Purification and characterization of Cyclophilin: a protein associated with protein folding in Salmonella Typhimurium

Salmonella Typhimurium (ST) is a Gram-negative zoonotic pathogenic bacterium that causes infectious disease in humans as well as in animals. It causes foodborne diarrheal or gastrointestinal illness and fever called salmonellosis, which is a leading cause of millions of deaths worldwide. Salmonellaenterica serovar Typhimurium (S. Typhimurium) during its pathogenesis take away the actin cytoskeleton of their host cells and this is the crucial step of its infection cycle. Cyclophilin A, a type of peptidyl-prolyl isomerase that’s encoded by the ppiA gene in ST, plays pleiotropic roles in maintaining bacterial physiology. In this investigation, the proteomic characterization of the peptidyl-prolyl cis–trans isomerase- A (Cyclophilin A) from Salmonella Typhimurium is reported. Cyclophilin A (CypA) protein from Salmonella Typhimurium proved to be highly conserved and homologous protein sequence compared to other organisms. This protein was expressed in Escherichia coli followed by its purification in a recombinant form protein exhibited a characteristic PPIases activity (Vmax = 0.8752 ± 0.13892 µmoles/min, Km = 0.9315 ± 0.5670 µM) in comparison to control. The mass spectrometry analysis of Cyp A protein-peptide showed a highest sequence similarity with the cyclophilin protein of Salmonella. PPIases proteins (enzyme) data suggest that Ppi-A has roles in the protein folding that may be contributing to the virulence of Salmonella by isomerization of protein outline. These results suggest an active and vital role of this protein in protein folding along with regulation in Salmonella Typhimurium.


Introduction
Salmonella enterica serovar Typhimurium (ST) is one of the most common pathogenic Gram-negative, non-typhoidal bacteria causing foodborne diarrheal and salmonellosis disease in humans. Salmonella Typhimurium is one of the widespread causative agents of human salmonellosis in the world and is among the top five most detected pathogens for each major food from animal origin (Rabsch et al. 2001). As per CDC zoonotic diseases database, human salmonellosis causing by Salmonella Typhimurium is one of the most frequent disease worldwide (Sabirovic et al. 2010). The global burden of foodborne Salmonella estimated around 93.8 million cases reported worldwide, out of which the disease claims 0.15 million lives (Majowicz et al. 2010). The common virulence strategy of the Salmonella genus is adhesion, invasion, intracellular replication and bacterial dissemination from the intestinal cells of the host. The ability of bacteria to sense and respond to unfavourable changes 1 3 in the host environment is important for their survival and infection (Foster and Spector 1995).
Peptidyl-prolyl isomerase (PPIases) a family of evolutionarily conserved enzymes, initially discovered in eukaryotes, catalyzes a diverse process required for protein folding at proline residues, which accelerates the rate of exchange between cis and trans isomers (Fischer et al. 1998). The catalysis of the cis-trans isomerization of peptidyl-prolyl bonds facilitated by the PPIases enzyme (Lang et al. 1987). PPIase includes four different families distinct in their amino acid sequence based on drug resistance and homology. These are cyclosporin A (CsA)-binding proteins, cyclophilins, the FK506 and rapamycin binding proteins, FKBPs, parvulins which do not bind immunosuppressant drugs (Fischer et al. 1998, Schiene-Fischer et al. 2013. Members of the cyclophilin family are also called immunophilins because of their involvement in the intermediate effects of immunosuppressive drugs, Cyclosporine A (Hamilton and Steiner 1998).
Peptidyl-prolyl isomerase protein is ubiquitously distributed in almost all living organisms, from bacteria, plants to humans. Bacterial PPIases have homologs with membraneassociated lipoproteins which involved in post-translocation secretion, protein folding and stability (Sarvas et al. 2004, Unal andSteinert 2014). Salmonella Typhimurium has identified peptidyl-prolyl cis-tans isomerase called "rotamase" a homolog of human cyclophilins. Protein Cyclophilin A (CypA), widespread with higher abundance in the cytoplasm (Dimou et al. 2017), is a secretary protein that belongs to a family of evolutionary protein preserved cis-trans isomerases (PPIases) that catalyze protein folding at prolyl amino acid and cellular trafficking (Nigro et al. 2013). The PPIases, especially Cyclophilin, are highly abundant proteins, while Cyclophilin account for about 0.4% of the dry mass of cellular proteins. Cyclophilin has been classified as an immunophilin and has a variety of intracellular functions, including intracellular signalling, protein trafficking, and the regulation of other proteins activity (Koletsky et al. 1986). Cyclophilin shows enzymatic activity required for optimal protein folding and also plays a vital role in multiple bacterial systems like the stress response, infectivity and virulence factors. Cyclophilin binds with high affinity to the clinically important immunosuppressive agent cyclosporin A (CsA) (Moochhala and Renton 1986).
These PPIase play an important role in protein conformation, which occurs during biological conditions like refolding of denatured proteins (Schiene-Fischer and Yu 2001). PPIase enzymes were initially thought to be limited to folding proteins only, but recent research established their role in other biological functions like signal transduction (Walsh et al. 1992), intracellular trafficking (Wintermeyer et al. 1995), gene transcription (Hanes 2015), cell cycle regulation (Baum et al. 2009), refolding of aggregated proteins (Zhang et al. 2013), regulation of reactive oxygen species (ROS) (Linard et al. 2009), apoptosis (Ding and Nam Ong 2003), proliferation and transformation (Zhu et al. 2015) and function as a molecular timer (Lu et al. 2007). Expression of Cyclophilin has been shown to be induced in the various stresses like heat shock, low temperature, salt stress, and pathogens (Chou and Gasser 1997).
In the bacterial membrane envelope, PPIases also play various essential biological functions. In Salmonella, SurA protein (PPIases type protein), found in the outer membrane, plays a key role in maintenance of the maturation of outer membrane proteins and resistance to various stress agents (like ROS and RNS), as this protein sustains the outer membrane protein biogenesis and assembly (Behrens-Kneip 2010). Other protein likeSurA from E. coli, Shigella flexneri, and Salmonella Typhimurium also helps in cell adhesion and invasion (Sydenham et al. 2000). In E. coli, PpiB also controls cell division by modulating the function of various proteins which are directly or indirectly associated with the cell division (Skagia et al. 2017). In this study, the role of Peptidyl-prolyl cis-trans isomerase-A type of cyclosporin is explored. Enzyme activity of the expressed and purified recombinant STPpiA was analyzed and identity was confirmed by the mass spectrometry sequencing. Finally, it was demonstrated that STPpiA is an active type protein that illustrates the function of PPIases, which could be a regulator to facilitate the survival and virulence factors of bacteria. Furthermore, the exact biological role and effects of the STP-piA function in Salmonella systems remain a challenge for future studies. The findings of this study facilitated a better understanding of the pathogenesis of Salmonella Typhimurium and led to an elucidation of the different mediators of the pathogenic mechanism resulting in the Salmonella Typhimurium infection.

