ]. In addition to its considerable arsenal of virulence factors, S. aureus must rapidly adapt to environmental conditions encountered during host colonization. Among the 16 TCSs encoded by the S. aureus genome [ 3 ], the SaeSR, AgrCA, and WalKR systems are particularly important for controlling virulence and innate immune evasion factors [ 4 – 8 ].

In order to identify which domain of SpdC interacts with the WalK and SaeS kinases, we compared interactions with full-length SpdC (pUT18c-spdC) and a carboxy-terminal truncated SpdC consisting only of the eight transmembrane domains (SpdC 1-252 ; pUT18c-spdC1-252). We noted self-interaction of SpdC following co-transformation of pKT25-spdC with either pUT18c-spdC or pUT18c-spdC1-252 ( Fig 8B ), indicating that the SpdC transmembrane domains are involved in these self-interactions. Similar results were obtained when testing interactions with WalK and SaeS, i.e. the transmembrane domains of SpdC are sufficient to allow interactions with the histidine kinases ( Fig 8B ).

A. Domains fused to either pUT18c or pKT25 for the BACTH assay. Hatched bars indicate transmembrane regions and functional domains are annotated according to the UniProt database ( http://www.uniprot.org/uniprot/Q2G2U4 for WalK, http://www.uniprot.org/uniprot/Q2G2U1 for SaeS and http://www.uniprot.org/uniprot/Q2FVT1 for SpdC). B. β-galactosidase activities of DHT1 E. coli strains co-transformed with pUT18c containing either the full-length spdC coding sequence or a fragment encoding only the first 252 amino-acids of SpdC, and pKT25 derivatives carrying DNA fragments encoding the indicated proteins. Positive interaction results are shaded. ND: Not determined.

We focused our analysis on the WalK and SaeS histidine kinases. WalK has two transmembrane domains (amino acids 14–34 and 183–203), with an extracellular loop of 148 amino acid residues, a HAMP domain involved in signal transduction (204–256), a PAS domain (261–331) a PAC domain (314–378) and a histidine kinase domain (382–600; Fig 8A ). In contrast, SaeS is a member of the intra-membrane sensing kinases [ 45 ], with two transmembrane domains (amino acids 9–29 and 40–60) separated by only ten amino acids, as well as a HAMP domain (61–114) and a histidine kinase domain (129–348; Fig 8A ). We tested the interactions of SpdC with the N-terminal domains of WalK and SaeS containing the transmembrane regions (WalK 1-203 and SaeS 1-64 , respectively). The truncated proteins were fused to the T25 domain of adenylate cyclase, and the resulting plasmids were co-transformed into E. coli strain DHT1 together with the pUT18c-spdC plasmid. As shown in Fig 8B , the first 203 amino acids of WalK are sufficient to allow stable interactions with SpdC. For SaeS, the N-terminal domain containing only the two transmembrane segments (SaeS 1-64 ) did not lead to interaction with SpdC, but a longer fragment of the protein (SaeS 1-120 ) gave rise to a stable interaction with SpdC and high β-galactosidase activity ( Fig 8B ). These results suggest that WalK and SaeS interact with SpdC through their transmembrane domains. The negative interaction results obtained with the truncated SaeS protein containing only the transmembrane domains suggest that since this kinase lacks an extracellular loop, the HAMP domain may be required for proper membrane insertion of the fusion protein.

Histidine kinases have different combinations of signaling domains such as HAMP or PAS domains [ 41 ] in addition to the conserved H, N, G1, F and G2 boxes of the phosphoacceptor/dimerization (HisKA) and catalytic (HATPase_C) domains [ 42 – 44 ]. We have shown that SpdC interacts with 10 of the 16 S. aureus histidine kinases, which do not share any strong amino acid sequence similarities other than the conserved histidine kinase HisKA and HATPase_C domains. Since SpdC interacts with some, but not all of the S. aureus histidine kinases, this protein-protein contact must involve some other domain.

Among the genes positively regulated by SpdC ( Table 1 ), the expression of at least fifteen is also activated by the SaeSR TCS, including the spl operon, sak, the hlgBC operon and chp [ 7 , 38 – 40 ]. This suggests that SpdC activates the SaeSR TCS, in contrast to its role in negatively controlling activity of the WalKR system.

