New Results

Sublethal β-lactam antibiotics induce PhpP phosphatase expression and StkP kinase phosphorylation in PBP-independent β-lactam antibiotic resistance of Streptococcus pneumoniae

Yan-Ying Huang, Yan-Hong Sun, Peng Du, Xiao-Xiang Liu, Jie Yan, Ai-Hua Sun
doi: https://doi.org/10.1101/342188

Abstract

StkP and PhpP of Streptococcus pneumoniae have been confirmed to compose a signaling couple, in which the former is a serine/threonine (Ser/Thr) kinase while the latter was annotated as a phosphotase. StkP has been reported to be involved in penicillin-binding protein (PBP)-independent penicillin resistance of S. pneumoniae. However, the enzymatic characterization of PhpP and the role of PhpP in StkP-PhpP couple remain poorly understood. Here we showed that 1/4 minimal inhibitory concentration (MIC) of penicillin (PCN) or cefotaxime (CTX), the representatives of β-lactam antibiotics, could induce the expression of stkP and phpP genes and phosphorylation of StkP in PCN/CTX-sensitive strain ATCC6306 and three isolates of S. pneumoniae (MICs: 0.02-0.5 μg/ml). The product of phpP gene hydrolyzed PP2C type Ser/Thr phosphotase-specific RRA(pT)VA phosphopeptide substrate with the Km and Kcat values of 277.35 μmol/L and 0.71 S−1, and the hydrolytic activity was blocked by sodium fluoride, a PP2C type Ser/Thr phosphatase inhibitor. The phosphorylation levels of StkP in the four phpP gene-knockout (ΔphpP) mutants were significantly higher than that in the wild-type strains. In particular, the MICs of PCN and CTX against the ΔphpP mutants were significantly elevated as 4-16 μg/ml. Therefore, our findings confirmed that sublethal PCN and CTX act as environmental inducers to cause the increase of phpP and stkP gene expression and StkP phosphorylation. PhpP is a PP2C type Ser/Thr protein phosphatase responsible for dephosphorylation of StkP. Knockout of the phpP gene results in a high level of StkP phosphorylation and PBP-independent PCN/CTX resistance of S. pneumoniae.

Importance Streptococcus pneumoniae is a common pathogen in human populations in many countries and areas due to the prevalence of β-lactam antibiotic-resistant pneumococcal strains. Production of β-lactamases and mutation of penicillin-binding proteins (PBP) have been considered as the major β-lactam antibiotic-resistant mechanisms in bacteria, but S. pneumoniae has not been confirmed to produce any β-lactamases and many pneumococcal strains present PBP mutation-independent β-lactam antibiotic resistance. StkP is a Ser/Thr kinase of S. pneumoniae to compose a signal-couple with PhpP protein. The present study demonstrated that the PhpP is a PP2C-type phosphotase for dephosphorylation of StkP and the sublethal penicillin (PCN) or cefotaxime (CTX) acted as environmental signal molecules to induce the expression of PhpP. The knockout of PhpP-encoding gene caused the PCN/CTX resistance generation of PCN/CTX-sensitive pneumococcal strains. All the data indicate that StkP-PhpP couple of S. pneumoniae is involved in PBP mutation-independent β-lactam antibiotic resistance by phosphorylation of StkP.

Introduction

Streptococcus pneumoniae is a major causative agent of bacterial pneumonia and tympanitis in children [1-3]. More importantly, in the recent years, S. pneumoniae-infected meningitis cases with high fatality have been frequently reported in many countries and areas [4-8]. Therefore, S. pneumoniae is a common pathogen for human beings with global importance.

β-lactam antibiotics are the first choice in clinic to cure S. pneumoniae-infected patients [9]. However, in the recent years, β-lactam antibiotic-resistance of S. pneumoniae isolates from patients is continuously increased and the antimicrobial-resistant S. pneumoniae strains became more epidemic in many countries and areas [10-15], which has been considered as the major reason for increased incidence of S. pneumoniae-infected diseases [15,16]. Bacterial β-lactamases have been confirmed to play a key role in generation of β-lactam antibiotic resistance in many bacteria including S. pneumoniae [17]. Mutation of penicillin-binding proteins (PBP), the receptors of β-lactam antibiotics located on surface of bacteria, has been reported as the major β-lactam antibiotic resistant mechanism in bacteria [18,19]. However, recent studies found that some of the S. pneumoniae strains had no PBP mutation but presented β-lactam antibiotic resistance [20-22], indicating that S. pneumoniae may have a PBP mutation-independent mechanism of β-lactam antibiotic resistance.

