Phosphorylation controls the functioning of Staphylococcus aureus isocitrate dehydrogenase – favours biofilm formation

Abstract

Isocitrate dehydrogenase (IDH) gene from Staphylococcus aureus ATCC12600 was cloned, sequenced and characterized (HM067707). PknB site was observed in the active site of IDH; thus, it was predicted as IDH may be regulated by phosphorylation. Therefore, in this study, PknB, alkaline phosphatase III (SAOV 2675) and IDH genes (JN695616, JN645811 and HM067707) of S. aureus ATCC12600 were over expressed from clones PV 1, UVPALP-3 and UVIDH 1. On passing the cytosloic fractions through nickel metal chelate column, pure enzymes were obtained. Phosphorylation of pure IDH by PknB resulted in the complete loss of activity and was restored upon dephosphorylation with SAOV 2675 which indicated that phosphorylation and dephosphorylation regulate IDH activity in S. aureus. Further, when S. aureus ATCC12600 was grown in BHI broth, decreased IDH activity and increased biofilm units were observed; therefore, this regulation of IDH alters redox status in this pathogen favouring biofilm formation.

• Biofilm
• dephosphorylation
• IDH
• phosphorylation
• PknB
• SAOV 2675

Introduction

Bacteria have positioned the metabolic requirement of TCA cycle according to their redox status. This cycle is largely regulated by the expression of isocitrate dehydrogenase (IDH)1. In bacteria, the activity of IDH is controlled through phosphorylation and dephosphorylation2–4. In Staphylococcus aureus, the expression of virulence factors, toxins and biofilm formation requires active functioning of TCA cycle and serine/threonine protein kinases (PknB)5–11. Krebs cycle in S. aureus is regulated by IDH whose expression regulates the carbon flow; thus, enabling the organism to adapt and form biofilms in different niches7^,9^,12–14. Earlier, we have cloned, sequenced, expressed and characterized IDH gene from S. aureus ATCC12600, the sequence revealed the presence of PknB site in the active site of IDH13.

PknB is also implicated in the cell wall and polysaccharide intercellular adhesin (PIA) synthesis, functioning of various metabolic pathways, resistance to antibacterial agents and autolysis which are all required for biofilm formation. This pathogen survives in the human host mostly as biofilms contributing resistance to several antibiotics making S. aureus as a multidrug resistant strain11^,15–20. Further, in this organism, PknB is actively present in the external secretions, and along with phosphatases they interact with vast range of host tissue proteins aiding this pathogen to colonize in any anatomical locales in human host21^,22.

In S. aureus, the dephosphorylation is carried out by phosphatases which are present along with phosphokinases. There are four different alkaline phosphatases which play distinct role in the organism, Pho R and Pho P genes are involved in signal transduction, while SAOV 255 and SAOV 2675 genes are involved in the regulation of metabolic process23. Thus, we predicted that in S. aureus IDH is probably regulated by phosphorylation and dephosphorylation. To establish this hypothesis, PknB, alkaline phosphatase (ALP) III (SAOV2675) and IDH genes from S. aureus ATCC12600 were cloned, expressed and characterized. Furthermore, the effect of phosphorylation and dephsophorylation on IDH activity was demonstrated.

Methods

Bacterial cultures

Staphylococcus aureus ATCC12600 was grown on modified Baird Parker agar. A black shiny colony with clear zone was picked and streaked on modified Baird Parker (BP) agar plate containing 0.001% phenolphthalein bisphosphate tetrasodium salt (Sigma) and 10% NaCl (w/v), as described by Barber and Kuper24. After incubation at 37 °C for 2 days, the pink-coloured colonies indicate the expression of phosphatases. Single colony from the above plate was inoculated in brain heart infusion (BHI) and Luria–Bertani (LB) broths. These cultures were grown overnight at 37 °C. After overnight incubation, the grown culture was subjected to isolation of chromosomal DNA25.

Biofilm assay

The staphylococcal biofilm assay was carried out as described earlier14.

Alkaline phosphatase enzyme assay

The enzyme reaction mixture contained 0.1 M carbonate–bicarbonate buffer, pH 10, 5 mM p-Nitro phenyl phosphate and mixed thoroughly just before the incubation at 37 °C crude or pure ALP enzyme was added. The absorbance was measured at 405 nm against blank (carbonate–bicarbonate buffer), and the kinetic parameters V_max and K_m were calculated from the Hanes–Woolf plot [[S] versus ([S]/V)]26.

IDH assay

Pure IDH assay was obtained from the cytosolic fraction of UVIDH1 clone, and the enzyme assay was carried out as described earlier13.

