Cytochrome P450 monooxygenase of Acanthamoeba castellanii participates in resistance to polyhexamethylene biguanide treatment

Acanthamoeba spp. are free-living parasites that can cause severe infections such as granulomatous amoebic encephalitis (GAE) and amoebic keratitis (AK). Polyhexamethylene biguanide (PHMB) is a topical application for AK treatment. However, PHMB is not entirely effective against all Acanthamoeba strains or isolates. The mechanisms by which Acanthamoeba protects itself against extreme drug conditions without encystation are still unknown. According to a previous study, cytochrome P450 monooxygenase (CYP450MO) plays an important role in the oxidative biotransformation of numerous drugs related to metabolism. In this study, a CYP450MO fragment was inserted into the pGAPDH-EGFP vector and transfected into Acanthamoeba castellanii. We found that CYP450MO-overexpressing Acanthamoeba had higher survival rates than those of the control cells after PHMB treatment. Moreover, we also found that encystation-related genes such as cellulose synthase I (CSI), encystation-mediating serine proteinase (EMSP), and autophagy-related protein 8 (ATG8) expression levels were not significantly different between Acanthamoeba transfected by pGAPDH-EGFP or pGAPDH-EGFP-CYP450MO. We suggest that Acanthamoeba transfected by pGAPDH-EGFP-CYP450MO may not induce encystation-related genes to resist PHMB treatment. In conclusion, these findings indicate that CYP450MO may be an additional target when PHMB is used for treatment of amoebic keratitis.


Introduction
Acanthamoeba spp. are free-living pathogenic protozoa that are distributed in several environments, including swimming lakes, pools, soil, and dust [6]. Acanthamoeba spp. cause severe sight-threatening infections such as granulomatous amoebic encephalitis (GAE) and amoebic keratitis (AK) [25,37]. AK has been increasing with contact lens misuse over the past two decades [1,4,6,7]. Acanthamoeba infects patients by causing lid edema, photophobia, epithelial defects, and ring-like stromal infiltrates through injury to the cornea [20,24]. Patients with AK have been treated effectively over the last two decades with topical biguanides; however, current therapy requires surgical intervention because of the failure of medical treatment [15]. Polyhexamethylene biguanide (PHMB) is a polymeric biguanide used as a disinfectant and antiseptic for patients with AK [19,22]. PHMB is effective against Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Candida albicans, and Aspergillus brasiliensis [2,13,26,38,39]. PHMB contains highly charged positive molecules that bind to the phospholipid bilayer of the cell membrane, which is negatively charged, causing penetration, damage, cell lysis, and death of the pathogens [21]. A previous study showed that 0.01% PHMB could not induce obvious corneal toxicity but lysed Acanthamoeba after treatment in vitro [10,22]. Combined AK treatment with propamidine, neomycin, and PHMB reduced pain in all patients within 2-4 weeks [36]. PHMB combined with H 2 O 2 is also used as an ingredient in contact lens-cleaning solutions to prevent corneal infections [30]. Corneal transplantation is another therapeutic approach when topical treatment fails. Nevertheless, corneal transplantation does not eliminate all trophozoites or cysts that can grow in the new cornea. Hence, there are no clinical therapeutic approaches recommended for incorporation into standard practice.
Cytochrome P450 enzymes (CYP450s) involved in drug metabolism are widely identified in different organisms ranging from protozoa to mammals [9,32,40]. CYP450s bind and activate two atoms of oxygen from substrates such as peroxide, and lead to hydroxylation [3]. CYP450s also depend on monooxygenase activity, catalyzing the oxidation of endogenous and exogenous substrates, and thereby cause drug degradation [35]. The metabolism of drugs by CYP450s contributes to the formation of products that are less toxic and are excreted easily into cells. Plasmodium berghei and Plasmodium falciparum can induce CYP450s to exhibit resistance to chloroquine treatment [28]. However, clinical isolates of Acanthamoeba with high resistance to PHMB are associated with serious health consequences in Taiwan [10]. Therefore, cytochrome P450 monooxygenase (CYP450MO) may play an important role in the oxidative biotransformation of numerous drugs during drug metabolism in Acanthamoeba. In this study, we overexpressed CYP450MO in Acanthamoeba to investigate its effects. CYP450MO-overexpressing Acanthamoeba had higher survival rates than those of the control cells after PHMB treatment. We suggest that CYP450MO in Acanthamoeba may catalyze PHMB drug metabolism to enhance survival rates after PHMB treatment. In conclusion, these findings may help to develop potential treatments for AK patients.
Total RNA isolation and cDNA synthesis A total RNA Extraction Miniprep System (Viogene, Taiwan) was used to isolate RNA. The total concentration and A260/A280 ratio of mRNA were measured using ND-1000 (NanoDrop, Thermo Fisher Scientific, USA). High-capacity cDNA Reverse Transcription kits (Thermo Fisher Scientific) were used in this study. The reverse transcription conditions were set at the following times and temperatures: 25°C for 10 min, 37°C for 120 min, and 85°C for 5 min; finally, the cDNA was kept at 4°C. The reaction volume was 20 lL.