The BACTH assay was used to test protein-protein interactions between SpdC and each of the S. aureus TCS histidine kinases. A. DHT1 E. coli strains co-transformed with each combination of plasmids (pUT18c-spdC and pKT25 derivatives carrying the genes for the designated kinases) were spotted on LB agar plates with X-gal as an indicator of β-galactosidase activity. Histidine kinases showing a positive result for interaction with SpdC are underlined. B. β-galactosidase activity of DHT1 strains co-transformed with the pUT18c-spdC and pKT25 derivatives carrying each of the histidine kinase encoding genes. β-galactosidase activity (Miller Units) of the strain carrying the empty vectors (pKT25-pUT18c) was arbitrarily fixed at 0 and that of the positive control (pKT25zip-pUT18czip) at 1000.

As shown above, SpdC localizes at the division septum and interacts with the WalK histidine kinase, negatively controlling WalKR activity. However, many of the SpdC-regulated genes identified by RNA-Seq do not belong to the WalKR regulon, but are known to be controlled by other two-component systems ( Table 1 ) suggesting SpdC may interact with other TCS regulatory pathways. The S. aureus HG001 genome encodes 16 two-component systems [ 33 ] and we constructed translational fusions for each of the histidine kinase genes with the carboxy-terminal region of the adenylate cyclase T25 domain. Each of the resulting plasmids was co-transformed in combination with the pUT18c-spdC plasmid into E. coli strain DHT1. As shown in Fig 7 , in addition to WalK, we detected interactions between SpdC and nine additional histidine kinases: YesM, GraS, SaeS, DesK, ArlS, SrrB, PhoR, VraS, and BraS. These interactions appear to be specific, since no interactions were detected between SpdC and the remaining six histidine kinases, as shown in Fig 7 (LytS, AirS, AgrC, KdpD, HssS, and NreB).

Cell surface modifications are known to impact virulence. Likewise, the capacity to form robust biofilms favors bacterial colonization of the host. Additionally, our RNA-Seq analysis revealed that the expression of at least 10 genes directly involved in bacterial virulence upon infection was lowered in the ΔspdC mutant, strongly suggesting that SpdC plays a role in virulence. We used a murine sepsis model to compare virulence of the HG001 and ΔspdC strains. SWISS mice were infected intravenously with 5.10 7 cfu and mortality was monitored over 9 days post infection. As shown in Fig 6 virulence of the ΔspdC mutant was strongly diminished. Indeed, following infection with the HG001 parental strain, significant mortality occurred in the first 3 days post-infection (greater than 60%), whereas only a single mouse out of 14 died within five days after infection with the ΔspdC mutant. After the sixth day, a moderate mortality was observed for the group infected with the ΔspdC mutant, with only 36% mortality at the end of the assay (compared to 72% mortality for mice infected with the parental HG001 strain). This significant difference indicates that SpdC is a novel virulence factor in S. aureus.

Biofilm assays were performed by growing cells in static cultures in PVC microtiter plates (TSB plus 0.75% glucose and 3.5% NaCl). Plates were incubated at 37°C for 24h and adherent biomass was quantified as described in Materials and Methods. Data were normalized to the OD 600nm of each culture and indicated as the n-fold change of the ΔspdC mutant strain compared to the HG001 parental strain. ***, P<0.0005 (Student’s t-test).

Since our results indicate that the ΔspdC mutation may modify the S. aureus cell surface, we tested whether the absence of SpdC may have an effect on biofilm formation. Strains were grown statically in TSB, supplemented with glucose and NaCl, for 24 h at 37°C in PVC microplates. As shown in Fig 5 , biofilm formation was strongly decreased in the absence of SpdC (approximately 7-fold). Complementation of the ΔspdC mutant with the pMK4Pprot-spdC plasmid restored biofilm formation to levels comparable to those of the parental HG001 strain ( Fig 5 ). These results are consistent with a modification of the S. aureus cell surface in the absence of SpdC, which could influence resistance against antimicrobial compounds targeting cell surface structures as well as the capacity to form biofilms. In order to determine which biofilm component is affected, biofilm detachment experiments were carried out ( S4 Fig ) by treatment with proteinase K, DNaseI and sodium metaperiodate (a carbohydrate-modifying agent). Under our conditions, biofilm production was lowered three-fold after treatment with DNaseI, and more than 10-fold when treated with proteinase K, but not significantly modified after treatment with sodium metaperiodate. Thus, biofilms formed under our conditions by the HG001 parental strain are essentially protein-based, and, to a lesser extent, due to extracellular DNA. We observed reduced biofilm formation for the ΔspdC mutant even after DNAseI treatment, but not after proteinase K treatment ( S4 Fig ), suggesting that SpdC affects the production of proteins important for biofilm formation.