Serine/threonine protein kinase (PknB) assay

PknB activity was determined at 30 °C using novel non-radiolabeled protein kinase spectrophotometric assay with synthetic peptide which acts as substrate in a Cyberlab spectrophotometer (Mill bury Town, MA). Serine/threonine protein kinase (PknB) assay mixture contained 0.1 M Tris-HCl at pH 7.5, 0.1 M ATP, 11.8 µM (30 µg/µl) peptide (stpks = NLCNIPCSALLSSDITASVNCAK), and 1 µg/µl enzyme fraction (pure His tag PknB) was mixed and incubated at 30 °C for 10 min. The phosphorylated peptide was purified by passing through Sephadex G-25 column (1 cm× 15 cm), the fractions were eluted with 0.1 M Tris-HCl pH 7.5 and 150 mM NaCl. The enzyme fraction appeared in the void volume, while phosphorylated peptide was obtained in the elution volume. The phosphate covalently bound to the proteins were estimated by adding freshly prepared reagent A: 3.4 mM of ammonium molybdate dissolved in 0.5 mM H₂SO₄, 10% SDS, 0.6 M l-ascorbic acid mixed in 6:1:1 (v/v/v) ratio and incubated at 30 °C for 15 min, and the absorbance was recorded at 820 nm against blank (0.1 M Tris-HCl, pH 7.5, 150 mM NaCl and reagent A)27. The enzyme activity was measured as amount of phosphorous added per microgram peptide at 30 °C/min/ml. For this, the calibration curve was developed using standard KH₂PO₄ for the estimation of inorganic phosphate, and free phosphate was determined by adding reagent A28. The phosphorylated peptide was further demonstrated by fractionating the eluted peptide in 12.5% SDS-PAGE. Upon staining the gel with reagent A, the blue-coloured band appeared in the gel indicating the peptide was phosphorylated by the enzyme fraction. Similarly, the auto-phosphorylation property of PknB was also determined for this, the reaction mixture composition was same, except stpks was not added. The enzyme activity was measured as amount of phosphorous added per microgram enzyme at 30 °C/min/ml. Substrate level phosphorylation was performed by taking different substrate concentrations of 10–120 µM of synthetic peptide keeping the ATP concentration constant, and the corresponding velocities were calculated and a graph of [[S] versus [S/V]] was plotted; from the graph obtained, K_m and V_max were determined. Similarly, the K_m and V_max for auto-phosphorylation of PknB was determined by the Hanes–Woolf plot. Protein concentrations in all steps were determined by the Bradford method29.

PknB gene amplification and sequencing

The PknB gene was amplified using the primers designed from the sequence of S. aureus USA 300 (Table 1). The PCR was performed using Eppendorf (Master gradient) thermocycler. The 50 -µl PCR reaction mixture contained 0.5 µg of chromosomal DNA, 100 µM of dNTPs mix, 100 pM of forward and reverse primers, 1× reaction buffer that contained MgCl₂ supplied by the manufacturer (Merck Biosciences, Bangalore, India) and 1 U of Hotstart Taq DNA polymerase (Merck Biosciences). PCR was performed with the following conditions: initial denaturation at 94 °C for 10 min, and 40 cycles of denaturation at 94 °C for 60 s of denaturation, 32 °C for 45 s of annealing, 72 °C for 90 s amplification followed by final elongation at 72 °C for 10 min. The amplified PCR products were resolved in 1% agarose gel along with supermix DNA ladder (Merck Bioscience), and resolved DNA bands were recorded in Vilber Lourmat gel documentation system. The purified PknB PCR product was sequenced by dye terminating method at commercial sequencing facility of Xcelris Labs Ltd, Ahmadabad, India. The obtained sequences were analyzed and deposited at NCBI-Gen Bank (http://www.ncbi.nlm.nih.gov/nuccore/JN695616).

Table 1. Primers used for PCR amplification.

