Inhibitory Effects of Fosmidomycin Against Babesia microti in vitro

Babesia microti, the main pathogen causing human babesiosis, has been reported to exhibit resistance to the traditional treatment of azithromycin + atovaquone and clindamycin + quinine, suggesting the necessity of developing new drugs. The methylerythritol 4-phosphate (MEP) pathway, a unique pathway in apicomplexan parasites, was shown to play a crucial function in the growth of Plasmodium falciparum. In the MEP pathway, 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) is a rate-limiting enzyme and fosmidomycin (FSM) is a reported inhibitor for this enzyme. DXR has been shown as an antimalarial drug target, but no report is available on B. microti DXR (BmDXR). Here BmDXR was cloned, sequenced, analyzed by bioinformatics, and evaluated as a potential drug target for inhibiting the growth of B. micorti in vitro. Drug assay was performed by adding different concentrations of FSM in B. microti in vitro culture. Rescue experiment was done by supplementing 200 μM isopentenyl pyrophosphate (IPP) or 5 μM geranylgeraniol (GG-ol) in the culture medium together with 5 μM FSM or 10 μM diminazene aceturate. The results indicated that FSM can inhibit the growth of B. microti in in vitro culture with an IC50 of 4.63 ± 0.12 μM, and growth can be restored by both IPP and GG-ol. Additionally, FSM is shown to inhibit the growth of parasites by suppressing the DXR activity, which agreed with the reported results of other apicomplexan parasites. Our results suggest the potential of DXR as a drug target for controlling B. microti and that FSM can inhibit the growth of B. microti in vitro.


INTRODUCTION
Parasites of the genus Babesia are prevalent apicomplexan pathogens transmitted by ticks and infect many mammalian and avian species (Yabsley and Shock, 2013). Human babesiosis is primarily caused by the parasite Babesia microti, with most people being infected by ticks and some by blood transfusion (Goethert et al., 2003;Hildebrandt et al., 2007;Young et al., 2012). The infection is characterized by fever and hemolytic anemia and can result in death in severe cases from complications, such as heart failure, respiratory distress, and pulmonary edema (Rosner et al., 1984). Due to the increasing number of people infected with Babesia, B. microti-related infection has been classified as a nationally notifiable disease since 2011 by the Center for Disease Control (United States) (Herwaldt et al., 2011). Babesiosis is usually treated with atovaquone and azithromycin, but resistance to these drugs has been reported (Krause et al., 2000;Wormser et al., 2010;Simon et al., 2017). Therefore, it is very urgent to develop new anti-Babesia drugs.
Isoprenoids comprise a large family and have an important function in membrane structure, cellular respiration, and cell signaling (Gershenzon and Dudareva, 2007). IPP in living organisms can be synthesized by two pathways [mevalonate (MVA) pathway and MEP pathway] (Odom, 2011). Humans use the MVA pathway to synthesize IPP from acetyl-CoA (Endo, 1992). However, there is no MVA pathway in the genus of Apicomplexa, which thus synthesizes IPP by the MEP pathway (Cassera et al., 2004). The MEP pathway was first reported to be present in Plasmodium falciparum in 1999 (Jomaa et al., 1999). With the deepening of research, the MEP pathway was found to be crucial for parasites (Cassera et al., 2004). For instance, the deoxyxylose-5-phosphate reductoisomerase (DXR) of P. falciparum was shown to contribute to the erythrocyte stage, and inhibiting the DXR activity reduced the growth and the development of the parasites (Odom and Van Voorhis, 2010;Zhang et al., 2011). Additionally, by knocking out the DXR genes of Toxoplasma gondii, the parasites were found unable to survive, proving the essentiality of the MEP pathway for their survival (Nair et al., 2011).
The first dedicated step in MEP isoprenoid biosynthesis is accomplished by the bifunctional enzyme DXR (Imlay and Odom, 2014). DXR is competitively inhibited in vitro by the antibiotic fosmidomycin (Koppisch et al., 2002;Sangari et al., 2010). Fosmidomycin has been shown to be a clinical prospect for antimalarial drugs due to its inhibition on the recombinant Plasmodium DXR to kill Plasmodium, and the current clinical trial of malaria treatment with clindamycin is in phase II (Olliaro and Wells, 2009). Babesia and Plasmodium have many similarities, and they both live in red blood cells (RBCs). In this study, we have found that B. microti DXR (BmDXR) has conserved binding sites of fosmidomycin (FSM), and FSM can inhibit the growth of B. microti in vitro, suggesting its potential as a new anti-Babesia drug.

Parasites
A B. microti strain ATCC PRA-99TM R (Ruebush and Hanson, 1979) was obtained from the National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention (Shanghai, China), and maintained in our laboratory (State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, China). The parasites were isolated at parasitemia of 30-40% as determined by Giemsa staining of thin blood smears.