Bacterial strains and plasmids
The E. coli strain DH5α and T7 Express lys γ were used for cloning and protein expression purposes. E. coli strains were cultured in Luria broth (LB). Kanamycin (sigma) was added, where necessary, to LB medium at a concentration of 30 μg/mL.

Expression and purification of STPpiA proteins
The recombinant clone PpiA_pET28c construct and conformation was done by double digestion and PCR amplification of STPpiA from the recombinant clone PpiA_pET28c plasmid. The recombinant PpiA_pET28c construct was transformed into competent E. coli T7 Express lys cells. Finally, 50 μl of the re-suspended pellets were plated on kanamycin (30 μg/ml) plus chloramphenicol (10 μg/ml) containing LB agar plate and incubated at 37 °C overnight.
The isolated colonies were inoculated and grown overnight in LB broth containing antibiotics at 37 °C at 180 rpm. The cultures were diluted (1:100) in fresh media and grew at 37 °C, 180 rpm (~ 3½ hrs) to an OD 600 of 0.5. About one ml of this culture was removed in a 1.5 ml tube as an uninduced culture. The remaining culture was induced using isopropyl-β-D-thiogalactopyranoside (IPTG) final concentration 1.0 mM. Both un-induced and induced cultures were incubated at 30 °C, 180 rpm overnight, the cells were harvested by centrifugation 7000 × g at 4 ºC, and the pellet was stored at −80 ºC.
The recombinant protein STPpiA was purified using the Ni-NTA affinity chromatography (Qiagen). The bacterial pellet was thawed on ice and suspended in a 10 ml chilled lysis buffer containing 50 mM sodium phosphate pH 8.0, 150 mM sodium chloride and 10 mM final imidazole concentrations. Lysozyme at a final concentration of 1 mg/ml was added to the suspension and incubated on ice for 30 min. The cells were also lysed by 15 cycles of sonication in ice. Each cycle has a 45 s pulse of amplitude 48 Hz and a 45 s gap before the next pulse. PMSF (1 mg/ml final concentration) as protease inhibitor was added to the cell suspension.
The lysate was centrifuged (10,000 g, 4 °C, 15 min) and the supernatant was filtered through a 0.45 μm syringe filter. The Ni-NTA column was equilibrated with 20 volumes of lysis buffer. The cell-free supernatant obtained by centrifugation was loaded in the Ni-NTA column. The unbound proteins from column were removed by washing with 50 mM sodium phosphate (pH 8.0), 150 mM sodium chloride and 40 mM imidazole. The protein bound to the Ni-NTA column was eluted with an elution buffer containing 50 mM sodium phosphate (pH 8.0), 150 mM sodium chloride and 200 mM imidazole. The fractions were analysed by spectrophotometer at 280 nm, and high absorbance peak fractions were pooled and dialyzed against 50 mM sodium phosphate (pH 7.5) buffer at 4 °C. The purity of eluted fractions was then analysed by SDS-PAGE and total protein was estimated by Bradford's total protein assay method using the BSA as standard. Finally obtained protein samples were stored at -80 °C for further experiments.