StkP is a sequence-conserved eukaryotic-type serine/threonine (Ser/Thr) kinase (STK) of S. pneumoniae that has been confirmed to be involved in PBP mutation-independent penicillin resistance [22]. In the chromosomal DNA of S. pneumoniae, StkP-encoding gene (stkP) and phpP gene compose a stkP-phpP operon and the product of phpP gene is annotated as a putative phosphatase [23,24]. A previous study demonstrated that the PhpP and StkP of S. pneumoniae composed a StkP-PhpP signaling couple [25]. It has been reported that both prokaryotic and eukaryotic STKs are activated through phosphorylation at Ser/Thr sites and some certain protein phosphatases can inactivate STKs by hydrolysis of phosphoryl groups at the Ser/Thr residual sites in STKs [26]. In particular, a previous study revealed that penicillin (PCN) could cause the gene expression profile change of S. pneumoniae [27]. Therefore, we presume that β-lactam antibiotics may act as environmental inducers to cause the change of PhpP and StkP expression and StkP dephosphorylation of S. pneumoniae as well as the PhpP may be involved in the StkP-associated PBP mutation-independent penicillin resistance by dephosphorylation of StkP.

In the present study, we used PCN and cefotaxime (CTX) as the representatives of β-lactam antibiotics to detect their induction of phpP and stkP gene expression and then identified the product of phpP gene as a PP2C type Ser/Thr protein phosphatase by virtue of its ability to hydrolyze Ser/Thr phosphatase-specific substrates. Moreover, the phpP genes of S. pneumoniae strains were inactivated to determine the role of PhpP in dephosphorylation of StkP in vivo and the change of β-lactam antibiotic resistance. The results of this study confirmed that the product of S. pneumoniae phpP gene is a Ser/Thr protein phosphatase that involved in β-lactam antibiotic resistance-associated StkP-PhpP signaling couple by StkP dephosphorylation during induction of sublethal PNC and CTX.

Materials and Methods

Bacterial strains and culture

S. pneumoniae ATCC6306 and three β-lactam antibiotic-sensitive S. pneumoniae isolates (No.: SP5, SP9 and SP14, belonging to serotype 3, 19F and 19A) from pneumonia children were kindly provided by the Department of Medical Microbiology and Parasitology, Zhejiang University School of Medicine. All the strains were cultured with Colombia blood agar (bioMerieux, France) or 0.5% yeast extract-containing Todd-Hewitt (TH) broth (Sigma, USA) at 37°C [28]. Besides, Escherichia coli EL21DE3 (Novagen, USA) was cultured in Luria-Bertani (LB) medium (Oxoid, England) at 37°C.

Animal

New Zealand white rabbits (3.0 to 3.5 kg per animal) were provided by the Laboratory Animal Center of Hangzhou Medical College (Certificate No.: SCXK[zhe]2012-0173). All the animal experimental protocols were approved by the Ethics Committee for Animal Experiment of Hangzhou Medical College.

Drug susceptibility test

Susceptibility of each of the four S. pneumoniae strains to PCN or CTX was detected by E-test (bioMerieux) according to the manufacturer’s instruction. The minimal inhibitory concentrations (MIC) against S. pneumoniae strains, ≤ 2 or ≥ 8 μg/ml of PCN (Sigma) and ≤ 1 or ≥ 4 μg/ml of CTX (Sigma), were considered to be sensitive or resistant [29].

Detection of sublethal PCN- and CTX-induced expression of phpP and stkP genes

Each of the four S. pneumoniae strains was inoculated into TH broth for a 200 rpm shaking incubation at 37°C. When the value of optical density at 600 nm (OD600) of pneumococcal culture turbidity reached 0.5, 1/4 MIC PCN or CTX was added and then incubated for 0.5, 1, 2, 4, 8 or 12 h as above. After centrifugation and washing with phosphate buffered saline (PBS), total RNA of each of S. pneumoniae strains was extracted using a TRIzol® MaxTM Bacterial RNA Isolation kit (Invitrogen) plus a gDNA Eraser Kit (TaKaRa, China) and then quantified by ultraviolet spectrophotometry [30]. Subsequently, cDNA from each of the total RNAs was synthesized using a PrimeScriptTM RT Reagent Kit (TaKaRa). Using each of the cDNAs as template, the phpP- or stkP-mRNA level was assessed by real-time fluorescence quantitative reverse transcription polymerase chain reaction (qRT-PCR) with the primers P-1F/P-1R or S-1F/S-1R (Table 1) using a SYBR® Premix Ex-TaqTM Kit (TaKaRa) in an ABI 7500 Real-Time PCR System (ABI, USA). The primers used were designed using Primer Premier 5.0 software according to the phpP or stkP gene sequences in GenBank (accession No.: NC_003098 and NC_003028). In the qRT-PCR, 16S rRNA gene of S. pneumoniae was used as the internal reference [31], while the PCN- or CTX-untreated S. pneumoniae strains were used as the controls. The obtained qRT-PCR data were analyzed using the ΔΔCt model and randomization test in REST 2005 software [32].