Cloning of cytochrome P450 monooxygenase
Two different protocols were used to clone the CYP450MO using two vectors: the pJET1.2/blunt cloning vector and pGAPDH-EGFP vector [5]. To confirm mRNA sequencing, the amplified CYP450MO was converted to blunt-ended using Pfu S + DNA polymerase and then ligated with the pJET 1.2/blunt cloning vector. The CYP450MO sequence was amplified by PCR using the ATCC_30010 cellular cDNA as the template. To amplify the cDNA encoding CYP450MO, forward CYP450MO _F (5 0 -ATG CTG TGG TCG CTG ATT GTT GCG G -3 0 ) and reverse CYP450MO _R (5 0 -GGG CAG TGG TAC GTT TGC GGC AAA -3 0 ) primers were used. The CYP450MO was cloned into the pJET1.2/blunt cloning vector using a CloneJET PCR Cloning kit (Thermo Fisher Scientific). A CYP450MO fragment was inserted into the pGAPDH-EGFP vector using NdeI/SpeI sites. To amplify the cDNA encoding CYP450MO, forward NdeI_CYP450MO_F (5 0 -AAC ATA TGC TGT GGT CGC TGA TTG TTG CGG -3 0 ) and reverse SpeI_CYP450MO_R (5 0 -ACA CTA GTG GGC AGT GGT ACG TTT GCG -3 0 ) primers were used. All plasmids were transformed to DH5a competent E. coli for replication and construction.

Phylogenetic analysis of AcCYP450MO
We conducted blastp with the peptide sequence of AcCYP450MO against the NCBI nr database (National Center for Biotechnology Information) and retrieved the sequences of the top 100 hits. These sequences were aligned with the "hmmalign" program of the HMMER package v.3.1b2, according to the "cytochrome P450" domain in the pfam database. With the best protein substitution model "JTT + G + I" predicted by MEGA v.7.0 [17], as well as a bootstrap analysis of 100, a maximum likelihood phylogeny was reconstructed with raxml v.8.2.12 [33]. In addition, the functional domain of cytochrome P450 was predicted with the "hmmscan" program of the HMMER package. Structural similarity was assessed by an online tool "Phyre2" [14].

Cell electroporation of A. castellanii
For electroporation, cells were counted using a hemocytometer and centrifuged at 3000 rpm for 3 min to remove the medium. Acanthamoeba cells were resuspended in PAS to a final count of 5 Â 10 6 cells/mL and placed in an Eppendorf tube. Ten micrograms of plasmid DNA were added to the Eppendorf tube, followed by PAS to a final volume of 800 lL. The mixture was gently mixed and dispensed into a 4-mm cuvette. Using Gene Pulser Xcell TM , the protocol was set as follows: 150 V, 10 ms. After electroporation, the cuvettes containing cells were placed on ice for 10 min, and cells were transferred to a T-75 flask containing PYG for incubation at 28°C overnight. Stable transformants were selected using 40 lg/mL Geneticin (G418).
Survival rates of CYP450MO-overexpressing A. castellanii CYP450MO-overexpressing amoeba cells were seeded at a density of 5 Â 10 6 cells/mL in a 6-well plate and treated with 0.01% PHMB for different times, counted using a hemocytometer, and stained using trypan blue.

Statistical analysis
Data are presented as mean ± standard deviation (SD) from three independent experiments. Student's t-test was used for statistical analysis. Statistical significance was set at p < 0.05.

Results
The sequencing of cytochrome P450 monooxygenase CYP450s are widely distributed throughout different organisms ranging from protozoa to mammals [9,32,40]. In Acanthamoeba, we found 27 CYP450 enzymes (Table 1); moreover, only one CYP450 contained a monooxygenase domain (cytochrome P450 monooxygenase, ACA1_277340) to catalyze a variety of substrates with one oxygen atom [35]. To confirm the mRNA sequence of CYP450MO, we amplified the cDNA using ATCC_30010 cellular cDNA as the template. Compared to the sequences in the NCBI-nr database, we found many differences in the CYP450MO of ATCC_30010 cellular cDNA. We conducted a phylogenetic analysis on CYP450MO and the most similar peptides in GenBank. All peptides of Acanthamoeba formed a monophyletic clade, next to sequences of Salpingoeca (a Choanoflagellate) (Fig. 1). In the clade, CYP450MO was closely related to ACA1_277340 (XP004344559.1). When comparing with the coding sequence with ACA1_277340, their 5 0 and 3 0 ends were identical, while the major difference occurred in the completeness of the cytochrome P450 domain (Fig. 2). CYP450MO possessed a full structure, but the domain was truncated in ACA1_277340 (Fig. 2B). Moreover, phyre2 analysis indicated that CYP450MO showed 99.9% confidence on a high similarity to the structure of human cytochrome P450 2a6. These results indicated that CYP450MO was more likely to show full function than that of ACA1_277340.