RNA Extraction and cDNA Synthesis
Total RNA was extracted from infected blood by using the TRIZOL reagent (Invitrogen, Shanghai, China) and treated with RNase-free DNase I (TaKaRa, Dalian, China). RNA concentration was measured by NanoDrop 2000 (Thermo, China). The cDNA was prepared from 1 µg of the total RNA using a PrimeScript TM RT reagent kit with gDNA eraser (TaKaRa, Dalian, China).

Cloning of the BmDXR Gene
Primer pairs of BmDXR were designed based on the sequences of the B. microti strain R1: BmDXR-F (5'-ATGACAAATTATTT AAAACTC-3') and BmDXR-R (5'-TTAACACTTAATTTTTTT TGC-3'). Complete sequences of the BmDXR were amplified by PCR from cDNA separately. The PCR reaction was performed at 95 • C for 5 min, followed by 35 cycles of 95 • C for 30 s, 47 • C for 30 s, 72 • C for 1 min 30 s, and finally at 72 • C for 10 min. The PCR products were purified and ligated into the cloning vector pEASY-Blunt (Trans, Beijing, China). Three positive colonies of each gene were sent for sequencing analysis by Invitrogen (Shanghai, China).

Sequence Analysis
The amino acid sequence of BmDXR was aligned with the selected amino acid sequences from other organisms by MAFFT online 1 , then edited by BioEdit v7.25, and phylogenetically analyzed by using the Maximum Likelihood method in MEGA 7 . The structure of BmDXR was predicted by SWISS-MODEL 2 (Guex et al., 2009;Bienert et al., 2017;Waterhouse et al., 2018). The 3D structure of BmDXR was virtually docked with FSM through Molecular Operating Environment (MOE) version 2014.09 (Chemical Computing Group).

Fosmidomycin Treatment and Rescue Assay
Drug stock solutions of FSM (Sigma-Aldrich, Shanghai, Chain) and diminazene aceturate (DA) (Sigma-Aldrich, Shanghai, Chain) were prepared in sterile water. Geranylgeraniol (Sigma-Aldrich, Shanghai, Chain) stocks were prepared in 100% ethanol. Isopentenyl pyrophosphate triammonium salt solution (Sigma-Aldrich, Shanghai, Chain) was used directly without any additional treatment. For the growth inhibition assay, B. microti cultures (20 µl of RBCs plus 100 µl of culture medium) were grown in 96-well flat-bottomed plates, and the susceptibility of B. microti in vitro to FSM was evaluated at concentrations up to 500 µM. The results were further confirmed by the IC50 values calculated using the Käber method. All the experiments were repeated three times.
In the rescue experiments, IPP or geranylgeraniol (GG-ol, alcohol of geranylgeranyl diphosphate) was added to the medium containing different drugs. IPP is one of the products in the MEP pathway (He et al., 2018), and GG-ol is the alcohol analog of the downstream isoprenoids (Yeh and DeRisi, 2011;Imlay and Odom, 2014). DA was used as a positive control, and ethanol was used as a negative control. The group of control is only medium. Each drug test was performed in triplicate.
In order to test the parasitemia, three smears were prepared from each well after 72 h of incubation. After air-drying, thin blood smears were fixed with methanol, followed by staining with Giemsa (Sigma-Aldrich, Shanghai, China), and measuring the parasitemia by microscopy. The data were analyzed using GraphPad Prism 7 (San Diego, CA, United States) by two-way analysis of variance (ANOVA), followed by Tukey's multiplecomparison test. The results are shown as mean ± SD (NS, P > 0.05 not significant at 5%; * P < 0.05 significant at 5%; * * P < 0.01 significant at 1%; and * * * P < 0.001 significant at 0.1%; error bars represent the standard deviations).

Cloning and Characterization of B. microti DXR
The open reading frame of BmDXR was cloned from B. microti PRA99 cDNA by conventional PCR. The results showed that BmDXR is 1,401 bp in length, encoding 466 amino acids with a predicted size of 51.8 kDa. The sequence was submitted to GenBank, with accession number MK673989. BLASTn indicated that BmDXR PRA99 (MK673989) is identical to that of B. microti R1 strain (XP_021338225).