View this table:
Table 1.

Sequences of primers used in this study.

Amplification and sequencing of PhpP and stkP gene segments

Genomic DNA of each of the four S. pneumoniae strains was extracted using a Bacterial Genomic DNA Preparation Kit (Axygen). By using a High Fidelity PCR Kit (TaKaRa), the entire phpP or stkP gene segments were amplified from the DNA templates by PCR using the primers P-2F/P-2R or S-2F/S-2R (Table 1). The PCR products were examined by 1.5% ethidium bromide-stained agarose gel electrophoresis and then cloned into pMD19-T plasmid using a T-A Cloning Kit (TaKaRa) to form recombinant pMD19-TphpP and pMD19-Tstkp plasmids for sequencing by Invitrogen Co. in Shanghai of China.

Bioinformatic analysis of phpP and stkP genes

Since the nucleotide and amino acid sequence identities of phpP and stkP genes from the four S. pneumoniae strains were as high as 98.7%-100%, the phpP and stkP genes of S. pneumoniae strain ATCC6306 were analyzed using TMHMM and NCBI Database Conserved Domain Database (CDD) software [33].

Generation of prokaryotic expression systems of phpP and stkP genes

The pMD19-TphpP or pMD19-TstkP plasmid from S. pneumoniae strain ATCC6306 and pET42a vector (Novagen) were digested with both Nde I and Xho I or Nde I and Hind III (TaKaRa). The recovered phpP or stkP gene segment was linked with the linearized pET42a using T4 DNA ligase (TaKaRa) and then transformed into E. coli BL21DE3 by CaCl2 transformation method to form E. coli BL21DE3pET42a-phpP or E. coli BL21DE3pET42a-stkP. The engineered strains were cultured in LB medium containing 50 μg/ml kanamycin (Sigma) and the pET42a-phpP and pET42a-stkP were extracted from the strains using a Plasmid Extraction Kit (Axygen) for sequencing again.

Expression of phpP and stkP genes and extraction of expressed products

The engineered strains, E. coli BL21DE3 pET42a-phpP and E. coli BL21DE3pET42a-stkP, were cultured in kanamycin-containing LB liquid medium to express the target recombinant proteins (rPhpP and rStkP) under induction of 0.5 mM isopropy-β-D-thiogalactoside (IPTG, Sigma). After ultrasonic breakage on ice and a 13,800×g centrifugation for 10 min (4°C), the supernatants of cultures were collected to extract soluble rPhpP and rStkP using a Ni-NTA affinity chromatographic column (BioColor, China). The extracted rPhpP or rStkP was quantified using a BCA Protein Assay Kit (Thermo Scientific, USA). Both the expressed and extracted rPhpP and rStkP were examined by sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) plus a Gel Image Analyzer (Bio-Rad, USA).

Preparation of rPhpP-IgG and rStkP-IgG

New Zealand rabbits were intradermally immunized on days 1, 14, 21 and 28 with Freund’s adjuvant-mixed 2 mg rPhpP or rStkP per animal. Fifteen days after the last immunization, the sera were collected to separate rStkP-IgG or rPhpP-IgG by ammonium sulfate precipitation plus a DEAE-52 column chromatography using 10 mM phosphate buffer (pH 7.4) for elution [34]. The titer of rPhpP-IgG or rStkP-IgG binding to rStkP or rPhpP was detected by immunodiffusion test.

Phosphatase activity assays

The enzymatic activity of rPhpP was detected using a pNPP-Phosphate Assay Kit (BioAssay Systems, USA) and a PP2C-specific Ser/Thr Phosphatase Assay Kit (Promega, USA) [35,36]. Briefly, 0.5, 1, 2.5, 5, 10 or 20 μg rPhpP was mixed with 500 nM para-nitrophenyl phosphate (p-NPP), an universal Ser/Thr phosphatase substrate, in 100 μl reaction buffer. After a 30-min incubation at 37°C, the OD405 values reflecting p-NPP hydrolysis were detected using type M5 spectrophotometer (Bio-Rad). On the other hand, 0.5, 1, 2.5, 5, 10 or 20 μg rPhpP was mixed with 100 μM RRA(pT)VA phosphopeptide, a PP2C type Ser/Thr protein phosphatase-specific substrate, in 100 μl reaction buffer. After incubation as above, the OD600 values reflecting RRA(pT)VA hydrolysis were detected by spectrophotometry.

