Purification and Characterization of an ATPase GsiA from Salmonella enterica

The coding sequence of Salmonella enterica gsiA was cloned and expressed in E. coli. The protein was purified and ATPase activity was characterized by NADH oxidation method. GsiA exhibited optimum activity at 30°C and at pH 8 in Tris/HCl buffer. GsiA protein was stable at 20°C. 66% and 44% activity remained after incubation at 30°C and 40°C for 30 min. pH 7 and pH 9 incubation would obviously reduce the ATPase activity. In vivo functionality of gsiA was determined by constructing gene deletion strains. gsiA was shown to be essential for GSI mediated glutathione uptake and gsiA deletion could decrease the virulence of Salmonella enterica. Interactions of glutathione import proteins GsiA, GsiB, GsiC, and GsiD were investigated by using bacterial two-hybrid system. GsiA could interact with itself and inner membrane proteins GsiC and GsiD. This report provides the first description of gsiA functions in Salmonella enterica. The results could help elucidating the glutathione uptake mechanism and glutathione functions in bacteria.


Introduction
Glutathione ( -L-glutamyl-L-cysteinyl-glycine; GSH) is a vital intracellular cysteine containing antioxidant across all kingdoms of life. Glutathione usually attains mM concentrations in cells and assumes a plethora of cellular roles, such as control intracellular redox homeostasis, and protects against oxidative and xenobiotic stresses by glutathionylation [1,2]. Glutathione also functions in cell signaling [3] and salvage of cysteine [4]. Glutathione manifests inside the cell predominantly in the thiol-reduced form (GSH, >98%) [5]. The remaining amounts of glutathione are in the oxidation form: glutathione disulfide (GSSG) or disulfides with target proteins [6]. Compounding evidence indicates that glutathione import by yeasts and bacteria could serve as a supply of organic sulfur [7][8][9]. However, physiological importance of glutathione import still needs further investigation.
H + symporter [10], Na + -dependent transporter [11], and glutathione S conjugate exporters [12] play important roles in transport glutathione and its derivatives in and out of cells. However, the first bacterial glutathione importer (GSI) with an ATP binding cassette was identified in 2005 [7]. This importer consists of gsiA, gsiB, gsiC, and gsiD, encoding the ATP binding protein, the periplasmic glutathione binding protein, and two plasma membrane components, respectively [13].
Usually, the ATP binding components could hydrolyze ATP, providing energy for substrate transport [14]. Glutathione import in Escherichia coli is energy dependent which is supported by GsiA. The ATP binding subunits of transporters usually worked as a dimer [15]. The nucleotide binding pocket will open and close for ATP or ADP binding, which make the protein intimately related to translocation [16] by regulating ATPase activity. Whether GsiA serves as a dimer and how it regulates the glutathione import are still unclear.
Herein, the spectrum of expression, purification, and characterization of gsiA from Salmonella enterica, a bacterial pathogen responsible for enteritis and typhoid fever, was described. MBP (maltose binding protein) was employed as 2 BioMed Research International The substrate specificity of GsiA and its stability as function of temperature and pH were investigated. Studies of GsiA will help to clarify the mechanism and functions of glutathione uptake in bacteria. The strain was grown in Luria-Bertani (LB) medium containing ampicillin (100 g/mL) at 37 ∘ C. Protein expression was induced by addition of 0.1 mM IPTG at an OD 600 of 0.5-0.6 and grown at 20 ∘ C for 20 h. Cells were harvested by centrifugation at 5000 for 15 min at 4 ∘ C.

Purification of GsiA Recombinant
Protein in E. coli. The cell pellet was resuspended in 50 mM Tris/HCl pH 8, 100 mM NaCl, 15 mM imidazole, 1 mM phenylmethanesulfonyl fluoride (PMSF), 1 M lysozyme, 1 M DNase, and a protease inhibitor cocktail (Sigma, one tablet per 100 mL). The cell was disrupted by homogenizer FB-110X (LiTu, China) at 1000 MPa. The cell debris was removed by centrifugation at 10,000 for 15 min at 4 ∘ C. The supernatant was loaded onto Ni 2+ -NTA affinity column (GE Healthcare) and washed with 30 mM imidazole, 50 mM Tris/HCl pH 8, and 300 mM NaCl. GsiA-MBP was eluted with 300 mM imidazole, 50 mM Tris/HCl pH 8, and 300 mM NaCl. The imidazole was removed by using desalting column (GE Healthcare, buffer containing 50 mM Tris/HCl pH 8, 300 mM NaCl). The protein was digested with TEV protease and purified by MBP column. The flow through GsiA protein was further purified by a size exclusion column Superdex 200 (GE Healthcare) with 50 mM Tris/HCl pH 8, 300 mM NaCl, and 5% (v/v) glycerol. The purity of GsiA was analyzed on 12% (v/v) SDS-PAGE. GsiA protein conformations were analyzed on 12% (v/v) native gel. The pure GsiA protein was concentrated (30 kDa molecular weight cutoff tube) (Sartorius) to about 10 mg/mL, which was measured by Nanodrop 2000 (Thermo Scientific).

