Samples from two independent RNA extractions were processed using Ambion MessageAmpTM Premier RNA Amplification Kit (Applied Biosystems, Foster City, CA, USA) according to manufacturer's instructions. Fragmented cRNA was then hybridized onto a probe array chip (Eh\_Eia520620F) that was custom-made by Affymetrix (Santa Clara, CA, USA) (Husain et al., 2011; Penuliar et al., 2012) using kits and protocols specified in the Affymetrix GeneChip Expression Analysis Technical Manual (Affymetrix, Santa Clara, CA, USA). Following hybridization, arrays were washed, stained with streptavidinphycoerythrin (Molecular Probes, Eugene, OR, USA) using Affymetrix GeneChip Fluidics Station 450, and scanned with an Affymetrix GeneChip Scanner 3000 at 570 nm. Each array image was visually screened to check for scratches, signal artifacts, and debris.

The microarray used in this study was made based on E. histolytica and E. invadens sequences stored at TIGR and Pathema databases (Loftus et al., 2005; http://amoebadb.org/ amoeba/). It contained 9230 probe sets for E. histolytica and an additional 25 and 81 control probe sets for Entamoeba and Affymetrix, respectively (Eh\_Eia520620F\_Eh). Nomenclature for the IDs was based on whether the probe set is unique to either Pathema (e.g., EHI\_123456) or TIGR (e.g., 12.m00345), or is found in both databases (e.g., 98.m00765\_234567). Probe sets labeled with "\_at" represent a single gene, while those labeled with "\_s\_at" represent probe sets that share all probes identically with at least two sequences. The "\_s\_at" probe sets represent highly similar transcripts, shorter forms of alternatively polyadenylated transcripts, or common regions in the 3′ ends of multiple alternative splice forms. Probe sets labeled with "\_x\_at," on the other hand, represent probe sets where it was not possible to select either a unique probe set or a probe set with identical probes among multiple transcripts. These probe sets could cross-hybridize with other genes in an unpredictable manner (Affymetrix, Santa Clara, CA, USA).

#### Data Normalization, Analysis, and Deposition

Raw probe intensities were generated by the GeneChip Operating Software (GCOS) and GeneTitan Instrument from Affymetrix. Resulting expression values were analyzed by R/BioConductor and GeneSpring GX Ver.11.5 to identify differentially expressed genes. Reproducibility of the experiments was determined by Pearson's correlation coefficient and confirmed by principal component analysis (Pearson, 1901; Stigler, 1989). Only genes that were considered "present" by GCOS in both arrays were used in further analysis. Gene probe sets were considered differentially expressed between samples if they had at least a 3-fold change and a P-value < 0.05, calculated using Welch's t-test (Welch, 1947), after multiple test correction by the Benjamini–Hochberg method (Benjamini and Hochberg, 1995). A post-hoc test using Tukey's Honestly Significant Difference test was conducted to determine significant differences between samples (Tukey, 1949). Probe sets ending with "\_x\_at" were removed together with redundant genes identified by sequence alignment using ClustalW2.48 Finally, RefSeq's annotated pseudogenes were omitted from the final list (ClustalW2; http://www.ebi.ac.uk/Tools/msa/ clustalw2/).