Western blotting of recombinant proteins
After the purification, STPpiA protein was resolved on SDS-PAGE followed by transferring onto polyvinylidene difluoride (PVDF) (0.2 µM) membrane (200 mA for 2 h). The PVDF membrane was blocked with 5% skimmed milk in PBST (w/v) overnight at 4 ºC. After that the membrane was washed with PBS and 0.05% Tween 20 (v/v) solution. After washing with PBST, the membrane was incubated with a 1:1000 dilution mouse anti-His antibody at 37 ºC for 3 h. After washing, the blot was incubated with 1:15,000 alkaline phosphatase-conjugated anti-rabbit IgG at 37 ºC for 2 h, which acted as the secondary antibody. The blotted membrane was developed using enzyme substrates like 5-bromo-4-chloro-3'-indolyphosphate and nitro-blue tetrazolium chloride.

Identification of proteins by mass spectrometry
The purified STPpiA protein was resolved on SDS-PAGE and was stained with CBB stain. After destining with 10% methanol and 10% acetic acid solution, the protein band was cut carefully and digested by trypsin followed by LC-MS, MS/MS was done to record mass spectrum (MS). The mass spectrometric data were analysed manually and with the

PPIases enzyme activity by recombinant proteins
In the present study, various assays were devised to measure PPIases activity based on Fisher 1990 (Fischer and Schmid 1990). The assay is based on the little difference in absorbance determined for the cis and trans isomers of Suc-Ala-Xaa-Pro-Phe-4-nitroanilide.To estimate enzyme activitywe use substrate concentration from 0.25 to 10 µM N-succinylala-ala-pro-phe-p-nitroanilidine as test peptide, assay buffer [50 mM HEPES (pH 7.8) and 150 mM NaCl prechilled] and 300 µg of the recombinant PPIase protein to made the final volume to 1 ml. The reaction was initiated by the addition of chymotrypsin (500 µg/ml) and the change in absorbance at 390 nm was monitored using a spectrophotometer (Perkin-ElmerLambda Bio20) for 3 min at 25 °C. The enzyme kinetic analysis and the relation between velocity (v) substrate concentrations (S) were done through the Michaelis-Menten equation.

Purification of recombinant proteins STPpiA
The expression of recombinant protein STPpiA was done using the transformed PpiA_pET28c plasmid into the T7 Express lys γ strain of E. coli. The induction of the recombinant STPpiA protein was confirmed by SDS-PAGE followed by CBB R-250 staining as shown in Fig. 1A. The full-length recombinant STPpiA protein was purified from the PpiA_pET28 containing E. coli culture using affinity chromatography by using nickel-NTA resin. A highly purified preparation of STPpiA was recovered by eluting the column with with 500 mM imidazole. The recombinant STPpiA protein had an apparent size of ~ 24 kDa on SDS-PAGE gel (Fig. 1A), reacted with anti-His antibody and showed ~ 24 kDa on western blot (Fig. 1B). A single band with good intensity was observed on the membrane suggesting the recombinant STPpiA protein with Histidine-tag. The size of the detected band was ~ 24 kDa with a Histidine Tag (~ 3 kDa), which predicted the molecular weight for STPpiA protein as ~ 21 kDa.

Identification of proteins by mass spectrometry
After the molecular weight confirmation by SDS-PAGE and Western blot analysis, the purified recombinant STPpiA was verified by using proteomic analysis with liquid chromatography-mass spectrometry. The full mass spectrum, as shown in Fig. 2a and peaks were marked with corresponding peaks of STPpiA peptides (Fig. 2a, b). The LC-MS and MS/MS were subjected to the MASCOT database search and identified the target protein band with a higher significance score. The result showed the identification of some Cyclophilin A-type PPIases isoforms. The peptide sequence coverage observed was more than 41%. Detailed peptide coverage information is provided in bold (Fig. 3). The LC-MS analyse strongly suggests the presence of recombinant STPpiA protein. These peptides matched the original sequence from NCBI, authenticating the protein identification. (Fig. 3a).
Perfectly matching LCMS data of peptides with NCBI sequence also suggest the appropriate identification of the protein (Fig. 3b).

