Virulence of <i>abx1</i> mutants.

<p>Mortality curves in neonate rats infected with the WT strain (black circle), the Δ<i>abx1</i> deletion mutant (blue square), and the K_Oe_abx1 overexpressing mutant (red triangle). Two days rat pups were inoculated by intraperitoneal injection of 5×10⁶ bacteria. The results shown are the mean of two independent experiments (2×10 rats per group).</p

The Abx1 (Gbs1532) protein is essential for hemolytic activity and pigment production in GBS.

<p>(A) ß-hemolytic and pigmentation phenotypes of NEM316 wild-type strain (WT) and of two independent insertional mutants (<i>abx1::Him</i>1 and <i>abx1::Him</i>2) obtained by random transposon mutagenesis. Strains contained the empty vector pTCV or the complementing vector pTCVΩ<i>abx1</i> where <i>abx1</i> is transcribed from its own promoter. Pigment production was assayed on Granada agar plates and hemolytic activities on Columbia horse blood (5%) agar plates. The clear halos surrounding bacterial colonies (black dots) on blood plates correspond to lysed erythrocytes. Plates are supplemented with erythromycin (10 µg/ml) for plasmid maintenance. (B) Phenotypes of in-frame <i>abx1</i> deletion mutant (Δ<i>abx1</i>). Strains contained the same vectors as in (A). Overnight cultures in TH were diluted in fresh media and approximately 10³ and 10² CFU were spotted on TH, Granada, and Columbia horse blood agar plates plus erythromycin. Pictures were taken after 24–36 h of growth at 37°C. (C) Hemolytic and pigmentation phenotypes associated with <i>abx1</i> overexpression. WT strain with the empty vector pTCV, the complementing vector pTCVΩ<i>abx1</i>, the overexpressing vectors pTCVΩP_cyl+_<i>abx1</i> or pTCVΩP_tet_<i>abx1</i>, the negative control vectors pTCVΩP_cyl+_<i>gbs1037</i> or pTCVΩP_cyl+_<i>EGFP</i> were spotted onto TH, Granada, and blood agar plates containing erythromycin. An overexpression mutant where the <i>abx1</i> chromosomal promoter was replaced by the strong P_cyl+ promoter (ΔP_abx1::P_cyl+) was similarly spotted on agar plates without antibiotic. TH and Granada plates were photographed after 24–36 h of growth, while blood agar plates were photographed after 16 h of growth at 37°C.</p

Abx1 function is dependent of the CylE ß-hemolysin through a protease-independent activity.

<p>(A) Abx1 is a member of a large family of proteins related to eukaryotic CaaX proteases <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003179#ppat.1003179-Pei1" target="_blank">[35]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003179#ppat.1003179-Pei2" target="_blank">[36]</a>. The three most conserved motifs of the Abi-domain (Pfam02517) are depicted for Abx1 and Gbs1037 from <i>S. agalactiae</i>, Spr1119 from <i>S. pneumoniae</i>, SagE from <i>S. pyogenes</i> and for two eukaryotic CaaX proteases, the budding yeast ScRCE1 and the human HsBRCA2. Conserved amino acids essential for CaaX protease activity are in red with the putative catalytic glutamic acid (E) residue shown by a plus (+). <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003179#ppat.1003179.s001" target="_blank">Figure S1</a> provides additional information on the Abi-domain family. (B) The CylE hemolysin is essential for Abx1 function on hemolysis and pigmentation. GBS hemolysis and pigmentation were observed on TH, Granada (Gr) and Blood agar (Bl). Strains are: (a) in-frame <i>cylE</i> deletion mutant (Δ<i>cylE</i>) with the empty vector pTCV or the <i>abx1</i> overexpression vector pTCVΩP_cyl+_<i>abx1</i>; and (b) WT, <i>abx1</i> deletion mutant (Δ<i>abx1</i>) and a mutant with a cysteyl to alanyl substitution in the CaaX motif of CylE (CylE C₆₆₄A), <i>i.e.</i>, the putative prenylation site of CylE. (C) Conserved amino acids critical for CaaX protease activity are not required for Abx1 activity. The non-hemolytic and apigmented Δ<i>abx1</i> mutant were complemented with pTCVΩ<i>abx1</i> vectors encoding a wild-type allele (Abx1) or several alanine substitution alleles in the predicted critical glutamic acid (E) or histidine (H) residues.</p

Abx1 physically interacts with the HK CovS.