We then tested sensitivity to antibiotics targeting the cell wall. As shown in Fig 4B the ΔspdC mutant displayed increased sensitivity to the β-lactam antibiotic oxacillin, whereas the parental and complemented strains were able to grow at the concentration tested (0.1 μg/ml). No difference in sensitivity between the strains was seen using fosfomycin, an antibiotic inhibiting MurA, which catalyzes the very first step of peptidoglycan biosynthesis ( Fig 4B ). These results suggested that the ΔspdC mutant strain may either be affected in the later steps of peptidoglycan biosynthesis or may exhibit a cell wall structure modification leading to a difference in accessibility of antibiotics acting extracellularly. Wall teichoic acids (WTAs) are anionic sugar rich cell surface polymers that can alter accessibility to the cell wall. We therefore tested resistance to tunicamycin, an antibiotic targeting biosynthesis of WTAs. As shown in Fig 4B the ΔspdC mutant strain was highly sensitive to tunicamycin, in contrast to the parental and complemented strains.

A. The ΔspdC mutant displays increased resistance to lysostaphin-induced lysis. Cells were grown in TSB until mid-exponential phase, harvested and incubated in PBS with lysostaphin (200 ng/ml) with aeration at 37°C. Bacterial lysis was measured by monitoring OD 600nm over time. Results are shown as the mean and standard deviation of three independent assays. HG001 parental strain ( ); ΔspdC mutant (■); ΔspdC/pMK4Pprot-spdC complemented strain (▲). B. The absence of SpdC leads to sensitivity to oxacillin and tunicamycin. Dilution series of the HG001, ΔspdC and ΔspdC/ pMK4Pprot-spdC strains on TSA plates with or without antibiotics. Oxacillin: 0.1 μg/ml; fosfomycin: 4 μg/ml; tunicamycin: 1 μg/ml.

We also tested sensitivity to lysostaphin, a glycyl-glycine endopeptidase that hydrolyzes the peptidoglycan pentaglycine interpeptide crossbridge, leading to cell lysis [ 37 ]. The HG001, ΔspdC mutant and complemented strains were grown in TSB until OD 600nm ≈ 1 and cells were then harvested and resuspended in PBS in the presence of lysostaphin. As shown in Fig 4A , the ΔspdC mutant was less sensitive to lysostaphin-induced lysis than the parental HG001 and complemented ΔspdC strains, suggesting that the absence of SpdC leads to cell wall modifications.

As shown above, the expression of several cell wall hydrolase genes is significantly increased in the ΔspdC mutant strain, suggesting that sensitivity to compounds targeting the cell wall might also be affected. We followed bacterial lysis during incubation in the presence of a non-anionic detergent, Triton X-100, thought to trigger cell lysis by favoring endogenous autolysin activity [ 36 ]. However, Triton X-100 induced lysis for the HG001 and ΔspdC mutant strains was not significantly different ( S3 Fig ).

We have previously shown that the S. aureus WalK histidine kinase is mainly localized at the division septum [ 30 ]. Since SpdC and WalK interact, we studied the subcellular localization of SpdC in S. aureus by constructing a translational fusion with the GFP fluorescent protein using the pOLSA vector (See Materials and Methods ), under the control of the cadmium-inducible P cad promoter. The resulting plasmid, pOLSA-spdC was then introduced into the HG001 strain. Expression of the gene fusion was induced by addition of CdCl 2 (0.25 μM), cells were harvested during exponential growth (OD 600nm = 1.5) and observed by fluorescence microscopy. As shown in Fig 3B , SpdC is preferentially localized at the division septum, with a mean septum/membrane fluorescence ratio of around 2.6. Taken together, our results indicate that SpdC and WalK interact and are localized at the division septum.