Nucleotide sequences of differentially expressed genes were searched against GenBank (http://www.ncbi.nlm.nih. gov/genbank/) and Amoeba DB (http://amoebadb.org/ amoeba/) by using Basic Local Alignment Search Tool (BLAST; http://blast.ncbi.nlm.nih.gov/Blast.cgi). Matches against hypothetical proteins (HP) with E-value < e −50 and maximal scoring pair alignment that covered at least 40% were considered as putative orthologs (McLysaght et al., 2003). Hierarchical clustering of modulated genes in MTZR, MTZR (−), and HM-1 (+) was performed with Cluster 3.0 (http://bonsai.hgc.jp/∼mdehoon/software/cluster/software.htm) using average linkage, while dendrograms and heatmaps were generated with Java TreeView version 1.1.6r2. (http:// jtreeview.sourceforge.net) Gene Ontology (GO) annotations were mined from the Gene Ontology Annotation Database (http://www.ebi.ac.uk), and Amoeba DB. GO term enrichment was computed using Fisher's exact test (Agresti, 1992), against a background consisting of all GO IDs for genes symbols beginning with EHI, which were downloaded from the QuickGO database of European Molecular Biology Laboratory - European Bioinformatics Institute (EMBL-EBI) (Taxon ID: 5759) (http:// www.ebi.ac.uk/QuickGO/). Genomic location of each gene was obtained from Pathema-Entamoeba (http://pathema.jcvi.org) while transmembrane domain prediction was performed using the following online servers: HMMTOP (http://www.enzim. hu), SOSUI (http://bp.nuap.nagoya-u.ac.jp), TMHMM (http:// www.cbs.dtu.dk), TopPred (http://mobyle.pasteur.fr), TMpred (http://www.ch.embnet.org), and Phobius (http://phobius.sbc. su.se). Signal peptide sequences were predicted using SignalP (http://www.cbs.dtu.dk), SOSUIsignal (http://bp.nuap.nagoya-u. ac.jp), and Phobius. Putative transmembrane domains and signal peptides were considered present only if positive hits were found in all programs used. Venn diagrams were constructed using Venn Diagram Generator (http://www.pangloss.com/seidel/ Protocols/venn.cgi).

The microarray data reported in this paper has been deposited in the Gene Expression Omnibus (GEO) database (http://www. ncbi.nlm.nih.gov/geo/) with accession number GSE35990.

#### Generation of Transgenic Ameba Overexpressing HP1 and ISFs

Full-length gene sequences were amplified from MTZR cDNA with sense- and anti-sense primers containing appropriate restriction sites (Table S2). PCR primers were designed as follows: sense primers contained 26∼30 nucleotides corresponding to a region of each gene encoding the amino-terminal portion of each target proteins, fused at the 5′ end with three additional nucleotides and SmaI restriction enzyme recognition site (tccCCCGGG); reverse primers contained 22∼33 nucleotides corresponding to a region of each gene encoding the carboxylterminal portion of each target proteins, fused at the 5′ end with three additional nucleotides and XhoI restriction enzyme recognition site (ccgCTCGAG). PCR was performed with the cDNA as a template using a DNA Engine Peltier Thermal Cycler (Bio-Rad, Hercules, CA, USA). The PCR cycling conditions consisted of an initial step of denaturation at 94◦C for 1 min, followed by 30 cycles of denaturation at 98◦C for 10 s, annealing at 55◦C for 30 s, and extension at 72◦C for 60 s with Phusion DNA polymerase (New England BioLabs, Tokyo, Japan). Resultant PCR fragments were precipitated with ethanol, digested with appropriate restriction enzymes, and then purified from an exised piece of agarose gel by using GENECLEAN kit (Funakoshi, Tokyo, Japan). After purification, the gene was inserted into an expression plasmid, pEhEx, as previously described (Penuliar et al., 2012). The plasmid was introduced into E. histolytica G3 strain by lipofection, with minor modifications as previously described (Penuliar et al., 2012). Briefly, E. histolytica G3 trophozoites in the mid- logarithmic growth phase were harvested as described above, and re-suspended with Opti-MEM medium supplemented with 5 mg/mL L-cysteine and 1 mg/mL ascorbic acid at 1 × 105 cells/mL. Approximately 5 mL of the suspension was dispensed into each well on a 12-well plate and the plate was incubated under anaerobic condition at 35.5◦C for 30 min. Following incubation, 4.5 mL medium from each well was removed and 500µL liposome–plasmid–mixture (5µg plasmid, 10µL PLUS reagent, 20µL Lipofectamine in Opti-MEM medium) was added. After 5 h of transfection, cells were harvested by placing the plate on ice for 15 min, then added to culture tubes with 5.5 mL cold BIS, and incubated at 35.5◦C for 24 h. Transformants were initially selected in the presence of 1µg/mL of G418, and drug concentration was gradually increased to 10µg/mL during the following 6 weeks before the transformants were analyzed.

