Strain-specific regulatory role of eukaryote-like serine/threonine phosphatase in pneumococcal adherence

Abstract

Streptococcus pneumoniae exploits a battery of virulence factors to colonize the host. Although the eukaryote-like Ser/Thr kinase of S. pneumoniae (StkP) has been implicated in physiology and virulence, the role of its cotranscribing phosphatase (PhpP) has remained elusive. The construction of nonpolar markerless phpP knockout mutants (ΔphpP) in two pathogenic strains, D39 (type 2) and 6A-EF3114 (type 6A), indicated that PhpP is not indispensable for pneumococcal survival. Further, PhpP also participates in the regulation of cell wall biosynthesis/division, adherence, and biofilm formation in a strain-specific manner. Additionally, we provide hitherto-unknown in vitro and in vivo evidence of a physiologically relevant biochemical link between the StkP/PhpP-mediated cognate regulation and the two-component regulatory system TCS06 (RR06/HK06) that regulates the expression of the gene encoding an important pneumococcal surface adhesin, CbpA, which was found to be significantly upregulated in ΔphpP mutants. In particular, StkP (threonine)-phosphorylated RR06 bound to the cbpA promoter with high efficiency even in the absence of the HK06-responsive and catalytically active aspartate 51 residue. Together, our findings unravel the significant contributions of PhpP in pneumococcal physiology and adherence.

Fig 1

Construction of nonpolar PhpP mutants in 6A and D39 strains of S. pneumoniae. (A) Schematic representation of the genome organization of the wild-type S. pneumoniae phpP-stkP operon with an intergenic region depicting a transcription terminator between SPD_1542 and SPD_1541. Forward and reverse arrows indicate the primers used for confirming gene deletion in the mutants. (B) PCR amplification confirming the deletion of phpP in the mutants by using SG1F/SG2R is described in the text and Table S1. The arrow (2.7 kb) and arrowhead (2.0 kb) indicate PCR products obtained from the wild-type and ΔphpP mutant strains. (C) PCR amplification with phpP-gene specific primers (see Table S1 in the supplemental material). DNA gel showing the presence of PCR product with genomic DNA templates from only wild-type 6A/D39 strains and not with those from the 6AΔphpP and D39ΔphpP strains. (D) Western blot analysis using anti-PhpP (α-PhpP) antibody showing the presence of PhpP in the total cell lysates of wild-type (lanes 1 and 4) and PhpP-complemented (lanes 2 and 6) strains and the absence of PhpP in the mutants (lanes 3 and 5). (E) Demonstration of PhpP expression by Western blot analysis using anti-PhpP antibody in the culture supernatants (extracellular) of the wild-type and mutant strains. MW, prestained molecular weight marker. (F) The level of cotranscribing StkP in the 6A-WT (lane 1) and D39-WT (lane 3) strains and in their corresponding ΔphpP mutants (lanes 2 and 4) as assessed by Western blot analysis using anti-StkP antibody. Expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH)/streptococcal surface dehydrogenase (SDH) (40) in the total cell lysates was used as a loading control (18). (G and H) Growth characteristics of the wild types (G) and D39 and their isogenic mutant strains (6AΔphpP [G] and 6AΔphpP [H]) in THY broth. Growth was monitored spectrophotometrically at A₆₀₀ over 10 h. The bar diagram underneath each growth curve represents the CFU of the indicated strain grown to A₆₀₀ of 0.8. Error bars represent means ± standard deviations obtained from at least three independent experiments.

Fig 2

PhpP is involved in cell division and chain formation. (A and B) Gram-stained (A) and LIVE/DEAD-stained (B) overlay images of green SYTO-9 and red PI of the 6A and D39 wild-type and 6AΔphpP and D39ΔphpP mutant strains. (C to F) Transmission electron micrographs showing the ultrastructure of the 6A (C) and D39 (E) wild types and 6AΔphpP (D) and D39ΔphpP (F) mutant strains. Black and white arrows indicate the outer electron-dense layer and inner membrane, respectively.