A: The SpdC protein was tested for pairwise interactions with the WalK histidine kinase and the WalR response regulator using the BACTH complementation assay by translationally fusing the full length corresponding coding sequences with those of the T25 or T18 adenylate cyclase domains (upper left panel). Specificity of the SpdC-WalK complex was confirmed by testing interactions with three other S. aureus Abi-domain proteins: SpdB, SpdA and SAOUHSC_02256 (lower left panel). Cultures of E. coli DHT1 strains co-transformed with the indicated plasmid combinations were spotted on LB agar plates with X-Gal as an indicator of β-galactosidase activity (see Materials and Methods ). Quantitative β-galactosidase activity assays for each strain are indicated in the right panel (expressed in Miller Units). The shaded cell indicates significant β-galactosidase activity resulting from positive protein-protein interactions. ND: Not determined. B: S. aureus HG001 cells producing a fluorescent SpdC-GFP protein fusion (HG001/pOLSA-spdC) were grown in TSB with 0.25 μM CdCl 2 and observed in exponential phase by fluorescence microscopy. Fluorescence ratios (septum/lateral membrane) were quantified for two biological replicates (33 cells from each) using ImageJ software and plotted using GraphPad Prism. The horizontal line indicates the mean fluorescence ratio and the error bars represent the SEM.

As shown above, we have uncovered a regulatory link indicating that SpdC negatively controls activity of the WalKR two-component system, strongly suggesting that the proteins interact. In order to test possible interactions between SpdC and the WalKR proteins, we used the bacterial adenylate cyclase two-hybrid system (BACTH) [ 35 ]. We fused the full-length membrane-bound WalK histidine kinase or the WalR cytoplasmic response regulator to the C-terminal domain of the T25 subunit of the Bordetella pertussis adenylate cyclase and full-length SpdC to the C-terminal domain of the T18 subunit, using plasmids pKT25 and pUT18c respectively. To probe putative interactions, E. coli strain DHT1 was co-transformed with combinations of the pKT25 and pUT18c derivatives carrying the translational fusions. Upon protein-protein interactions, the close proximity between the T18 and T25 subunits restores adenylate cyclase activity, leading to cAMP synthesis and activation of the lactose operon. Interactions were tested both by spotting the resulting strains on LB plates containing X-Gal and by measuring β-galactosidase activity. To determine pair-wise interactions, we chose a cut-off value of 100 Miller Units as indicating a positive interaction between the protein fusions. As shown in Fig 3A , strong β-galactosidase activity was only observed for the plasmid combination co-producing the membrane anchored proteins SpdC and WalK while no interactions between SpdC and the cytoplasmic regulator WalR could be detected. SpdC is annotated as an Abi domain protein (CAAX protease self-immunity) in genome databases. The S. aureus HG001 genome encodes 4 Abi domain proteins, three of which, SpdA (SAOUHSC_01900), SpdB (SAOUHSC_02587), and SpdC, have been reported as being involved in surface protein display, whereas the fourth (SAOUHSC_02256) has no known function [ 31 ]. In order to test whether WalK also interacts with the other three Abi domain proteins, we constructed translational fusions for the remaining Abi proteins with the T18 domain of adenylate cyclase. As shown in Fig 3A , the combinations of SpdA, SpdB or SAOUHSC_02256 with WalK did not generate significant levels of β-galactosidase activity, demonstrating that SpdC is the only S. aureus Abi domain protein specifically interacting with WalK.

For studying levels of protein A, known to be covalently anchored to the cell wall (LPxTG sortase motif), identical quantities of cell wall fractions of the HG001 parental strain, ΔspdC deletion mutant, and complemented strain (ΔspdC/pMK4Pprot-spdC) were subjected to SDS/PAGE and compared by Western blot. As expected, protein A levels were significantly lower in the ΔspdC mutant than in the parental and complemented strains ( Fig 2C , lower panel), in agreement with the RNA-Seq and qRT-PCR results.