Phosphatase inhibition test

Okadaic acid (OA) is an inhibitor of PP1, PP2A and PP2B type Ser/Thr phosphatases while sodium fluoride (NaF) is a PP2C type Ser/Thr phosphatase inhibitor [37,38]. Briefly, 5 μg rPhpP was mixed with 0.1, 0.5, 1, 5 or 10 μM OA (Sigma) or 0.1, 0.5, 1, 5 or 10 mM NaF (Sigma) in 100 μl reaction buffer and then incubated at 37°C for 30 min. The activity of OA- or NaF-treated rPhpP to hydrolyze RRA(pT)VA substrate was detected as described above.

Determination of Km and Kcat values

According to the results of phosphatase activity assays, 5 μg rPhpP was mixed with 50, 100, 150, 200 or 250 μM RRA(pT)VA substrate and then detected by spectrophotometry as described above. According to the OD600 values reflecting RRA(pT)VA hydrolysis and free phosphate concentration standard curve, the Km and Kcat values of rPhpP hydrolyzing the substrate were calculated by double reciprocal Lineweaver-Burk plot [39].

Generation and identification of phpP gene-knockout mutant

pEVP3 plasmid has been used to generate the target gene-knockout mutant of S. pneumoniae [40,41]. Briefly, a 495-bp phpP gene segment (phpP-495) from S. pneumoniae strain ATCC6306 was amplified using a High Fidelity PCR Kit (TaKaRa) with the primers P-3F/P-3R and then cloned into pMD19-T to form pMD19-TphpP−495 for sequencing as above. The pMD19-TphpP−495 and pEVP3 plasmid were digested with both Xho I and BamH I (TaKaRa). The recovered phpP-495 segment was linked with the linearized pEVP3 using T4 DNA ligase (TaKaRa) to form suicide plasmid pEVP3phpP495 for sequencing again. Each of the four S. pneumoniae strains was inoculated in TH broth containing 0.01% CaCl2 and 0.2% BSA (Sigma) for a 250-rpm incubation at 37°C to the OD600 value as 0.25 and then collected by centrifugation. The competent pneumococcal cells were suspended in 200 µl TH broth containing 0.01% CaCl2, 0.2% BSA and 200 ng/ml competence stimulation peptide (CSP, AnaSpec, USA) and then added with 200 ng pEVP3phpP−495 for transformation. The mixtures were smeared on 5% sheet blood Columbia agar (bioMerieux) plates containing 2.5 µg/ml chloromycin (CM, Sigma) for a 48-h incubation at 37°C in a 5% CO2 atmosphere to obtain phpP gene-knockout colonies [42]. The strategy for generating phpP gene-knockout mutants (ΔphpP-6306, ΔphpP-SP5, ΔphpP-SP9 and ΔphpP-SP14) is summarized in Fig. 1.

Fig. 1
Fig. 1

Strategy for generation of S. pneumoniae ΔphpP mutant. See section of materials and methods in text for details.

Identification of phpP gene-knockout mutants

Growth of the ΔphpP-6306, ΔphpP-SP5, ΔphpP-SP9 or ΔphpP-SP14 mutant was assessed by spectrophotometry. The phpP gene knockout in the ΔphpP mutants was determined by PCR using the primers P-4F/P-4R and P-5F/P-5R (Table 1) and sequencing of the 5’-phpP-pEVP3 and pEVP3-3’-phpP segment amplification products. Using 1:200 diluted rabbit anti-rPhpP-IgG as the primary antibody and HRP-conjugated goat anti-rabbit-IgG (Abcam, USA) as the secondary antibody, Western blot assay was performed to detect the PhpP form the ΔphpP mutants, in which the wild-type strains were used as the controls.