Fig 3

The growth patterns of 6A and D39 wild types and their corresponding ΔphpP mutants grown under different stress conditions. The 6A and D39 wild types and corresponding ΔphpP mutants were grown in C+Y medium either under nonstress conditions (A and B) or various stress conditions (C to J) for a period of 10 to 12 h. The growth patterns of the indicated wild-type and mutants strains were spectrophotometrically measured at elevated temperatures (C and D), under high-salt conditions (E and F), at low pH (G and H), and under oxidative stress (I and J). The impact of oxidative stress on pneumococcal survival was measured based on the percentage of survival of the wild type and ΔphpP mutants (OD₆₀₀, 0.4) exposed to 50 mM H₂O₂ for different time periods. Each data point represents an average of three independent readings plus the standard deviation.

Fig 4

Effect of deletion of the phpP gene in pneumococcal adherence to human pharyngeal cells and biofilm formation. (A) The ability of 6A and D39 pneumococcal wild type strains and their corresponding 6AΔphpP and D39ΔphpP mutants to adhere to Detroit 562 human nasopharyngeal cells. (B) Ability to form biofilms by the wild type (6A and D39) strains and their corresponding ΔphpP mutant and phpP-complemented strains. The data represent means ± standard deviations of A₅₉₅ values from two independent experiments performed in 10 wells.

Fig 5

In vitro and in vivo StkP- and PhpP-mediated reversible phosphorylation of the response regulator Pn-RR06 of TCS06. (A and B) The StkkP-PhpP couple-mediated reversible phosphorylation of the wild-type Pn-RR06 (A) and its catalytic variant RR06D51A (B) as revealed in an in vitro kinase assay. The outcomes under various reaction conditions (shown on top) were measured by resolving proteins in the reaction mixtures by using 16% SDS-PAGE gels stained with Coomassie stain (lower panels) and corresponding autoradiograms (upper panels). MW, molecular mass markers, in kDa. (C and D) The specific phosphorylation at the threonine residues in both StkkP-phosphorylated wild-type Pn-RR06 (C) and RR06D51A (D), as determined by TLC. The TLC plates were sprayed with ninhydrin prior to autoradiography to detect phosphorylated Ser (pS), Thr (pT), and Tyr (pY) amino acids as, indicated by the arrows. (E) Determination of in vivo Thr phosphorylation of RR06 in 6A-WT and 6AΔphpP mutant strains. Western blot analysis showing the reactivities of immunoprecipitated RR06 from the whole-cell lysates of log-phase-grown 6A wild-type and 6AΔphpP mutant strains to mouse anti-RR06 antibody and anti-phospho-Thr antibodies. MW, molecular mass markers, in kDa.

Fig 6

The ability of untreated and Thr-phosphorylated RR06 and its catalytic variant, RR06D51A, to bind to the promoter of cbpA. (A) Electrophoretic mobility shift assay (EMSA) showing slow-migrating DNA-protein complex (marked with a C), formed by different concentrations (in μM) of RR06 and RR06D51A with γ-³²P-labeled 26-bp PcbpA_26bp (marked with an F). (B) EMSA showing the ability of 20 μM and 40 μM RR06 to form DNA-protein complexes with PcbpA_26bp in the presence and absence of StkkP and PhpP. (C and D) EMSA- (C) and densitometry-based kinetic analysis (D) depicting the binding abilities of untreated and StkkP-phosphorylated RR06 (0.3 to 20 μM) to PcbpA_26bp. (E and F) EMSA- (E) and densitometry-based kinetic analysis (F) depicting the ability of untreated and StkkP-phosphorylated RR06D51A (0.3 to 20 μM) bind to PcbpA_26bp. Arrows in panlels D and F indicate the amount of native RR06 or RR06D51A required for achieving 50% of the maximum binding obtained by their corresponding phosphorylated forms.