In order to confirm SpdC-dependent regulation at the protein level, we chose two genes that were positively or negatively controlled by SpdC, spa and lytM, respectively, and performed Western blot analyses. Whole cell extracts were prepared from cultures of strains HG001, the ΔspdC mutant and the complemented mutant strain (ΔspdC/pMK4Pprot-spdC) and subjected to SDS-PAGE and immunoblotting. As shown in the top panel of Fig 2C , LytM levels are higher in the ΔspdC strain compared to the parental strain (lane 2), and in the complemented strain the LytM level is reduced to a level lower than in the parental strain (lane 3), likely reflecting the higher production of SpdC in the complemented strain. Indeed, under these conditions, spdC mRNA levels were increased more than 100-fold as measured by qRT-PCR as compared to the parental HG001 strain.

We used qRT-PCR to verify SpdC-dependent regulation for 3 positively (spa, hlgC, sdrD) and 3 negatively (atlA, sceD, lytM) controlled genes, in the ΔspdC strain compared to the HG001 parental strain, grown under the same conditions as for the RNA-Seq analysis ( Fig 2B ). We observed a perfect correlation with the RNA-Seq data: expression of the spa gene encoding protein A was very strongly lowered in the ΔspdC mutant strain (about one hundred-fold), while sdrD and hlgC expression levels were 5- to 10-fold less. The atlA, sceD and lytM cell wall hydrolase genes were more highly expressed in the absence of SpdC (2-, 12- and 5-fold, respectively) in agreement with the RNA-Seq analysis ( Fig 2B ).

As shown above, spdC expression is controlled by the WalKR system. The RNA-Seq data analysis of SpdC-regulated genes reveals that 25 of these belong to the WalKR regulon (indicated by an asterisk in Table 1 ). In particular, the expression of 4 WalKR-dependent cell wall hydrolase genes (sceD, ssaA, lytM and atlA) is increased in the ΔspdC mutant, suggesting that SpdC negatively controls WalKR activity ( Table 1 ). It is interesting to note that among the genes positively controlled by SpdC, many are also WalKR-activated genes ( Table 1 ). All of these are classified as virulence genes, however they are not preceded by the WalR consensus binding site, and we have previously shown that several of these are not directly regulated by the WalKR system but through the SaeRS two-component system instead [ 6 ].

A. Ontological grouping of SpdC-regulated genes according to their annotated functions. Variations in gene expression were identified by RNA-Seq analysis of a ΔspdC strain compared to the HG001 parental strain grown in TSB until mid-exponential phase. B. qRT-PCR comparison of gene expression in a ΔspdC strain and the HG001 parental strain. Strains were grown in TSB until mid-exponential phase and RNA was extracted and treated as described in Materials and Methods. Expression levels were normalized using 16S rRNA as internal standard and presented as the n-fold change of the ΔspdC mutant strain compared to the HG001 parental strain. C. Western blot analysis of LytM (upper panel) and Spa (lower panel) production. Crude extracts (LytM) and cell wall extracts (Spa) were prepared from stationary phase cultures. Lanes: 1: Purified Staphylococcus aureus Protein A (50 ng) 2: HG001 strain; 3: ΔspdC mutant strain; 4: ΔspdC/pMK4Pprot-spdC complemented strain.

Three biological replicates were analyzed by RNA-Seq for each strain and the data presented as the mean fold-change. Using a value cut-off greater than 2 with a P value less than 0.05, we found that the expression of 42 genes was lowered in the ΔspdC mutant strain and that of 65 increased ( Table 1 ). In order to perform a general analysis of the transcriptomic data we generated an ontological grouping of SpdC-regulated genes ( Fig 2A ). Among the genes positively controlled by SpdC, 10 are known virulence factors, suggesting that SpdC may influence S. aureus pathogenicity, and 11 are involved in capsular biosynthesis. The S. aureus capsule is known to impede phagocytosis and promote host colonization, however although the HG001 strain used in this study carries the serotype 5 capsule gene cluster, a missense mutation in the cap5E gene prevents capsular biosynthesis [ 32 – 34 ].