GsiA Interacts with Other Components.
The interaction of GsiA with other components of glutathione importer was analyzed by using GFP fragments reassembly protocol [18,19]. gsiA, gsiC, and gsiD genes were cloned into pET11alink-NGFP. gsiA, gsiB, gsiC, and gsiD genes were cloned into pMRBAD-link-CGFP. Recombinant plasmids carrying Nand C-fragment of GFP were simultaneously transformed into BL21(DE3) using 10 ng of each construct. The cells were plated on LB plates containing kanamycin (35 g/mL) and ampicillin (100 g/mL). Single colony harboring two plasmids was obtained and incubated overnight with shaking at 37 ∘ C. Fresh overnight culture was diluted (1 : 100) and 10 L medium was plated onto screening media, containing 10 M IPTG and 0.2% arabinose. The plates were incubated at 20 ∘ C for 2 days, which will give reproducible green-fluorescent colonies.

gsiA In Vivo Function Assay.
Glutathione is supposed to be imported into Gram-negative bacteria mainly through -glutamyltranspeptidase (GGT) or GSI method [7]. To characterize function of GisA, the gsiA and ggt gene deletion mutant of Salmonella enterica was constructed with Red recombination system [21,22]. The kanamycin fragment in pKD4 was amplified by using primers gsiA del-F and gsiA del-R ( Table 1). The PCR product with 58 bp upstream and 58 bp downstream homologous to adjacent regions of gsiA was digested with DpnI and gel-purified. pKD46 was transformed into Salmonella enterica by traditional CaCl 2 method. The cell was grown in SOB medium and grown at 30 ∘ C to an OD 600 of 0.5-0.6. 2 mM L-arabinose was added 1 h before cell collection. Competent cells were obtained by washing the pellet with ice-cold 10% glycerol. The cells were subjected to electroporation by MicroPulser (Bio-Rad) using a 0.1 cm chamber with 50 L of the competent cell and 50 ng of PCR product.
gsiA gene deletion was verified by PCR with primers gsiA-F1 and gsiA-R1. The gsiA deletion strain was made competent and ggt gene was deleted as above. The chloramphenicol fragment in pKD3 was amplified with primers ggt del-F and ggt del-R (Table 1), generating a product with 56 bp upstream and 58 bp downstream homologous to adjacent regions of ggt.
The cell growth and glutathione uptake curves of gsiA deleted Salmonella enterica strains were measured. M9 medium [23] was used as minimal medium with MgSO 4 replaced by MgCl 2 . Reduced glutathione (1 mM, ≥98%) was served as the only sulfur source. Plasmid pBAD24-gsiA was constructed (pBAD24 plasmid and gsiA primers F1 and R1 were used) and transformed into gsiA deleted strain to compensate this defection.
6-to 8-week-old male Kunming mice were employed in present study, which were provided by Experimental Animal

Expression and Purification of GsiA.
The coding sequence of the gsiA gene was amplified from Salmonella enterica subsp. enterica serovar Typhimurium LT2: STM0848 genome and cloned into pMAL-c2X derived plou3 vector. To facilitate protein purification, a 6×His tag was added before MBP and a TEV protease cleavage site was added between MBP and polylinker. The resultant plasmid was denominated by plou3-gsiA. The insertion of gsiA was confirmed by DNA sequencing. The expression conditions of GsiA-MBP in BL21(DE3) were optimized. 0.1 mM IPTG induced at 20 ∘ C for 20 h will give high productivity of soluble GsiA (Figure 1(a)). SDS-PAGE analysis showed that the molecular mass of GsiA was about 70 kDa, in accordance with prediction (Figure 1(c)).
GsiA-MBP fusion protein was firstly purified by Ni 2+ column. Incubation of GsiA-MBP with TEV protease could separate GsiA from MBP. After MBP and gel filtration column purification, the purity level of GsiA was in excess of 95% (Figure 1(c)). The purified GsiA was shown to have two conformations in gel filtration buffer (Figure 1(d)). The pure protein was concentrated to 12 mg/mL and used for ATPase activity assay. Approximately 1.6 mg of GsiA protein was obtained from 1 L bacteria culture.
Western blot was carried out to confirm the expression of GsiA. As there was a 6×His tag at the N terminal of the fusion protein, anti-His antibody could be used to detect the expression of GsiA-MBP (Figure 1(b)).

Characterization of ATPase
Activity of GsiA. The purified GsiA exhibited optimum activity at 30 ∘ C and at pH 8 in Tris/HCl buffer (Figure 2). The GsiA protein was stable at 20 ∘ C. 66% and 44% activity remained after incubation at 30 ∘ C and 40 ∘ C for 30 min, respectively (Figure 2(b)). The protein was pH sensitive. pH 7 and pH 9 incubation would obviously reduce the ATPase activity (Figure 2(d)). One enzyme unit was defined as the amount of protein converting 1 M ATP to ADP per min at pH 8 and 30 ∘ C. 1 M GsiA could transform 78.8 M ATP into ADP in 1 h (Figure 3). The virulence of Salmonella enterica was determined by recording the CFU counts of viable bacteria from livers and spleens.