Enzymatic activity of the purified STPpiA protein
After The purified STPpiA protein exhibited PPIases enzymatic activity with V m = 0.8752 ± 0.13892 µmoles/min, K m = 0.9315 ± 0.5670 µMin comparison to control (Fig. 4). The purified protein found to be a first-order rate constant, and the presence of purified STPpiA showed an increase with an increase in the protein concentration (Table 1), thus, implying that the STPpiA specifically contributed to the observed PPIases activity.

Discussion
Salmonella Typhimurium is one of the major serovars commonly isolated from the human gastrointestinal tract that causes gastrointestinal infection. The PPIases proteins are associated with a broad range of pathogenic bacteria. The initial function known to cyclophilin was participation in protein folding only. Among the PPIases, CypA appears to play a pivotal role in the proper protein folding in many 1 3 biological conditions. Studies showed that CypA expression to several cellular processes that require action polymerization and cytoskeletal remodelling (Calhoun 2008, Saleh et al. 2016. Some research study also showed that Cyclophilin protein plays a role as an antifungal, antiviral, and antioxidant agents (Wong et al. 2010). The recombinants from of Cyclophilin from Pyropiayezoensis express and purified in E. coli showedcell proliferation (Jung et al. 2019). Some studies showed that PPIases are play vital role for proteins function/folding in bacterial/viral infection and diseases (Theuerkorn et al. 2011, Unal and Steinert 2014, Zhang et al. 2014). In the pathogenic Brucella cyclophilins are also playing a vital role in virulence and survival in the host cells (Roset et al. 2013). PPIases also play an important role in the secretion of virulence factor. In E. coli are combined protein Cyclophilin from Trypanosoma cruzi expressed and the purified recombinant protein exhibited PPIases activity (Bua et al. 2001). Some bacterial cyclophilins, also play important chaperone activity in different environmental conditions (Dimou et al. 2011, Pandey et al. 2016 which are disposing of independent cis-trans activity.
In this study, we have characterized Cyclophilin A protein from Salmonella that showed high protein peptides sequence homology to CyPs from other organisms and demonstrated the PPIase activity of recombinant protein exhibited by artificial enzyme substrate in vitro. Earlier finding on bacterial cyclophilins showed cypA gene have PPIase as well as chaperone activity and its role in many biological processes, like cellular signal transduction, adaptation to stress, control of pathogens virulence, and modulation of host immune response during the infection (Dimou et al. 2017). Our previous finding has showed that Salmonella Typhimurium cyp-A gene also have high sequence similarities with other virulence bacteria (Manoj et al. 2016, Kumawat et al. 2020.
Another study showed that ppiA gene that is highly conserved among all Salmonella genomes, has a critical role in the growth of Salmonella Typhimurium in the examined stress conditions, and may play a role in its responses and virulence (Kumawat et al. 2020). Thus, this study will help to understand the importance of Cyclophilin protein in Salmonella Typhimurium which may Be involved in various biological activities. This will lead to know how Salmonella regulates virulence factors. Some previous study has confirmed that cyclophilin A protein plays a role in modulating virulence of bacterial species resulting in attenuation in vivo (Wang et al. 2001, Roset et al. 2013, Dimou et al. 2017, Bzdyl et al. 2019. However biological functions of Salmonella CyPA, their natural substrate and their role in host-parasite relationships are still unclear. PPIases proteins enzyme data suggest that Ppi-A has roles in the protein folding that may be contributing to the virulence of Salmonella by isomerization of protein outline. Peptidyl-prolyl cis-trans isomerisation showed more specificity for their protein targets than other chaperones, the purposeful importance of the interplay isn't always frequently clear. In this study, we performed recombinant form of STPpiA protein expression and purified in vivo. The proteomic analysis of recombinant form STPpiA proteins showed the significant acceleration of peptidyl-prolyl cis-trans isomerisation, a rate-limiting step in protein folding. Possibly these PPIAase enable the survival of ST under uncomplimentary conditions. Other PPIases protein activity data suggest that PPIase plays role in the protein folding that may be contributing to the survival of Salmonella. In conclusion, The findings of the present study demonstrate that CypA is an active cellular protein which has the critical role in salmonella infection. Therefore, CypA may be a potential therapeutic target for salmonellosis. Furthermore, experimental studies are also required to assess how alterations in protein conformation mediated by PpiA affect cell, during infections caused by Salmonella, which in turn could prove beneficial for the development of potential therapeutic and prophylactic candidates against this infection.