As shown above, the WalKR TCS controls spdC expression. In Streptococcus agalactiae, another bacterial Abi-domain protein, Abx1, has been shown to inhibit activity of the CovSR two-component system [ 25 ]. In order to determine whether SpdC has a regulatory role in S. aureus, we generated a ΔspdC mutant in strain HG001 and performed a comparative RNA-Seq analysis. The ΔspdC mutant did not display any gross morphological changes or growth defects. Indeed, although it had a slight lag during the first hour post inoculation, the growth rate and final OD 600nm were not significantly different from those of the parental strain ( S1 Fig ). Doubling times ( http://www.doubling-time.com/compute.php ) calculated during the exponential growth phase ( S1 Fig , 90 min to 210 min) gave identical values of 32 min for both strains. The HG001 strain and ΔspdC mutant were grown in TSB until early exponential phase (OD 600nm = 1) and total RNA was extracted for RNA-Seq analysis (See Materials and Methods ). We verified that spdC is well expressed under these conditions using a lacZ reporter fusion with the spdC promoter region ( S2 Fig ).

A. Predicted membrane topology of the SpdC protein generated using the Protter prediction tool ( http://wlab.ethz.ch/protter/start/ ). The eight predicted transmembrane segments are numbered. Amino acids forming the putative Abi domain (based on Pfam database annotation) are shaded in green. B. Relative levels of walR and spdC transcripts were measured by qRT-PCR during growth of the HG001 PspacwalRKHI strain (ST1017). Bacteria were grown to exponential phase in TSB with or without increasing concentrations of IPTG to induce expression from the Pspac promoter. Expression levels were normalized using 16S rRNA as an internal standard and are indicated as n-fold change with respect to the control condition (absence of IPTG).

We previously performed an extensive transcriptome analysis in order to define the scope of the S. aureus WalKR regulon [ 6 ]. Our results showed that expression of the spdC gene (SAOUHSC_02611) was increased 3.5-fold in a S. aureus strain producing a constitutively active form of the WalR response regulator (D55E) [ 6 ]. SpdC, a membrane-anchored protein with 8 predicted transmembrane segments and an Abi domain (CAAX protease self-immunity), ( Fig 1A ), was previously reported as playing a role in the display of surface proteins such as protein A [ 31 ]. In order to confirm that spdC is a member of the WalKR regulon, we used quantitative real-time PCR (qRT-PCR) to measure its expression in a S. aureus strain where the walRKHI operon is placed under the control of the IPTG-inducible Pspac promoter [ 9 ]. Cells were grown overnight in TSB with 0.05 mM IPTG, and cultures were inoculated at OD 600nm = 0.05, with or without different IPTG concentrations (0.05 and 1 mM) to induce expression from the Pspac promoter. RNA samples were prepared from exponentially growing cells harvested at OD 600nm = 0.5, before cessation of growth of the culture lacking IPTG, and walR and spdC mRNA levels were measured by qRT-PCR. As shown in Fig 1B , walR transcription is increased approximately 4-fold when cells are grown with 0.05 mM IPTG and 8-fold at 1 mM IPTG. Under the same conditions, spdC expression followed that of walR, and was increased 2-fold and 5-fold with 0.05 or 1 mM IPTG, respectively ( Fig 1B ), confirming positive regulation by the WalKR system.

Discussion

Abi-domain proteins constitute a large family whose functions are mostly unknown. SpdC was initially designated LyrA for Lysostaphin resistance A and identified by screening a bursa aurealis transposon mutant library for increased lysostaphin resistance [46]. An independent study aiming at characterizing proteins involved in the display of surface proteins led to the identification of three proteins, SpdA, SpdB and SpdC (Surface protein display A, B and C), playing a role in protein A levels at the staphylococcal cell surface [31]. These proteins share a similar structural organization with 6 to 8 transmembrane domains and an Abi-domain embedded within the hydrophobic region. An additional protein with a similar organization is encoded by the S. aureus genome, SAOUHSC_02256, but appears to have no role in controlling protein A levels [31].