Detection of sublethal PCN- or CTX-induced StkP phosphorylation

The ΔphpP mutants and their wild-type strains were treated with 1/4 MIC PCN or CTX for 1, 2, 4 or 8 h at 37°C. After centrifugation and washing with PBS, the pneumococcal pellets were suspended in deionized water and then ultrasonically broken on ice. The lysates were centrifuged at 14,000×g for 10 min (4°C) and then the supernatants were collected to detect protein concentrations using a BCA Protein Assay Kit (Thermo Scientific). 200 µg of each of the total pneumococcal proteins was mixed with 20 µg rabbit anti-rStkP-IgG for a 2-h incubation in a 90-rpm rotator (4°C). The mixture was added with 600 µg protein-A-coated agarose beads (Millipore, USA), followed by a 60-min incubation as above. After a 14,000×g centrifugation for 5 min and washing thoroughly with PBS, the precipitated beads were suspended in 200 µl 50 mM Tris-HCl buffer (TB, pH7.5) and then mixed with 200 µl 2M NaOH solution for a 30-min water-bath at 65°C to release phosphate groups from the captured StkP according to the instruction of Phosphoprotein Phosphate Detection Kit (Sangon Biotech, Canada). The mixture was neutralized with 200 µl 4.7 M HCl solution and then added with 200 µl detection buffer for a 30-min incubation at room temperature. The OD620 value reflecting phosphate group concentration released from IgG-captured StkP were detected using a spectrophotometer (Molecular Devices, USA) [43]. In the detection, the PCN- or CTX-untreated ΔphpP mutants and their wild-type strains were used as the controls.

Detection of β-lactam antibiotic resistance of ΔphpP mutants

Susceptibility of the ΔphpP-6306, ΔphpP-SP5, ΔphpP-SP9 and ΔphpP-SP14 mutants to PCN or CTX was detected by E-test as described above. In the detection, the wild-type S. pneumoniae strains were used as the controls.

Statistical analysis

Data from a minimum of three experiments were averaged and presented as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test were used to determine significant differences. Statistical significance was defined as p <0.05.

Results

Increase of stkP- and phpP-mRNA levels induced by sublethal PCN and CTX

The phpP- and stkP-mRNA levels of each of the four tested S. pneumoniae strains were relatively lower. When the strains were treated with 1/4 MIC PCN or CTX, the phpP- and stkP-mRNA levels were rapidly elevated (Fig. 2A-2D). The data suggested that sublethal PCN and CTX can act as the stimulators to induce the expression of S. pneumoniae phpP and stkP genes.

Fig. 2
Fig. 2

Increase of phpP- and stkP-mRNAs after treatment with sublethal PCN and CTX.

(A). Increase of phpP-mRNA induced by 1/4 MIC PCN, detected by qRT-PCR. Bars show the mean ± SD of three independent experiments. The phpP-mRNA levels in the PCN-untreated S. pneumoniae strains (before treatment) were set as 1.0. *: p<0.05 vs the phpP-mRNA levels in the PCN-untreated strains.

(B). Increase of phpP-mRNA induced by 1/4 MIC CTX, detected by qRT-PCR. The legend is the same as shown in A but for CTX-induced phpP-mRNA detection.

(C). Increase of stkP-mRNA induced by 1/4 MIC PCN, detected by qRT-PCR. Bars show the mean ± SD of three independent experiments. The stkP-mRNA levels in the PCN-untreated S. pneumoniae strains (before treatment) were set as 1.0. *: p<0.05 vs the stkP-mRNA levels in the PCN-untreated strains.

(B). Increase of stkP-mRNA induced by 1/4 MIC of CTX, detected by qRT-PCR. The legend is the same as shown in C but for CTX-induced stkP-mRNA detection.

Characterization of phpP and stkP genes

The product of S. pneumoniae phpP or stkP gene was predicted as a secretary cytosolic or a transmembrane protein (Fig. 3A and 3B). The phpP gene contains a PP2Cc type protein phosphatase domain (6-238 aa) with five enzymatic active sites (Fig. 3C). The stkP gene contains a C type STK domain belonging to STKc_PknB superfamilay containing twelve ATP-binding sites, six dimer interface sites, two activation loops, eight polypeptide substrate-binding sites and sixteen enzymatic active sites as well as four penicillin-binding protein and STK-associated (PASTA) superfamily domains (Fig. 3D).

Fig. 3
Fig. 3

Predicted characteristics of S. pneumoniae phpP and stkP genes.

(A). Structure and location of phpP gene product, predicted using TMHMM software.

(B). Structure and location of stkP gene product, predicted using TMHMM software.

(C). PP2C type phosphatase domain in phpP gene product (PhpP), predicted using NCBI Database CDD software.

(D). STK and PASTA domains in stkP gene product (StkP), predicted using NCBI Database CDD software.

Effects of expression and extraction of stkP and phpP genes

The nucleotide and amino acid sequence identities of phpP or stkP genes from the four S. pneumoniae strains were 99.3%-100% and 99.1%-100% or 99.2%-99.7% and 98.7%-99.9%, compared to the same two genes in GenBank (accession No.: NC003098) (data not shown). The two engineered strains expressed the target recombinant proteins rPhpP and rStkP, respectively, and the extracted rPhpP or rStkP was showed as a single band in gels (Fig. 4A-4D).