#### Reverse Transcriptase (RT)-PCR and Quantitative Real-Time (qRT)-PCR

Total RNA from semi-confluent cultures, about 2×105 cells/mL, of HM-1, MTZR, and transformants harboring one of the following plasmids: pEhEx-HA (mock plasmid), HP1-HA, ISF1- HA, ISF2-HA, and ISF4-HA, cultured in 25 cm2 flasks, was extracted and quantified as described previously (Penuliar et al., 2012). cDNA was reverse transcribed from 5µg RNA with the SuperScript III First-Strand Synthesis System, and RT-PCR was performed with the resulting cDNA as template and primers listed in Table S2 on a DNA Engine Peltier Thermal Cycle (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The PCR parameters used were: an initial denaturation step at 95◦C for 2 min followed by 30 cycles of denaturation at 94◦C for 15 s, annealing at 55◦C for 30 s, and extension at 72◦C for 30 or 60 s. The products were resolved on 1.5% [w/v] agarose gel with 0.5µg/mL ethidium bromide, visualized, and photographed under ultraviolet illumination.

The Fast SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA) was used for qRT-PCR in accordance with the manufacturer's instructions. The list of primers for genes whose expression was quantified by qRT-PCR can be found in Table S1, and includes a housekeeping gene, RNA polymerase II gene (rpoII), as control. Each PCR reaction contained 5µL (1:50 dilution) of cDNA and 15µL primer mix, composed of 10µL 2X Fast SYBR Green Master Mix, sense and antisense primers, and nuclease-free water, to bring the volume to 20µL. qRT–PCR was performed using StepOne Plus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) with the following cycling conditions: enzyme activation at 95◦C for 20 s, followed by 40 cycles of denaturation at 95◦C for 3 s and annealing/extension at 60◦C for 30 s. All test samples were run in triplicate including an RT-negative control for each sample set along with a blank control consisting of nuclease-free water in place of cDNA. Quantification for each target gene was determined by the 11Ct method with rpoII as reference gene (Livak and Schmittgen, 2001).

#### Statistical Analysis

Correlation coefficients were calculated using the Student's ttest function of Microsoft Excel statistical package (Microsoft Corp., Redmond, WA, USA). Probability levels (P) < 0.05 were considered significant.

#### Results

#### Generation and Phenotypic Characterization of MTZR

Exposure of HM-1 to MTZ concentrations up to 3µM for 24 h did not result in any significant changes in cell morphology. Cells continued to grow and cultures formed confluent lawns after 72 h. Starting at 6µM MTZ, however, cells became rounder than usual and were slightly bigger in size. Cells also took longer to attach to surfaces and for cultures to form confluent lawns. Cell viability, however, was verified by cell adhesion and trypan blue exclusion assay. After 29 weeks, cells growing at 12µM MTZ (MTZR) were obtained. We tried to increase the concentration of MTZ further, but this resulted in further increase in doubling time.

The population doubling time of MTZR was significantly longer compared to HM-1 (26.8 ± 0.9 and 18.2 ± 0.5 h, respectively; P-value < 0.001, **Figure 1**). MTZR also appeared rounder, bigger, and more granular than HM-1. In contrast, parental cells exposed to 12µM MTZ showed signs of morphological changes as early as 2 h, rounding in 5 h, cell shrinkage, and detachment within 24 h. The slight enlargement of MTZR (+) compared to HM-1 was suggested when we measured the total protein content of MTZR and HM-1, 4.81 ± 0.04 and 4.24 ± 0.03 pg/cell, respectively. Flow cytometric analysis also confirmed that MTZR was slightly larger than HM-1 (data not shown).

The IC50 of MTZ in MTZR and HM-1 was 12.9 ± 0.3 and 6.5 ± 0.3µM, respectively, and its resistance index was 2.0 (**Table 1**). When MTZR was cultured without the drug for 1 week followed by MTZ challenge for 24 h, percent survival was only 28%, indicating that resistance to the drug was reversible (data not shown). To determine if MTZR was cross-resistant with other amebicides, the IC50 of the drugs listed in **Table 1**, against MTZR and HM-1 was determined. Results showed that MTZR had reduced sensitivity to the drugs.