We previously characterized the essential WalKR two-component system in S. aureus and highlighted its major role in controlling cell wall homeostasis [9, 47]. Transcriptome analysis revealed that spdC expression is positively controlled by WalKR [6]. We show here that the SpdC membrane protein and the WalK histidine kinase interact and that SpdC negatively controls WalKR activity and expression of WalKR-regulated genes. Interaction of the WalK histidine kinase with SpdC is specific since no interaction was seen with the other Abi-proteins (SpdA, SpdB and SAOUHSC_02256). Accordingly, we also showed that SpdC and WalK are both localized preferentially at the division septum.

Interestingly, an RNA-Seq analysis of a ΔsdpC mutant revealed that the expression of 107 genes varied compared to the parental strain. Among these, 24 (more than 20%) are controlled by the WalKR system. Since SpdC appears to negatively control WalKR activity by interacting with WalK, this septal localization is consistent with the previously suggested cell wall metabolism-related activation signal of the WalK histidine kinase in Bacillus subtilis [11, 48]. Indeed, in cocci, cell wall synthesis has been shown to exclusively occur at the division septum in an FtsZ-dependent manner [49], suggesting that a peptidoglycan metabolism related signal at the septum may relieve negative control of WalK activity by SpdC.

Histidine kinases often act as phosphoprotein phosphatases towards their associated response regulator. WalK was previously classified as a kinase/phosphatase «bifunctional sensor» [23] and the PAS domain of Streptococcus pneumoniae WalK plays a role in its phosphatase activity [50, 51]. In S. aureus, we have previously shown that WalK acts as a WalR phosphoprotein phosphatase upon entry into stationary phase in order to shut off WalR activity [6]. Interactions between SpdC and WalK can either interfere with signal perception by the sensor histidine kinase, inhibit its kinase activity or increase its phosphatase activity towards WalR, thus negatively controlling WalKR-dependent gene expression. SpdC is unlikely to directly regulate gene expression since it is a membrane protein lacking any typical DNA-binding domain.

To understand how the other SpdC-dependent genes were controlled (83 of the genes identified by RNA-Seq are not regulated by the WalKR system), we tested interactions of SpdC with the other S. aureus histidine kinases and found that it interacts with 10 of the 16 encoded in the genome. No obvious structural motifs or domain sequences were common to those that interacted with SpdC compared to those that did not. Two histidine kinases, WalK and SaeS, were chosen for further analysis of their interactions with SpdC. Our results indicate that the two transmembrane domains of WalK are sufficient to allow interaction with SpdC, whereas a greater amino-terminal fragment of SaeS was required, encompassing the HAMP domain. This result suggests that although the transmembrane domains of SaeS are likely involved in interactions with SpdC, a longer fragment may be necessary to ensure proper membrane insertion of the truncated protein. We also showed that a truncated form of SpdC, containing only the eight transmembrane domains, was sufficient for self-interaction and interaction with WalK and SaeS, indicating that transmembrane domains are involved in the interactions between SpdC and the histidine kinases. Interestingly, the only two S. aureus cytoplasmic histidine kinases which lack transmembrane domains, AirS and NreB, did not interact with SpdC under our conditions, in agreement with our results indicating transmembrane segments are involved in the interactions. The only other example of an Abi domain protein interacting with a histidine kinase is Abx1 of Streptococcus agalactiae, which interacts with the CovS kinase [25]. The two transmembrane domains of CovS were shown to be necessary and sufficient for these interactions [25].

At least fifteen genes belonging to the SaeSR regulon, including the spl operon, sak, the hlgBC operon and chp [7, 38–40], are also positively controlled by SpdC (Table 1), indicating that SpdC likely activates the SaeSR TCS, in contrast to its role in negatively controlling activity of the WalKR system. Since WalR controls spdC expression, this is consistent with our previous results showing that constitutive activation of WalR generates a signal leading to activation of the SaeSR TCS and a corresponding increase in SaeSR regulon expression [6].