Bioinformatic Analysis
The obtained BmDXR sequence was characterized by bioinformatic analysis. SignalP4.1 analysis indicated that BmDXR has a 22-amino-acid signal peptide in N-terminus 3 , and a 48-amino-acid transit peptide right after the signal peptide. The amino acid sequence of BmDXR was aligned with the DXR amino acid sequences of other apicomplexan parasites by MAFFT. The results showed that BmDXR has the highest similarity to the DXR sequence of P. falciparum (AAD03739) with a percent identity of 41.71%, and the lowest similarity to that of Mycobacterium tuberculosis (NP_217386), with a percent identity of 36.59% (Figure 1A).
DXR amino acid sequences were characterized by phylogenetic analysis with MEGA6, and B. microti was shown to fall in the piroplasma clade in the same category of Plasmodium. In contrast, bacteria, plant, algae, and sarcocystis are grouped in the same category ( Figure 1B). In the piroplasma clade, B. microti is significantly different from the other species, including B. bigemina, B. ovata, B. bovis, T. equi, T. orientalis, T. parva, and T. annulata.
The 3D structure of BmDXR was predicted by SWISS-MODEL, and BmDXR is shown as a dimeric structure with a metal ion binding site consisting of amino acids D216, E218, and E298. The 3D structure of BmDXR was virtually docked with FSM using MOE2014.0901. The results showed that FSM can form hydrogen bonds with Ser217, Asp216, Cys253, Met281, Ser289, Asn294, and Lys295 of BmDXR ( Figure 1C).

Fosmidomycin Inhibits the Growth of B. microti in vitro
The effect of FSM on the growth of B. microti in vitro was tested by adding different concentrations of FSM into the in vitro culture medium at an initial percent parasitized erythrocytes (PPE) of 3%. Parasitemia was counted at 72 h post-treatment by microscopy. The parasitemia of the FSM groups is 4.27 ± 0.28%, 3.60 ± 0.16%, 3.09 ± 0.25%, 2.49 ± 0.33%, 1.67 ± 0.18%, and 1.45 ± 0.45% at the concentration of 5, 50, and 500 nM and 5, 50, and 500 µM, respectively, in contrast to an increase from 3% to 4.83 ± 0.8% for the negative control group (the group without drug) after 72 h of culture. After the 72h treatment, the parasitemia is significantly lower (P < 0.05) in the 50 nM FSM group than in the negative control group, with a significant difference (P < 0.01) between 5 and 50 or 500 nm FSM groups, but no difference between the 50-and 500-µM FSM groups (Figure 2). The test results indicated that the drug efficacy is dose dependent, and FSM could not completely inhibit the growth of B. microti even at a drug concentration as high as 500 µM (inhibition rate of 70%). Compared to the negative control group, FSM exhibited a potential anti-B. microti activity at a low micromolar concentration, with an IC50 of 4.63 ± 0.12 µM.

IPP and GG-ol Can Rescue B. microti Treated by Fosmidomycin
The inhibition of FSM on the growth of B. mitroti was investigated through rescue experiments in B. microti in vitro cultivation with 200 µM IPP and 5 µM GG-ol added separately into 5 µM FSM and 10 µM DA using the latter as a positive control. The 5 µM FSM and 10 µM DA showed 53.8 and 58.6% inhibition on the growth of the parasites (Figure 3A) in the rescue experiment. The growth in 5 µM FSM could be restored by adding 200 µM IPP or 5 µM GG-ol into culture media as indicated by having no difference (P < 0.001) in the relative growth rate among FSM + IPP, GG-ol, and the control (Figures 3B,C). However, the growth in 10 µM DA could not be rescued by adding IPP or GG-ol, as shown by a significant difference (P < 0.001) in the relative growth rate among DA + IPP, DA + GG-ol, and the control [ANOVA, F(2, 6) = 259.2, P < 0.0001; ANOVA, F(2, 6) = 65.1, P < 0.0001] (Figures 3B,C).