Gene name	Orientation	Primer sequence (5′ to 3′)	Reference	Amplicon size
PknB-F1	Forward	CATGATAGGTAAAATA	Present Study	2000
PknB-R1	Reverse	TTATACATCATCATA	Present Study
ALP-F1	Forward	TGCAGTTGCTAACCT	Present Study	1425
ALP-R1	Reverse	TGCGCTTTCTGCATT	Present Study
IDH-F1	Forward	CATGCTGATGTAGAAACT	Prasad et al.13	902
IDH-R1	Reverse	TCAAGGAACTGAAGGAT	Prasad et al.13

ALP gene amplification and sequencing

The ALP gene was amplified from the chromosomal DNA using the primers designed manually from the ALP gene sequence of S. aureus Mu 50 strain30 (Table 1). The cocktail mixture contained 100 pM of each primer, 100 μM of dNTPS mix, Tris with 15 mM MgCl₂, 1 U of hot start Taq DNA polymerase (Merck Bioscience Pvt. Ltd) and 0.5 µg chromosomal DNA. Thermocycler conditions included an initial denaturation step for 10 min at 94 °C; 35 cycles of each having denaturation at 94 °C for 60 s, annealing at 39 °C for 35 s and amplification at 72 °C for 90 s which was followed by a final extension step at 72 °C for 10 min. Amplicons were purified with NP-PCR purification kit (Taurus Scientific, Cincinnati, OH) and sequenced by chain terminating method at MWG Biotech India Ltd. Thus, obtained ALP gene sequence was deposited at Gen Bank (http://www.ncbi.nlm.nih.gov/nuccore/JN645811).

Multiple sequence alignment

The multiple sequence alignment was performed between S. aureus alp, Mycobacterium tuberculosis (NCBI ID: WP_006242595.1), Bacillus subtilis (NCBI ID: WP_009879253.1) Streptococcus (NCBI ID: YP_002746311) and Homo sapiens (NCBI ID: NP_000469.3) using ClustalX software. Similarly, using the same software, the multiple sequence alignment was performed between S. aureus PknB, M. tuberculosis (NCBI ID: NP_214528.1), B. subtilis (NCBI ID: NP_00748102.1), Streptococcus (NCBI ID: NP_720924.1) and H. sapiens (NCBI ID: AAQ_02456.1)14.

Cloning and expression of PknB and alkaline phosphatase III genes

The amplified PCR products of 2 kb and 1.425 kb corresponding to PknB and alkaline phosphatase III genes were electroeluted from the agarose gel and were made into proper blunt ends using Klenow fragment (New England Bio Labs, Ipswich, MA) following the manufacturer’s protocol. The blunt-ended PCR products were cloned into the SmaI site of plasmid pQE-30 (QIAGEN, Valencia, CA) and were transformed into the E. coli DH5α. Thus, obtained clones were named as PV 1 and UVP-ALP3. These clones were induced with 0.1, 0.2, 0.5, 0.75, 1 and 1.5 mM IPTG and we found at 0.75 mM IPTG concentration PV 1 clone cells survived and expressed PknB similarly, at 1 mM IPTG concentration UVP-ALP3 cells survived, and optimum expression of alkaline phosphatase III was achieved. The recombinant enzymes were purified by passing the cytosolic fractions of PV1 and UVP-ALP3 clones through nickel metal chelate agarose column. The pure recombinant alp was analyzed in 10% SDS-PAGE, and kinetics of purified recombinant proteins were determined as described earlier13^,31^,32.

In vitro regulation of IDH

Phosphorylation of IDH with PknB was performed in the assay mixture containing 0.1 M Tris-HCl, pH 7.5, 0.1 M ATP, 1 µg/ml pure IDH and 3 µg/ml pure PknB, and incubated at 30 °C for 10 min. The phosphorylated IDH was purified by passing through Sephadex G-25 column (GE Healthcare Bio-Sciences, Pittsburgh, PA) (1 cm× 15 cm), the fractions were eluted with 0.1 M Tris-HCl, pH 7.5 and 150 mM NaCl. The enzyme fractions appeared in the void volume, the bound phosphorous was estimated by adding freshly prepared reagent A and incubated at 30 °C for 15 min, and the absorbance was read at 820 nm against blank (0.1 M Tris-HCl, pH 7.5, 150 mM NaCl and reagent A). The phosphorylated IDH was used to carry out the enzyme assay as described earlier. Similarly, the phosphorylated IDH was incubated with pure alkaline phosphatase III enzyme, and this dephosphorylated IDH was used for enzyme assay13.