The localization of SpdC at the division septum and its role in gene regulation through interactions with sensor kinases of two-component systems led us to speculate that SpdC may interfere with bacterial division sensing and impact cell wall metabolism. Accordingly, the ΔspdC mutant displays increased resistance against lysis when treated with lysostaphin, in agreement with the original phenotype characterized by transposon insertion [46]. Additionally, the absence of SpdC was reported to lead to increased cross wall abundance and thickness [31]. We tested sensitivity of the ΔspdC mutant to antibiotics targeting the cell wall. The ΔspdC mutant is highly sensitive to oxacillin and tunicamycin, but not to fosfomycin, which inhibits the first step of cell wall biosynthesis. In agreement with the sensitivity of the ΔspdC mutant to tunicamycin, which inhibits wall teichoic acid synthesis, the spdC gene was also identified as a candidate using a screen to identify synthetically lethal mutations with teichoic acid biosynthesis defects [52]. We have shown that expression of cell wall hydrolase genes is increased in the ΔspdC strain (sceD, ssaA, lytM, atlA). This may lead to increased cell wall degradation, which could explain the lowered resistance to oxacillin. This could also lead to the mutant’s increased sensitivity to tunicamycin. Indeed, teichoic acids are key elements for the proper localization of AtlA to the division septa, where cell wall biosynthesis takes place, since cell wall plasticity is essential for cell division [53]. In the presence of tunicamycin, the absence of wall teichoic acids results in a delocalized distribution of AtlA across the cell surface. Since atlA is more highly expressed in the ΔspdC stain, this could explain why this strain is more sensitive to tunicamycin.

Taken together with the strong links to the WalKR TCS, these results indicate that SpdC is involved in bacterial cell envelope homeostasis. The importance of the bacterial cell envelope in host-pathogen interactions cannot be over-emphasized: it is the first layer of contact between the bacterium and its host, containing an array of cell wall-linked or associated toxins and virulence factors, the first and major bacterial line of defense against threats from the host or environment, and is also both the target of choice for antibiotic treatment and the source of many antibiotic-resistance pathways. We have shown that SpdC is required for biofilm formation, an important step in S. aureus pathogenesis. We also showed that SpdC is a novel S. aureus virulence gene, required for the infectious process in a mouse septicemia model. The loss of virulence observed with the ΔspdC mutant may involve both its diminished capacity to form biofilms as well as the lowered expression of multiple virulence genes as shown by RNA-Seq analysis.

The regulatory mechanism mediated by SpdC remains to be determined. Abi-domain-containing proteins have been extensively studied in eukaryotes. They are involved in CAAX-protein maturation by cleaving the C-terminal AAX tripeptide after addition of an isoprenyl group on a cysteine, the last step consisting in methylation of the new C- terminus. These three modification steps are termed prenylation [54]. This post-translational maturation has a crucial role in maintaining cellular homeostasis by controlling the localization and activity of a large range of proteins. In particular, by adding a lipid group at the carboxy-terminal end of proteins, it favors their interactions with membranes, which have a high concentration of signaling molecules [55]. Prenylation has been recently described in prokaryotes and a geranyltransferase, IspA, has been identified in S. aureus [56]. Putative methyltransferase and CAAX-protease encoding genes (including spdC) are also present in the S. aureus genome. Deletion of the ispA gene has pleiotropic effects such as a growth defect, increased sensitivity to oxidative stress and an altered cell envelope [56]. Of note, the absence of IspA or SpdC both lead to increased cell wall antibiotic sensitivity. The RNA-Seq transcriptome analysis of the ΔispA strain [56] shows similarities with that of the ΔspdC mutant. Indeed, one of the most regulated genes in both cases is spa, encoding the immunoglobulin G binding protein A. We also noted that 21 Φ13 genes are up-regulated in the ΔispA mutant whereas we characterized 11 Φ13 genes up-regulated in the ΔspdC mutant, with several in common. These data suggest that SpdC and IspA could be involved in the same cellular pathway.

Interestingly, the transcriptome analysis of the ΔispA mutant revealed modified expression of a large number of genes involved in regulatory circuits and particularly increased expression of the walR, walH, walI and walJ genes of the wal locus [56]. While no direct link between prenylation and Abi domain proteins has been shown in prokaryotes, there are several links with the WalKR system, either by physical interaction and negative control of activity, for SpdC, or at the transcriptional level for the IspA geranyltransferase.

This study identifies a membrane-bound protein with an Abi domain, SpdC, at the core of an interaction network that coordinates bacterial division with cell envelope metabolism and host interactions. Further studies are required in order to decipher the molecular mechanism and consequences of these interactions.