Fig. 4
Fig. 4

Amplification and expression of S. pneumoniae phpP and stkP genes.

(A). Amplification fragments of phpP genes from S. pneumoniae strains, determined by PCR. Lane M: DNA marker. Lanes 1-4: amplicoms of phpP genes from S. pneumoniae strains ATCC6306, SP5, SP9 and SP14 (738 bp). Lane 5: blank control.

(B). Amplification fragments of stkP genes from S. pneumoniae strains, determined by PCR. The legend is the same as shown in A but for stkP gene amplification (1977 bp).

(C). Expression and extraction effects of rPhpP, detected by SDS-PAGE. Lane M: protein marker. Lane 1: wild-type E. coli BL21DE3. Lane 2: the expressed rPhpP (~28.3 kDa). Lane 3: the extracted rPhpP by Ni-NTA affinity chromatography.

(D). Expression and extraction effects of rStkP, detected by SDS-PAGE. The legend is the same as shown in C but for rStkP expression and extraction (~75.8 kDa).

Powerful Ser/Thr protein phosphatase activity of rPhpP

The rPhpP from S. pneumoniae ATCC6306 hydrolyzed p-NPP, an universal Ser/Thr phosphatase substrate, and RRA(pT)VA, a PP2C type Ser/Thr protein phosphatase-specific substrate, with concentration-dependent manners (Fig. 5A). The Km and Kcat values of rPhpP hydrolyzing RRA(pT)VA substrate were 277.35 μmol/L and 0.71 S−1, respectively (Fig. 5B). Moreover, the PP2C type Ser/Thr protein phosphatase inhibitor NaF, but not the PP1, PP2A or PP2B type Ser/Thr phosphatase inhibitor OA, inhibited the hydrolytic activity of rPhpP (Fig. 5C). The data suggested that the product of S. pneumoniae phpP gene is a PP2C type Ser/Thr protein phosphatase.

Fig. 5
Fig. 5

Powerful protein phosphatase activity of rPhpP from S. pneumoniae.

(A). Ability of rPhpP hydrolyzing phosphatase substrates p-NPP and RRA(pT)VA, determined by spectrophotometry. Bars show the means ± SD of three independent experiments. p-NPP is a universal Ser/Thr phosphatase substrate while RRA(pT)VA is a PP2C type Ser/Thr protein phosphatase-specific substrate.

(B). Km and Kcat values of rPhpP hydrolyzing RRA(pT)VA substrate, determined by spectrophotometry. 5 μg rPhpP and 50, 100, 150, 200 or 250 μM RRA(pT)VA phosphopeptide were used.

(C). Enzymatic activity of rPhpP after treatment with phosphatase inhibitors, determined by spectrophotometry. NaF is a PP2C type Ser/Thr protein phosphatase inhibitor while OA is an inhibitor of PP1, PP2A or PP2B type Ser/Thr phosphatases.

PCN/CTX-induced StkP phosphorylation and PhpP-caused StkP dephosphorylation

The ΔphpP mutants could grow persistently in the TH broth similarly to the wild-type strains (Fig. 6A). The PCR plus sequencing and Western Blot assay confirmed the phpP gene knockout and no PhpP expression in all the four ΔphpP mutants (Fig. 6B and 6C). 1/4 MIC PCN or CTX could cause the increase of StkP phosphorylation levels in the ΔphpP mutants and their wild-type strains (Fig. 6D and 6E). However, the ΔphpP mutants presented significantly higher PCN- or CTX-induced StkP phosphorylation levels than the wild-type strains (Fig. 6D and 6E). The data suggested that sublethal PCN and CTX can induce phosphorylation of StkP and PhpP can cause dephosphorylation of StkP in vivo.

Fig. 6
Fig. 6

PCN/CTX-induced StkP phosphorylation and PhpP-caused StkP-dephosphorylation.

(A). Growth curves of the ΔphpP mutants and wild-type strains in TH broth, determined by spectrophotometry. Bars show the means ± SD of three independent experiments.

(B). Schematic diagram of PCR and sequencing results of the ΔphpP mutants.

(C). PhpP absence in the ΔphpP mutants, determined by Western Blot assay. Lanes 1-4: immunoblotting bands of PhpP proteins from the wild-type S. pneumoniae strain ATCC 6306 and S. pneumoniae isolates SP1, SP5 and SP9. Lanes 5-8: no immunoblotting bands of PhpP proteins in the ΔphpP mutants.