Protein Interaction of GsiA with Other Components.
To determine the interaction of GsiA with other proteins of glutathione import system, gsiA, gsiC, and gsiD in pET11alink-NGFP and gsiA, gsiB, gsiC, and gsiD in pMRBADlink-CGFP were pairwise simultaneously transformed into BL21(DE3). 10 M IPTG and 0.2% arabinose were used to induce the protein expression, which make GFP reassembly possible. The assembled GFP usually showed fluorescence in vivo (Figure 4), especially when activated by UV light. Protein interaction assay showed that GsiA could interact with the trans-membrane proteins GsiC and GsiD ( Figure 4). The most interesting phenomenon was that GsiA showed interaction with itself. Native gel showed that GsiAcould oligomerize in gel filtration buffer. This might be explained that GsiA worked as a dimer (Figure 1(d)), like most of ATP binding proteins in ABC cassette superfamily. GsiA did not interact with GsiB. It might be because of their different cell location. GsiA and GsiB were predicted to be located in the periplasm and cytoplasm of cell, respectively [13].

gsiA gene Was Essential for GSI Mediated Glutathione
Import. The gsiA and ggt gene were replaced by kanamycin and chloramphenicol resistant gene, respectively. The resulting Salmonella enterica strain was named ΔgsiA and ΔgsiAΔggt. The gene deletion was verified by PCR (data not shown). M9 medium was used to cultivate the mutant with glutathione as the sole sulfur source. The cell growth and glutathione uptake curves of wild type and mutant strains were measured. Compared with cultivation in LB broth, wild type strain grown in glutathione containing M9 medium was not affected, showing that glutathione could serve as sulfur source ( Figure 5(a)). The growth of ΔgsiA was also not affected. However, ΔgsiAΔggt strain which grew in glutathione containing M9 medium was slower than in LB medium. While pBAD24 empty vector had no effect on cell growth, pBAD24-gsiA transformed ΔgsiAΔggt strain could completely compensate this defection ( Figure 5(a)). The glutathione uptake in ΔgsiAΔggt strain was undetectable ( Figure 6). gsiA gene deletion could affect the glutathione uptake, which was compensated by pBAD24-gsiA. The results showed that GsiA was essential for GSI mediated glutathione uptake. GGT might mediate more glutathione uptake than GSI system.

Discussion
Glutathione is the most important antioxidant in cell and plays a plethora of cellular roles. Salmonella enterica can synthesize glutathione. However, there is a glutathione import like system in Salmonella enterica, which has never been investigated. GsiA is the ATP binding protein of glutathione importer. Putting deep insights into GsiA will help to elucidate the mechanism of glutathione import. plou3 is derived from pMAL-c2X and used as expression vector. MBP fusion can assist protein folding and be purified specifically by MBP column. MBP used here can promote soluble expression of GsiA and make GsiA purification more convenient.
Lower IPTG concentrations and inducing temperature could contribute to the yield of soluble protein. When The Ni 2+ -NTA column was efficient at enriching the His-tagged GsiA-MBP, comprising more than 90% of total proteins. By skillfully using tags and columns, the purification of proteins could be convenient and efficient. Achievement of sufficient GsiA made future biochemical and biophysical studies possible. The ATPase activity of GsiA was measured by recording NADH oxidation. Characterization of GsiA would facilitate future determination of active sites.
To determine the in vivo function of gisA, the gene was deleted in Salmonella enterica. The growth condition of ΔgsiA was not affected when glutathione was used as sole sulfur source. This might be because GGT could hydrolyze glutathione to liberate glutamic acid and cysteinylglycine [25]. Cysteinylglycine was then taken up into cytoplasm and cleaved into cysteine and glycine by aminopeptidases A, B, and N and dipeptidase D, which could be utilized as a source of cysteine and glycine. However, the growth of ΔgsiAΔggt strain was affected when glutathione served as sole sulfur source. The glutathione uptake in ΔgsiAΔggt strain was undetectable. The defects could be compensated by pBAD24-gsiA. The results showed that GsiA was essential for glutathione import by GSI.
It is surprising that compared with wild type strain the CFU counts of viable bacteria of ΔgsiA and ΔgsiAΔggt in livers and spleens both undergo pronounced decrease. This might be related to the virulence of Salmonella enterica. Considering the functions in other pathogens [3], glutathione here was supposed to work as a signal involved in strain invasion. The pathogen might sense the host environment by importing glutathione. As Salmonella enterica could synthesize glutathione, it was supposed that the glutathione concentration might be used as the signal.
Collectively, an ATPase GsiA from Salmonella enterica was identified and characterized for the first time. Investigation of protein interaction and biological functions of GsiA might help to elucidate the glutathione import mechanism. Glutathione import involved in the virulence of Salmonella enterica was determined for the first time. The mechanism by which glutathione was imported and how glutathione promotes virulence of Salmonella enterica still need further investigation.