Results

Cloning, sequencing and expression of ALP gene

In the present study, all phosphatase-producing colonies of S. aureus ATCC12600 exhibited pink colour, and from this culture the chromosomal DNA was extracted and used to amplify SAOV 2675 gene. The 1.425 kb PCR product was sequenced (JN645811) and the sequence when BLAST searched showed complete homology with (SAOV 2675) alkaline phosphatase III present in all the strains of S. aureus. This gene was cloned in the SmaI site of pQE 30 in “-1” frame and transformed into E. coli DH5α, the resultant clone UVP-ALP3. The expression of alkaline phosphatase III was induced with 1 mM IPTG, and the recombinant alp3 was purified from the cytosolic fraction of UVP-ALP3 by passing through Nickel metal chelate column. The enzyme kinetics of recombinant protein showed V_max 4.703 ± 0.62 µM/mg/ml/min, K_m 7.1 ± 0.2 mM and exhibited single band in SDS-PAGE with molecular weight of 52 kDa (Figure 1; Table 1). The annotated protein sequence of S. aureus (SAOV 2675) when scanned in PROSITE showed one active site that contains following amino acids 104I, 105T, 106D, 107S, 108A, 109A, 110G, 111G and 112T which are also conserved in human alkaline phosphatase (halp) (PDB ID: 2GLQ). Multiple sequence alignment using Clustal X showed very low-sequence identity with human and other bacterial alkaline phosphatase (Figure 2).

Figure 1. (A) Lane 1 PCR amplification of PknB gene (2.0 kb) from the chromosomal DNA of S. aureus ATCC12600, Lane M supermix DNA ladder obtained from Merck Bioscience Pvt. Ltd. (B) SDS-PAGE (10%) analysis of rPknB expression. Lane 1 cytosolic fraction of IPTG-induced PV1 clone, Lane 2 Nickel metal agarose column purified PknB and Lane 3 cytosolic fraction of uninduced PV1 clone and Lane M molecular size marker obtained from Merck Bioscience Pvt. Ltd. (C) Separation of pure phosphorylated PknB identified by reagent-A. L1: Pure phosphorylated PknB. L2 & L3: phosphorylated stpks. (D) Lanes 2 and 3 PCR amplification of alp gene (1.425 kb) from the chromosomal DNA of S. aureus ATCC12600, L1 supermix DNA ladder obtained from Merck Bioscience Pvt. Ltd. (E) SDS-PAGE (10%) analysis of rALP-III expression. Lane 1 uninduced cell lysate of UVPALP-3 clone, L2 IPTG-induced cell lysate of UVP-ALP3 clone and L3 pure rUVP-ALP3 eluted from nickel metal chelate agarose chromatographic column, and Lane M molecular size marker obtained from Merck Bioscience Pvt. Ltd. (F) Separation of pure phospharylated IDH identified by reagent-A. L1: Pure IDH. Lane M: Marker. L2 & L3: phosphorylated IDH.

Figure 2. (A) Multiple sequence alignment of ALP sequence with other bacterial ALP. (B) Multiple sequence alignment of PknB sequence with other bacterial PknB.

Cloning, sequencing and expression of PknB gene

The gene encoding PknB (2.0 kb) was amplified from S. aureus ATCC 12600 chromosomal DNA and sequenced, and the sequence showed complete homology with PknB of several S. aureus strains in the reported databases. The sequence analysis of PknB enzyme consisting of catalytic domain was distributed between 10th and 267th residues which contain 12 specific Hanks motifs, they contained both ATP and substrate-binding regions which are also conserved in eukaryotic protein kinases. Transmembrane domain was followed by three different duplicated forms of PASTA domains: PASTA1 distributed between 377th and 440th residues, PASTA2 distributed between 445th and 508th residues, PASTA3 distributed between 514th and 577th residues in the annotated protein sequence of PknB gene, and between these two domains is a single transmembrane segment. These unique characters are the features exhibited by a several PknB enzymes expressed in different strains of S. aureus (Figure 2).

The PknB gene was cloned in pQE 30 vector in “-1” frame and the cloned named as PV 1. The insert in the clone was confirmed by sequencing (JN695616); the PknB gene was expressed with 0.75 mM IPTG and purified by passing through nickel metal chelate column chromatography. The molecular weight of the pure rPknB was found to be 73 kDa, which corresponds to the inserted cloned and is equivalent to the monomeric form of PknB protein (Figure 1). The enzyme kinetics of PknB is shown in Table 2. IDH of S. aureus ATCC 12600 was expressed from UVIDH 1 clone12, and the kinetics of IDH are shown in Table 3. The rate of S. aureus biofilm formation was higher in BHI broth compared to LB broth with decreased IDH activity (Table 3).

Table 2. Enzyme kinetics of rPknB and rALP III.

Source of enzyme	Protein concentration (µg/µl)	Enzyme activity (µM/ml/min)	Km (mM)	Vmax (µM/mg/min)
rPknB (Substrate level)	4	0.162 ± 0.08	0.720 ± 0.08	1.98 ± 0.2
rPknB (Auto phosphorylation)	4	0.316 ± 0.090	0.380 ± 0.07	8.0 ± 0.43
rALP III	3.89	0.203 ± 0.013	4.703 ± 0.619	7.1 ± 0.96

Values are the mean ± SD from three determinations.