(D) Sublethal PCN-induced increase of StkP phosphorylation and decrease of StkP phosphorylation in the ΔphpP mutants, detected by spectrophotometry. Bars show the means ± SD of three independent experiments. *: p<0.05 vs the StkP phosphorylation levels of the PCN-untreated wild-type strains and ΔphpP mutants. #: p<0.05 vs the StkP phosphorylation levels of the wild-type strains.

(E) Sublethal CTX-induced increase of StkP phosphorylation and decrease of StkP phosphorylation in the ΔphpP mutants, detected by spectrophotometry. The legend is the same as shown in D but for detection of CTX-induced StkP phosphorylation.

Increased PCN and CTX resistance of ΔphpP mutants

The MICs of PCN and CTX against wild-type ATCC6306, SP5, SP9 and SP14 strains of S. pneumoniae were 0.02-0.5 μg/ml, indicating all the strains were sensitive to both PCN and CTX. However, the MICs of PCN and CTX against the four ΔphpP mutants were significantly elevated as 4-16 µg/ml (Table 2). The data suggested that PhpP is involved in β-lactam antibiotic resistance of S. pneumoniae.

View this table:
Table 2

MICs of PCN and CTX against S. pneumoniae strains.

Discussion

Kinases play key roles in signal transduction in both eukaryotes and prokaryotes and are activated by either phosphorylation or dephosphorylation [44]. Unlike eukaryotes, histidine kinases but not serine/threonine/tyrosine (Ser/Thr/Tyr) kinases act as the major signal sensors and transducers in prokaryotic bacteria [45]. Previous studies confirmed that StkP of S. pneumoniae is an eukaryotic-type Ser/Thr protein kinase containing PBP-like domains in its extracellular region and Ser/Thr phosphorylation sites in its intracellular region [46], and PhpP and StkP of S. pneumoniae compose a StkP-PhpP signaling couple [25]. Operon is a genic unit for transcription in prokaryotic genome that composed of promoter, operator and function-associated structural genes [47]. Our bioinformatic analysis showed that PhpP- and StkP-encoding genes of S. pneumoniae are located in the same one operon for co-expression, implying the close functional association between the two genes. As described in the introduction, StkP of S. pneumoniae is involved in PBP mutation-independent penicillin resistance [22]. Therefore, the product of phpP gene, PhpP, may act as a protein phosphotase to down-regulate StkP phosphorylation level by dephosphorylation to participate in StkP-involved β-lactam antibiotic resistance.

According to the differences of amino acid sequences and structures, Ser/Thr protein phosphatases are classified into MPP and PPP families as well as the former contains Mg2+ or Mn2+-dependent PP2C type phosphatases while the latter includes PP1, PP2A and PP2B type phosphatases [48]. PP2C type phosphatases has an extensive distribution in bacteria, yeasts, plants and mammalian cells to play various and complicated functions such as signaling transduction by dephosphorylation, cellular generation cycle regulation, monitoring DNA damage and RNA splicing [48-50]. Our bioinformatic prediction showed that phpP gene of S. pneumoniae contains a PP2C type protein phosphatase domain. The recombinant expression product of phpP gene (rPhpP) hydrolyzed both the general Ser/Thr phosphatase substrate p-NPP and PP2C type Ser/Thr protein phosphatase-specific substrate RRA(pT)VA in a concentration-dependent manner but its RRA(pT)VA-hydrolyzed ability was blocked by the PP2C type Ser/Thr protein phosphatase-specific inhibitor NaF. Previous studies confirmed that StkP and PhpP of S. pneumoniae compose a StkP/PhpP signaling couple in which StkP is activated by Ser/Thr residual phosphorylation and PhpP could hydrolyze phosphoryl groups in StkP in vitro [25,51]. However, we confirmed that the sublethal PCN and CTX induced the phosphorylation of StkP and the StkP phosphorylation levels in the ΔphpP mutants were significantly higher than that in the wild-type strains. All the data suggested that the product of S. pneumoniae phpP gene is a PP2C type Ser/Thr protein phosphatase to play a reverse regulation role in StkP/PhpP signaling couple by dephosphorylation of StkP.

Previous studies reported that penicillin-binding protein and STK-associated (PASTA) superfamily domains in some proteins from many Gram-positive bacteria have a potential ability to bind to β-lactam antibiotics [52,53], and our bioinformatic analysis also revealed that StkP of S. pneumoniae possesses four PASTA domains located its carboxyl terminal, implying that the StkP may served as the β-lactam antibiotic receptor and β-lactam antibiotics may cat as environmental activators of StkP-PhpP signaling couple. In the present study, the sublethal PCN or CTX (1/4 MIC) caused the significant increase of stkP- and phpP-mRNA levels and StkP phosphorylation. The data suggested that StkP-PhpP couple is a β-lactam antibiotic-associated signaling pathway of S. pneumoniae.

