Figure 1.
The Abx1 (Gbs1532) protein is essential for hemolytic activity and pigment production in GBS.
(A) ß-hemolytic and pigmentation phenotypes of NEM316 wild-type strain (WT) and of two independent insertional mutants (abx1::Him1 and abx1::Him2) obtained by random transposon mutagenesis. Strains contained the empty vector pTCV or the complementing vector pTCVΩabx1 where abx1 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 abx1 deletion mutant (Δabx1). 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 abx1 overexpression. WT strain with the empty vector pTCV, the complementing vector pTCVΩabx1, the overexpressing vectors pTCVΩPcyl+_abx1 or pTCVΩPtet_abx1, the negative control vectors pTCVΩPcyl+_gbs1037 or pTCVΩPcyl+_EGFP were spotted onto TH, Granada, and blood agar plates containing erythromycin. An overexpression mutant where the abx1 chromosomal promoter was replaced by the strong Pcyl+ promoter (ΔPabx1::Pcyl+) 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.
Table 1.
Hemolytic titer is dependent of abx1 expression level.
Figure 2.
Abx1 function is dependent of the CylE ß-hemolysin through a protease-independent activity.
(A) Abx1 is a member of a large family of proteins related to eukaryotic CaaX proteases [35], [36]. The three most conserved motifs of the Abi-domain (Pfam02517) are depicted for Abx1 and Gbs1037 from S. agalactiae, Spr1119 from S. pneumoniae, SagE from S. pyogenes 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 (+). Figure S1 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 cylE deletion mutant (ΔcylE) with the empty vector pTCV or the abx1 overexpression vector pTCVΩPcyl+_abx1; and (b) WT, abx1 deletion mutant (Δabx1) and a mutant with a cysteyl to alanyl substitution in the CaaX motif of CylE (CylE C664A), i.e., 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 Δabx1 mutant were complemented with pTCVΩabx1 vectors encoding a wild-type allele (Abx1) or several alanine substitution alleles in the predicted critical glutamic acid (E) or histidine (H) residues.
Figure 3.
The two-component system CovSR is epistatic to Abx1.
(A) Control of hemolysin and pigment production by Abx1 is dependent on the two-component system CovSR. Phenotypic comparison of WT and abx1 mutant without or with mutations in CovR (ΔcovR), CovSR (ΔcovSR), and in the CovR binding sites of the cyl operon (ΔCBScyl), as assayed on TH, Granada (Gr) and Blood agar (Bl) plates. (B) CovR-regulated genes are similarly expressed in abx1-overexpressing and covR-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).
Figure 4.
Abx1 inhibits the CovSR system.
Transcriptome analysis of deletion (Δabx1, ΔcovS, ΔcovR, and ΔcovRS) and overexpressing (Oe_abx1) mutants. The latter mutants were obtained either by chromosomal replacement of the abx1 WT promoter by the strong constitutive promoter Pcyl+ (K_Oe_abx1) or by using the overexpression vector pTCVΩPcyl+_abx1 (v_Oe_abx1). 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 abx1 overexpression vector. (A) Pairwise comparisons of Log2 expression ratios for 1,905 genes between abx1 overexpressing mutant (K_Oe_abx1) and ΔcovR (blue dots) or ΔcovS (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 figure S3. (D) qRT-PCR expression analysis of selected genes in abx and cov mutants. The relative expression level of abx1, of 5 negatively-regulated CovR genes (cylE, cylJ, bibA, gbs0791 and gbs1037), and of one positively-regulated CovR gene (cfb coding the CAMP factor) were measured in Δabx1, ΔcovS, and ΔcovR deletion mutants, in the WT and Δabx1 mutant containing the abx1 complementing vector (+ abx1; plasmid pTCVΩabx1), and in two abx1 overexpressing mutants (K_Oe_abx1 = chromosomal abx1 overexpression; v_Oe_abx1 = vector-based abx1 overexpression with the pTCVΩPcyl+_abx1 plasmid). Results are the relative expression level between mutant and WT strains expressed as the means value (+/− SEM) from three independent cultures in triplicates.
Figure 5.
Abx1 physically interacts with the HK CovS.
The physical interactions between proteins were assayed using a two-hybrid system in E. coli. 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).
Figure 6.
CovS activities are Abx1-dependent.
(A) CovS is epistatic to Abx1. Phenotypes of the WT strain, Δabx1, Δabx1ΔcovS, and ΔcovS deletion mutants. (B) CovS kinase/phosphatase activities are essential for CovR regulation. Phenotypes of ΔcovS deletion mutant were compared with those of critical point mutants of CovS (H278A and T282A) or CovR (D53A). The histidine residue number 278 (H278) is the conserved phospho-accepting residue essential for the autophosphorylation of CovS. The threonine number 282 (T282) is a conserved residue necessary for the phosphatase activity of several HKs. The aspartate number 53 (D53) is the conserved residue of CovR phosphorylated by CovS. (C) Abx1 is dependent of CovS activities. Phenotypes of WT, ΔcovS, and CovS H278A or CovS T282A alanine substitution mutants overexpressing abx1 (+Pcyl+_abx1 = plasmid pTCVΩPcyl+_abx1). The hyper-pigmentation and hyper-hemolysis associated to abx1 over-expression in WT strain are suppressed in ΔcovS and CovS H278A mutants. Reversion of CovS H278A and T282A substitution back to the WT CovS sequence (indicated by _WT) restores the abx1 over-expression associated-phenotypes. All assays were performed on TH, Granada (Gr) and blood agar (BI) plates. (D) abx1 overexpression effects are dependent of CovS enzymatic activities. Relative expression levels of selected genes were measured by qRT-PCR in logarithmic growing cells in TH media. Relative expression levels of abx1, of five negatively regulated CovR genes (cylE, cylJ, bibA, gbs0791 and gbs1037), and of one positively regulated CovR gene (cfb coding the CAMP factor) were measured in CovS and CovR alanyl substitution mutant, and in WT and CovS alanyl substitution mutants overexpressing abx1 (+Pcyl+_abx1 = plasmid pTCVΩPcyl+_abx1). Results are means +/− sem from three independent cultures in triplicates.
Figure 7.
CovR-dependent phenotypes are regulated by convergent signaling pathways mediated by Abx1-CovS and Stk1.
Phenotypes associated to the inactivation of the serine/threonine kinase Stk1 in a WT background (Δstk1) and in combination with CovS inactivation (Δstk1ΔcovS). All strains were transformed with the empty vector pTCV or derivatives. To complement the Δstk1 mutant, the two-gene operon containing stk1 was cloned with its promoter into the pTCV vector (pTCVΩstp-stk). To compare the effect of Abx1 on CovR-dependent phenotypes, the Abx1 complementing vector (pTCVΩabx1) or the Abx1 over-expression vector (pTCVΩPcyl+_abx1) were introduced into relevant strains. Increased pigmentation and hemolysis due to the presence of the abx1 vectors are indicated with a plus sign (+), absence of effect of the abx1 vectors are indicated by a minus sign (−), and condition-dependent toxicity of abx1 overexpression specifically on Granada medium is indicated by a mark (#). All assays were performed on TH, Granada (Gr) and blood agar (BI) plates.
Figure 8.
Proposed model of virulence gene regulation by the CovS/Abx1/Stk1/CovR network.
(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 cyl 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 cyl 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 cyl 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 cyl 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 [15], [25]. 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).
Figure 9.
Mortality curves in neonate rats infected with the WT strain (black circle), the Δabx1 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).