DISCUSSION
The MEP pathway, an essential route in apicomplexan parasites, plays a vital role in the growth of parasites by synthesizing IPP (Imlay and Odom, 2014); however, very few effective inhibitors have been studied. Currently, the MEP inhibitors with lower IC50 for Plasmodium are FSM and 1R, 3S MMV008138 (Ghavami et al., 2018). DXR is the second and also a ratelimiting enzyme in the MEP pathway (Imlay and Odom, 2014). The inhibitors of DXR enzymes, such as FSM, suppress the synthesis of IPP in the MEP pathway of multiple organisms in vitro (Figure 4; Jomaa et al., 1999). It has been reported that FSM can inhibit B. divergen, B. bovis, and B. orientalis in vitro  (Baumeister et al., 2011;Caballero et al., 2012;He et al., 2018). As shown by the reported P. falciparum and M. tuberculosis crystal structures of inhibitor-free and FSM-bound complete quaternary complexes of DXR (Mac Sweeney et al., 2005;Andaloussi et al., 2011;Umeda et al., 2011), a large cleft was closed between NADPH-binding and catalytic domains upon inhibitor binding, which means that FSM inhibits DXR activity by competing with DOXP. The FSM binding site is conservative, and BmDXR is similar in structure to PfDXR and EcDXR. We speculate that FSM can inhibit the DXR activity in B. microti due to its inhibition on the growth of B. microti in in vitro culture with an FSM IC50 FIGURE 4 | Schematic of isoprenoid metabolism through the non-mevalonate pathway. The methylerythritol 4-phosphate pathway is a unique route to isoprenoid biosynthesis in apicomplexan, and fosmidomycin is a specific inhibitor to 1-deoxy-D-xylulose 5-phosphate reductoisomerase.
value of 4.63 ± 0.12 µM, which is higher than that of B. bovis and B. bigemina (3.87 and 2.4 µM, respectively) (Sivakumar et al., 2008). The growth of B. microti can be rescued by adding IPP or GG-ol in the culture medium, which agreed with the report that GG-ol can rescue the growth of B. orientalis inhibited by FSM (He et al., 2018). These results further suggest that FSM inhibits B. microti growth by suppressing the MEP pathway. It is reported that B. microti, an obligate parasite of red blood cells (Silva et al., 2016), obtains most of the nutrition materials for parasite survival from host red blood cells, but it cannot obtain IPP from host cells due to the small amount of IPP in RBCs (Wiback and Palsson, 2002). In this case, FSM may inhibit the growth of B. microti by suppressing the synthesis of IPP.
FSM can cause the death of P. falciparum in the first life cycle (Howe et al., 2013), but we failed to observe the death of B. microti after 24 h of treatment at 5 µM FSM. According to the results of Giemsa staining (Supplementary Figure S1), we selected the parasites in the period of merozoites and compared their morphologies. All the merozoites in the control group have an obvious contour and a complete shape, while those treated by FSM have lost their contour and complete shape which, however, can be recovered upon addition of IPP or GG-ol in the medium. This is consistent with the observation in B. bovis and B. bigemina treated with FSM, with obvious changes in the shape of the parasites (Sivakumar et al., 2008). These results indicate that IPP is important for Babesia to keep a normal shape. Meanwhile, the merozoites treated by DA present a pointed shape, which could not be restored to a normal shape after adding IPP or GG-ol. These morphologies indicate a milder efficacy of FSM than DA because DA made the merozoites of B. microti point-like, while FSM caused the parasite to lose its normal form. FSM-treated P. falciparum was shown to reduce protein prenylation, leading to marked defects in food vacuolar morphology and integrity (Howe et al., 2013). However, no food vacuole has been reported in B. microti (Rudzinska et al., 1976), suggesting that the impact of FSM on B. microti may be different from its influence mechanism on malaria parasites.
Traditionally, azithromycin + atovaquone was used to treat babesiosis in humans and clindamycin + quinine as a treatment strategy for patients with resistance to atovaquone (Simon et al., 2017). Meanwhile, many patients have adverse reactions to chloroquine (Krause et al., 2000;Rozej-Bielicka et al., 2015). Generally, traditional treatments cannot eliminate B. microti parasitemia completely, suggesting the high recurrence potential of B. microti. Despite being a safe and effective inhibitor, FSM has some limitations to clinical applications. First of all, it is an unmodified compound which is very costly. Secondly, FSM has a poor pharmacokinetics profile with a plasma halflife of 3.5 h (Na-Bangchang et al., 2007); it will need multiple shots for clinic use. This limitation of FSM can be solved by drug modification. For example, FR9008 is a derivative of FSM, which has a better effect on P. falciparum than FSM. Currently, it is necessary to improve the ability of FSM in entering cells and extend its half-life for clinical applications. We believe that the limitations of FSM can be overcome by drug modification. For drug development, modified drugs have better clinical results; for example, dihydroartemisinin has a better effect than artemisinin in treating Plasmodium (Li et al., 1983). About combination therapy, clindamycin + FSM can play a better effect in the treatment of Plasmodium (Borrmann et al., 2006), but clindamycin has less effect to B. microti in vitro (Lawres et al., 2016). Other drugs could be used as combination therapy with FSM if required. Our future work will focus on modifying FSM and the combination therapy of FSM.

CONCLUSION
The MEP pathway is a favorable target for drug development. In this study, it is shown that FSM can inhibit the growth of B. microti in vitro, which can be rescued by a medium supplemented with IPP or GG-ol. These results indicate that DXR is a potential drug target for designing anti-Babesia drugs and that the DXR function and FSM structure contribute to the design of such drugs.

DATA AVAILABILITY STATEMENT
The datasets generated for this study can be found in the NCBI GenBank under the accession number MK673989.

ETHICS STATEMENT
This study was approved by the Scientific Ethic Committee of Huazhong Agricultural University (permit number: HZAUMO-2017-040). All mice were handled in accordance with the Animal Ethics Procedures and Guidelines of the People's Republic of China.