Table 3. Enzyme kinetics of S. aureus IDH grown in LB and BHI media.

	Aerobic media (LB)				Anaerobic media (BHI)
Source of enzyme	Vmax (µM/mg/min)	Km (µm)	Kcat	BU	Vmax (µM/mg/min)	Km (µm)	Kcat	BU
IDH in the cytosolic Fraction  of S. aureus ATCC12600	751 ± 58.18	8.2 ± 0.28	3.75 ± 0.06	0.075 ± 0.045	550 ± 38.18	8.2 ± 0.28	2.75 ± 0.07	0.091 ± 0.06

Values are the mean ± SD from three determinations.

Regulation of IDH by phosphorylation and dephosphorylation

The deduced amino acid sequence of IDH showed the presence of PknB site in the active site of enzyme where NADP binds and regulates the catalysis process (Figure 3), IDH was phosphorylated with PknB and the bound phosphate was identified by its ability to react with reagent A, which was identified spectrophotometrically. On fractionating these phosphorylated enzymes in 10% SDS-PAGE and immersing in reagent A, appearance of blue-coloured bands indicated IDH was phosphorylated (Figure 1). The mobility of phosphorylated IDH was lower than the native pure IDH (Figure 1). The phosphorylated IDH lost its activity completely, and the activity was restored on treatment with ALP III (Table 4). Thus, it is clear that in S. aureus IDH is regulated by phosphorylation and dephosphorylation.

Figure 3. Effect of phosphorylation and dephosphorylation in S. aureus biofilm formation.

Table 4. IDH regulation by phosphorylation and dephosphorylation.

IDH regulation	Enzyme activity units
Pure rIDH	23.5 ± 0.28
IDH-P by PknB	Inactive
Dephosphorylation by ALP	20.4 ± 0.24

Values are the mean ± SD from three determinations.

Discussion

Phosphorylation and dephosphorylation of proteins are the most conserved biochemical process used for intercellular and intracellular communication in both prokaryotes and eukaryotes33. This plays crucial role in the regulation of various metabolic processes in the organism as well as expression of various virulence factors and by passing the host immune system, thus helping the pathogen in colonization in any anatomical locales in human host. This condition is further aided by the presence of secretary PknB and alkaline phosphatase21–23.

In S. aureus cell wall, exopolysaccharide and PIA molecule biosynthesis are primarily regulated by PknB. In anaerobic conditions, increased PknB and PIA expression downregulates TCA cycle and purine biosynthesis in S. aureus, wherein it facilitates rapid biofilm formation. The results of the present study too concur with low-IDH activity and increased biofilm formation in anaerobic conditions (Table 3). Such features are commonly observed in all multi-drug resistant strains of S. aureus which also show high resistance to vancomycin8^,11^,14–17.

In the present study we have cloned, sequenced, expressed and characterized PknB and alkaline phosphatase III (SAOV 2675) genes of S. aureus ATCC12600. The PknB sequence showed well-conserved PASTA domains which sense environmental signals by triggering eukaryote-like N-terminal catalytic domain to autophosphorylate and hence activate different downstream signalling cascades which help to regulate physiology of the organism11^,15–17^,21^,34^,37. The results of the current study also explain unambiguously how IDH become completely inactive on phosphorylation with PknB and are restored upon dephosphorylation with alp III (Table 4). This shift in phosphorylation and dephosphorylation plays vital role in maintaining the redox status in S. aureus, which in turn has profound effect in the formation of biofilms11^,15–17^,35^,36^,38^,39 (Figure 3).

Conclusion

In S. aureus, high-reductive condition coupled with increased expression of PknB is one of the prime reasons for increased rate of biofilm formation. In this study IDH enzyme expression was assessed in S. aureus ATCC12600 grown in anaerobic and aerobic media, which revealed decreased IDH activity and increased biofilm formation correlated with complete loss of IDH activity on phosphorylation with PknB. Thus, explaining in reductive conditions, biosynthesis phase is higher than energy generation leading to the increased cell wall, PIA and exopolysaccharide synthesis which facilitated increased rate of biofilm formation, which is one of the key pathogenic factors of S. aureus.

Declaration of interest

Authors report no conflict of interest. We sincerely acknowledge Sri Venkateswara Institute of Medical Sciences University for providing funds and facilities under SBAVP scheme (SBAVP/Ph. D/02) to carry out this work, and this paper is a part of Ph.D. work going to be submitted to SVIMS University.