MTZR had a slower rate of CHO monolayer destruction, especially at early time points, indicating a slight decrease in virulence (P-value < 0.05 for 30, 45, 60, and 75 min; **Figure 2A**). This was consistent with the observed decrease in intensity of bands corresponding to cysteine protease 1 and 2 (CP1 and

CP2, synonymous to EhCP-A1 and EhCP-A2, respectively) in the zymogram (**Figure 2B**), and by an ImageJ (http://rsbweb. nih.gov/ij/) analysis of the same bands that indicated an approximately 33% decrease in band areas. The band for CP5 (EhCP-A5) and control bands (arrow head) were comparable between HM-1 and MTZR. The decrease in cytopathy in MTZR was also indicated by the lower the number of engulfed beads (**Figure 2C**) and percentage of cells with engulfed beads in the phagocytosis assay (**Figure 2D**). MTZR also consumed glucose at a reduced rate compared to HM-1 (P-value < 0.05, **Figure 2E**). In addition, MTZR adhered to plastic surfaces, and to fibronectinand collagen-coated plates at relatively comparable levels, but not as efficiently as HM-1 (Supplementary Figure S2).

#### Transcriptomic Analysis of MTZR

Our expression data showed variations in the number of differentially expressed genes between MTZR and HM-1 cultured with and without MTZ (GEO ID: GSE35990). To identify genes whose expression was most highly affected, a general filtering of the expression data, as indicated in Materials and Methods, was used. This resulted in the identification of 142 probe sets for MTZR, 58 probe sets for MTZR (−), and 37 probe sets for HM-1 (+) that showed at least 3-fold changes compared to HM-1 (−) (**Figure 3A**, Table S3). In MTZR, 95 (66.9%) of the genes were upregulated, while 47 (33.1%) genes were downregulated (**Tables 2**, **3**). Only 55 (38.7%) genes were annotated after conducting BLAST search, while 87 (61.3%) genes encoded for proteins of unknown function, i.e., hypothetical proteins (Table S4). The most upregulated gene was a hypothetical protein (HP) (EHI\_006850) with some similarity to a zinc finger protein in E. histolytica (E-value = 1.0e−46), designated as HP1 in this study. Its expression in MTZR was increased by 36.9 folds. Genes for DNA polymerase (EHI\_164190), iron-sulfur flavoprotein (ISF) (EHI\_025710), AIG1 family protein [XP\_648192 (522.m00018)], and four other HPs were also upregulated by more than 10 folds. The most downregulated genes were three leucine-rich proteins [EHI\_033560, EHI\_077280, EHI\_124070 (371.m00031)] which had fold changes of more than 160, and two AIG family

proteins [XP\_648014 (628.m00011), EHI\_180390] and two CPs (EHI\_160330, EHI\_121160) genes that were downregulated by at least 5 folds. The two CPs identified had high similarity to CP7 (synonymous to EhCP-B1) (81%) based on amino acid sequence (Tillack et al., 2007). The differential expression of 16 genes was validated by qRT-PCR, using rpoII as an endogenous control (**Table 4**). Pearson's correlation coefficient (0.81) between microarray and qRT-PCR results indicated a reasonable degree of concordance between the fold-changes of the genes selected for this comparison.

indicates comparable protein loading per lane as estimated by CBB

To determine whether the genes we identified were important in MTZ resistance or resulted from a general stress response, a 3-way comparison of differentially expressed genes in MTZR, MTZR (−) and HM-1 (+) was performed. **Figures 3A,B**, and Table S3 show that out of 142 genes modulated in MTZR, 48 genes (33.8%) were also modulated in MTZR (−), while only 8 genes (5.6%) were differentially transcribed in HM-1 (+). Among the genes specifically modulated in MTZR were DNA polymerase (EHI\_164190), 4 AIG1 family proteins [EHI\_026000, EHI\_126550, EHI\_126560, XP\_648127 (554.m00020)], four iron-sulfur flavoproteins (EHI\_022270, EHI\_022600, EHI\_181710, EHI\_067720, EHI\_103260), metal-dependent hydrolase (EHI\_054690), dUTP nucleotidohydrolase domain-containing protein (EHI\_072960), three serine-rich 25 kDa antigen protein [EHI\_073980, EHI\_072000, EHI\_072000 (82.m00164)], two Ras family protein (EHI\_074750, EHI\_045600), Rho guanine nucleotide exchange factor (EHI\_006140), and a competence/damage-inducible protein (EHI\_090260). In MTZR (−), AP-2 complex protein (EHI\_014390), serine acetyltransferase (EHI\_021570), zinc finger protein (EHI\_091050), cysteine protease (EHI\_117650), and a cell division control protein (EHI\_154270) were specifically modulated. In HM-1 (+), on the other hand, genes for heat shock proteins [EHI\_005657 (181.m00064), EHI\_156560 (482.m00014), EHI\_034710)] were specifically upregulated together with genes putatively involved in drug resistance