<p>The physical interactions between proteins were assayed using a two-hybrid system in <i>E. coli</i>. Fusion proteins were constructed with the T18 or the T25 adenylate cyclase fragments. The activity of the ß-galactosidase cAMP-dependent reporter gene, expressed in arbitrary units (AU), was determined as described in Materials and Methods. Means and standard deviations are calculated from at least three independent cultures. (A) Interactions between Abx1 and the transcriptional regulator CovR, the histidine kinase CovS, and two others GBS histidine kinase (Gbs2082 and Gbs0430). The T18 and the T25 tags are located at the C-terminal end of tested proteins. Interactions of Abx1-T18 (dark blue) were measured against the indicated T25 tag protein. Reciprocally, interactions of Abx1-T25 (light blue) were measured against the indicated T18 tag protein. (B) Homo-dimerizations were measured by the interaction between the T18 and T25 fusions of the same proteins and are show with light grey bars. The specific interaction between the HK CovS and its RR CovR is shown with dark grey bars. (C) Schematic representation of the CovS histidine kinase domains and of the truncated alleles used in (D). The amino-terminal part of CovS (residues 1–211) is made of two transmembrane (TM) domains flanking an extracellular loop. The cytoplasmic carboxy-terminal part of CovS (residues 211–501) contains the typical HAMP, HisKA and HATP regulatory and catalytic domains. Truncated alleles (I to VI) were tag with either T18 or T25 at the C-terminal end (I to III) or at the N-terminal end (IV to VI). A full length CovS allele tagged at the N-terminal end (VII) was added as a control. (D) Interactions between Abx1 and truncated alleles of CovS. The CovS alleles with a C-terminal tag were tested against the corresponding Abx1 tagged at its C-terminal end (blue). Similarly, the CovS alleles with a N-terminal tag were tested against the corresponding Abx1 tagged at its N-terminal end (red).</p

Hemolytic titer is dependent of <i>abx1</i> expression level.

*<p>As measured by qRT-PCR and reported as expression ratio against the WT strain. Mean +/− SD of three independent experiments. n.d. = not determined.</p

Proposed model of virulence gene regulation by the CovS/Abx1/Stk1/CovR network.

<p>(A) Simplified wiring diagram of the regulatory network controlling GBS hemolysin and pigment production. In a WT context, the Abi-domain protein Abx1 maintains the equilibrium between the kinase (green arrow) and the phosphatase activity (red lines) of the HK CovS. Activation of the RR CovR by CovS-dependent phosphorylation increases its inhibitory activity on <i>cyl</i> operon transcription. Inhibition of CovR is dependent on its dephosphorylation by CovS and its phosphorylation by the Stk1 serine/threonine kinase. In this condition, the <i>cyl</i> operon is expressed at a level defining the WT β-hemolytic and pigmentation phenotype. (B) In the absence of Abx1, the HK CovS is locked in its kinase-competent form that activates CovR, thus inhibiting the expression of the <i>cyl</i> operon. Phosphorylation of CovR by CovS precludes the Stk1-dependent CovR phosphorylation, leading to minimal CovR inhibition by Stk1. In this condition, GBS are non-hemolytic and non-pigmented. (C) In the presence of an excess of Abx1, the kinase-competent form of CovS is inhibited and/or the phosphatase-competent form is stabilized. The kinase Stk1 is necessary to fully inactive CovR, thereby allowing a strong expression of the <i>cyl</i> operon. In this condition, GBS are hyper-hemolytic and hyper-pigmented. (D) An integrated model for CovSR signaling with modular schematization of CovS (blue), Abx1 (green), Stk1 (red) and CovR (purple). The CovR activity reflects the equilibrium between the mutually exclusive ATP-dependent phosphorylation of two critical residues: threonyl 65 (T₆₅) by Stk1 and aspartyl 53 (D₅₃) by CovS, as proposed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003179#ppat.1003179-Lembo1" target="_blank">[15]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003179#ppat.1003179-Lin1" target="_blank">[25]</a>. This equilibrium is dependent on Abx1, which is essential for CovS-dependent inactivation of CovR (red arrow). Modulation of CovR affinities for DNA by phosphorylation regulates the expression of target genes, among which those necessary for stress resistance, host adhesion and damages characteristic of invasive bacterial forms. Potential connections on the core of the signaling network are in grey (other Stk1 targets, putative Stp phosphatase activity, putative interaction of CovS-Abx1 with other HKs, putative CovR phosphorylation by small phosphate donor as acetyl-phosphate).</p

Abx1 inhibits the CovSR system.