The function of CYP450MO in Acanthamoeba
To determine whether CYP450MO of Acanthamoeba can affect PHMB drug degradation, the enzyme was overexpressed by the pGAPDH-EGFP vector. A CYP450MO fragment was inserted into the pGAPDH-EGFP vector using NdeI/SpeI sites (Fig. 3A). After transfection in Acanthamoeba by electroporation for 14 days, the pGAPDH-EGFP-CYP450MO vector was expressed. To confirm that the pGAPDH-EGFP-CYP450MO vector was transfected into Acanthamoeba, the DNA extracted from Acanthamoeba was amplified using the pGAPDH-EGFP primers (Fig. 3B). The EGFP-CYP450MO fusion protein was also expressed in Acanthamoeba using a Cell R microscope (Olympus America, Inc., USA) for 7 days (Fig. 3C).
Acanthamoeba-transfected pGAPDH-EGFP-CYP450MO vectors were treated with 0.01% PHMB. The results showed that the survival rates of Acanthamoeba-transfected pGAPDH-EGFP-CYP450MO vector were higher than those of the control at 1, 16, and 24 h (Fig. 4). Hence, we suggest that Acanthamoeba overexpressing CYP450MO may be resistant to PHMB drug, enhancing survival rates.

Discussion
Acanthamoeba castellanii has 27 CYP450 genes compared to the 57 CYP450 genes in the human genome [29]. The CYP450 genes related to drug metabolism in humans are CYP2C9, CYP2C19, CYP2D6, and CYP3A4 [11]. In nematodes, Caenorhabditis elegans encodes 80 CYP450 genes. Some CYPs in C. elegans such as cyp35a2, cyp35a5, and cyp35c1 play a role in albendazole (ABZ), an anti-helminthic medication [8,18]. However, in protozoa such as Toxoplasma gondii, the CYP450 gene exists as a single copy. The CYP450 of T. gondii plays an important role in developing resistance to drugs such as quinine, mefloquine, and clarithromycin [40]. In this study, we found 27 related CYP450 enzymes in A. castellanii (Table 1). A previous study showed that CYP450 genes in humans were observed to enhance gene diversity by alternative RNA splicing [34]. Therefore, it is likely that CYP450s are produced from the Acanthamoeba gene by alternative splicing to metabolize different drugs.
In this study, CYP450MO induced PHMB drug metabolism for the survival of Acanthamoeba, as CYP450MO overexpression enhanced the resistance of Acanthamoeba. Moreover, in previous studies, strains resistant to encystation were also transformed into pseudocysts or cysts under the effects of PHMB drug stress [10,23]. ATG8 in Acanthamoeba encystation plays an important role in autophagy against drug therapy [12]. CSI and EMSP have also been identified in Acanthamoeba and are involved in the encystation mechanism [16,27]. However, ATG8, CSI, and EMSP levels were not significantly different between Acanthamoeba-transfected pGAPDH-EGFP and pGAPDH-EGFP-CYP450MO (Fig. 5). Hence, we suggest that Acanthamoeba may not express encystation-related genes against PHMB drug lysis.
CYP450s are known to catalyze a variety of chemical reactions and attack substrates from electron transfer chains. On the electron transfer chains, CYP450s incorporate oxygen atoms into the substrate molecule by transferring electrons from NAD(P)H [31]. Monooxygenase systems depend on monooxygenase activity catalyzing one oxygen atom in the substrate molecule. Many drug metabolic processes catalyzed by monooxygenase involve the oxidation of endogenous and exogenous substrates [35]. In this study, we also found that the survival rates of Acanthamoeba-transfected pGAPDH-EGFP-CYP450MO vector were higher than those of the control after PHMB treatment (Fig. 4). Hence, we suggest that CYP450MO in Acanthamoeba may catalyze PHMB drug metabolism to exogenous substrates and be secreted into the extracellular environment. In the future, we aim to focus on CYP450MO as a drug target to potentially treat AK.

Conclusions
In this study, we overexpressed CYP450MO in Acanthamoeba to investigate PHMB drug resistance. Acanthamoeba with CYP450MO-overexpression had higher survival rates than those of the control cells after PHMB treatment. We suggest that CYP450MO in Acanthamoeba may catalyze PHMB drug metabolism to enhance survival rates after PHMB treatment.