Bacterial PBP is a group of peptidoglycan biosynthesis-associated transpeptidases, carboxypeptidases and endopeptidases that located on surface of bacteria [54]. β-lactam antibiotics can bind to the peptidases to cause them inactivation by due to enzymatic molecular allosterism but PBP mutation can cause β-lactam antibiotic resistance due to the decrease of β-lactam antibiotic-binding ability of PBP [55,56]. However, some S. pneumoniae strains presented PBP mutation-independent β-lactam antibiotic resistance [20-22]. In the present study, all the tested S. pneumoniae strains were sensitive to both PCN and CTX (MICs: 0.02-0.5 μg/ml). When the phpP genes were knockout, the ΔphpP mutants became resistant to the two antibiotics (MICs: 4-16 μg/ml). The data imply that StkP phosphorylation promotes but PhpP-based StkP dephosphorylation inhibits the generation of PBP mutation-independent β-lactam antibiotic resistance.

Conclusions

Sublethal PCN and CTX can act as environmental inducers to cause the increase of stkP and phpP gene expression and StkP phosphorylation of S. pneumoniae. The product of phpP gene is a PP2C type Ser/Thr protein phosphatase to cause dephosphorylation of StkP that plays a reverse regulating role in StkP/PhpP signaling couple. Sublethal β-lactam antibiotics can act as environmental inducers of phpP and stkP gene expression and knockout of phpP gene caused the significant increase of β-lactam antibiotic resistance of S. pneumoniae.

Additional file

This manuscript has no additional files.

Abbreviations

S. pneumoniae
Streptococcus pneumoniae
PNC
penicillin
CTX
cefotaxime
qRT-PCR
quantitative reverse transcription polymerase chain reaction
ΔphpP
phpP gene-knockout mutant
MIC
minimal inhibitory concentration
mRNA
messenger ribonucleic acid
PBP
penicillin-binding proteins
STK
serine/threonine kinase
DNA
deoxyribonucleic acid
TH
Todd-Hewitt
LB
Luria-Bertani
OD
optical density
PBS
phosphate buffered saline
RNA
ribonucleic acid
SDS-PAGE
sodium dodecyl sulfate polyacrylamide gel electropheresis
CDD
conserved domain database
IPTG
isopropy-β-D-thiogalactoside
OA
Okadaic acid
NaF
sodium fluoride
SD
standard deviation
PASTA
penicillin-binding protein and STK-associated
TB
Tris-HCl buffer

Acknowledgements

We are grateful to Dr. Morrisson (University of Illinois, Chicago, USA) for kindly providing the pEVP3 plasmid used in this study.

Funding

This work was supported by a grant from the National Natural Science Foundation of China (81772232) and a grant from Zhejiang Provincial Program for the Cultivation of High-Level Innovative Health Talents (2012-241).

Availability of data and materials

The datasets analyzed during the current study are available from the corresponding author on reasonable request.

Author’s contributions

A.H.S. and J.Y. conceived and designed the experiments. Y.Y.H., Y.H.S. and P.D. performed the experiments. Y.Y.H., Y.H.S. and X.X.L. analyzed the data. Y.Y.H., A.H.S. and J.Y. wrote the manuscript. A.H.S. obtained the funding. All authors have read and approved the manuscript.

Ethics approval and consent to participate

This study has no medical ethic problems. All the animal experimental protocols were approved by the Ethics Committee for Animal Experiment of Hangzhou Medical College.

Consent for publication

Not applicable.

Competing interests

All the authors have no potential conflict of interest to declare.

Footnotes

  • E-mail addresses: Yan-Ying Huang: 1842725667{at}qq.com, Yan-Hong Sun: syhsarah1123{at}163.com, Peng Du: pdu1145{at}163.com, Xiao-Xiang Liu: 494957023{at}qq.com, Jie Yan: Med_bp{at}zju.edu.cn, Ai-Hua Sun: sunah123{at}126.com

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Posted June 08, 2018.
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Sublethal β-lactam antibiotics induce PhpP phosphatase expression and StkP kinase phosphorylation in PBP-independent β-lactam antibiotic resistance of Streptococcus pneumoniae
Yan-Ying Huang, Yan-Hong Sun, Peng Du, Xiao-Xiang Liu, Jie Yan, Ai-Hua Sun
bioRxiv 342188; doi: https://doi.org/10.1101/342188
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