<p>Transcriptome analysis of deletion (Δ<i>abx1</i>, Δ<i>covS</i>, Δ<i>covR</i>, and Δ<i>covRS</i>) and overexpressing (Oe<i>_abx1</i>) mutants. The latter mutants were obtained either by chromosomal replacement of the <i>abx1</i> WT promoter by the strong constitutive promoter P_cyl+ (K_Oe_<i>abx1</i>) or by using the overexpression vector pTCVΩP_cyl+_<i>abx1</i> (v_Oe<i>_abx1</i>). The WT strain with the empty vector (NEM_ery) was included to take into account the potential effect of erythromycin (10 µg/ml) used to maintain the <i>abx1</i> overexpression vector. (A) Pairwise comparisons of Log₂ expression ratios for 1,905 genes between <i>abx1</i> overexpressing mutant (K_Oe_<i>abx1</i>) and Δ<i>covR</i> (blue dots) or Δ<i>covS</i> (red dots) deletion mutants. Pearson's correlations (r²) were calculated to estimate similarities in gene expression change. (B) Heatmap of the genes (N = 147) with an absolute log₂ ratio >2 in at least one strain. Gene expression changes were color-coded (blue = down; yellow = up). Hierarchical clustering (uncentered; average linkage) was done for genes (upper tree) and strains (right tree). Genes of the CovSR regulon conserved in 3 sequenced GBS strains (NEM316, A909, and 2603V/R) are highlighted below the heatmap (blue line = down-conserved; yellow line = up-conserved). (C) Highlight of the gene cluster in (B) containing the conserved negatively regulated genes of the CovSR regulon (in dark red letters). The gene tree is shown on the left of the heatmap and the corresponding gene identifiers and protein annotations are indicated on the right. Genes marked with a star were selected for confirmation of gene expression by qRT-PCR. Enlarge version of (B) and (C) heatmaps at a lower threshold ( = absolute log₂ ratio >1 in at least one strain) are presented in supplementary <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003179#ppat.1003179.s003" target="_blank">figure S3</a>. (D) qRT-PCR expression analysis of selected genes in <i>abx</i> and <i>cov</i> mutants. The relative expression level of <i>abx1</i>, of 5 negatively-regulated CovR genes (<i>cylE</i>, <i>cylJ</i>, <i>bibA</i>, <i>gbs0791 and gbs1037</i>), and of one positively-regulated CovR gene (<i>cfb</i> coding the CAMP factor) were measured in Δ<i>abx1</i>, Δ<i>covS</i>, and Δ<i>covR</i> deletion mutants, in the WT and Δ<i>abx1</i> mutant containing the <i>abx1</i> complementing vector (+ <i>abx1</i>; plasmid pTCVΩ<i>abx1</i>), and in two <i>abx1</i> overexpressing mutants (K_Oe<i>_abx1</i> = chromosomal <i>abx1</i> overexpression; v_Oe<i>_abx1</i> = vector-based <i>abx1</i> overexpression with the pTCVΩP_cyl+_<i>abx1</i> plasmid). Results are the relative expression level between mutant and WT strains expressed as the means value (+/− SEM) from three independent cultures in triplicates.</p

CovR-dependent phenotypes are regulated by convergent signaling pathways mediated by Abx1-CovS and Stk1.

<p>Phenotypes associated to the inactivation of the serine/threonine kinase Stk1 in a WT background (Δ<i>stk1</i>) and in combination with CovS inactivation (Δ<i>stk1</i>Δ<i>covS</i>). All strains were transformed with the empty vector pTCV or derivatives. To complement the Δ<i>stk1</i> mutant, the two-gene operon containing <i>stk1</i> was cloned with its promoter into the pTCV vector (pTCVΩ<i>stp-stk</i>). To compare the effect of Abx1 on CovR-dependent phenotypes, the Abx1 complementing vector (pTCVΩ<i>abx1</i>) or the Abx1 over-expression vector (pTCVΩP_cyl+<i>_abx1</i>) were introduced into relevant strains. Increased pigmentation and hemolysis due to the presence of the <i>abx1</i> vectors are indicated with a plus sign (+), absence of effect of the <i>abx1</i> vectors are indicated by a minus sign (−), and condition-dependent toxicity of <i>abx1</i> overexpression specifically on Granada medium is indicated by a mark (#). All assays were performed on TH, Granada (Gr) and blood agar (BI) plates.</p

The two-component system CovSR is epistatic to Abx1.

<p>(A) Control of hemolysin and pigment production by Abx1 is dependent on the two-component system CovSR. Phenotypic comparison of WT and <i>abx1</i> mutant without or with mutations in CovR (Δ<i>covR</i>), CovSR (Δ<i>covSR</i>), and in the CovR binding sites of the <i>cyl</i> operon (ΔCBS_cyl), as assayed on TH, Granada (Gr) and Blood agar (Bl) plates. (B) CovR-regulated genes are similarly expressed in <i>abx1</i>-overexpressing and <i>covR</i>-deletion mutants. Immunoblots of the surface-exposed adhesin BibA (Gbs2018) and secreted CAMP factor (Gbs2000) in cell wall extracts and concentrated supernatants, respectively. The Bsp protein was used as a loading control. Specificities of BibA and CAMP factor antibodies were confirmed with extracts from deletion mutants of the corresponding genes (negative controls).</p