HM-1 and MTZR. (E) Glucose consumption assay.

like P-glycoprotein 5 (EHI\_075410, EHI\_125030) and ATPbinding cassette protein (EHI\_134470, EHI\_178580). Two iron-sulfur flavoproteins (EHI\_025710, EHI\_138480) and an ABC transporter [EHI\_084730 (36.m00218)] were upregulated in both MTZR and HM-1 (+), although their expression in HM1 (+) was 2 to 3 folds higher. It is also interesting to note that in all three strains, induction of gene transcription clearly prevailed over inhibition. This was particularly true for HM-1 (+) where almost 95% of the modulated genes were upregulated.

#### Go Classification, Features, and Clustering on the Genome

To have a general view of the cellular functions regulated as a result of MTZ resistance, we performed a GO enrichment analysis. A total of 132 GO terms were mined from Amoeba DB representing 63 unique GO IDs (Table S5). However, based on Fisher's exact test, only 24 terms were significantly enriched in MTZR (**Table 5**). Genes involved in GTP binding and proteolysis were the most highly enriched, followed by genes associated with cysteine-type peptidase activity, small GTPase-mediated signal transduction, oxidoreductase activity, protein kinase activity, and electron carrier activity. Roughly, the enriched terms could be classified into four functional categories, namely (1) nucleotide binding, (2) metabolism, (3) oxidative stress response, and (4) signal transduction.

Only a fraction of the differentially expressed genes in MTZR (about 5%) encoded for membrane proteins, based on positive results for transmembrane and signal peptide predictions (Table S6). Only 21 genes (14.8%) contained at least one transmembrane domain. Eleven of these were upregulated, while 10 were downregulated. Proteins encoded by 25 genes (17.6%) also had predicted signal peptides. Only seven genes have both transmembrane domains and signal peptides. Only one, however, was annotated as tyrosine kinase (EHI\_118410), while the rest were all genes for HPs.

Most of the modulated genes were encoded on different scaffolds. Thirty-four genes (30.6% of EHI-probe sets), however, formed 15 sets of dyads or triads that occupied the same contig (Table S7). Included were genes for four AIG1 family proteins (EHI\_126550, EHI\_126560, EHI\_176700, EHI\_176580) and two Ras family proteins (EHI\_045450, EHI\_045600). In four sets of genes, the members were found to be located on opposite strands, but the regulation of their expression was the same and their foldchanges were comparable. For some genes found on the same strand, the distance between the genes was less than 1 kb. It was therefore possible for these genes to be co-regulated by the same transcription factor.

#### Functional Analysis

To study further the relationship between the expression of some of the modulated genes in MTZR with drug resistance, generation of stable transformants overexpressing HA-tagged HP1, ISF1, ISF2, and ISF4 was performed (**Figure 4A**). The mRNA level of these proteins was estimated by qRT-PCR. The levels of mRNA of HP1 and ISFs were about 5.5 or 2.5 to 3 fold, respectively, higher in MTZR compared to their expression in HM-1 transfected

#### TABLE 2 | List of annotated genes that were upregulated in MTZR by >3 fold compared to HM-1.

with mock vector (pEhEx-HA). Transformants cultured with 10µM G418 were challenged with MTZ for 48 h (**Figure 4B**). The highest concentration shown was based on the IC50 of MTZ against HM-1, performed under the same conditions (6.5µM). No significant difference in percent survival was observed at all concentrations tested between the overexpressors and control (**Figure 4B**). Neither was significant difference in survival against hydrogen peroxide, observed, among the transformants (**Figure 4C**).

#### Discussion

#### Differences in MTZR Generation in This Study and Previous Works

Currently, only 4 studies have been published regarding E. histolytica and its response to or resistance to MTZ (Samarawickrema et al., 1997; Wassmann et al., 1999; Leitsch et al., 2007; Tazreiter et al., 2008). These studies either exposed wild-type cells to serum level concentrations of the drug (70–100µM) or generated drug resistant trophozoites, similar to what we did in this study. However, the MTZ concentrations used in these studies and ours differ, which might explain some of the differences observed. The concentration used here is slightly higher compared to those used by Samarawickrema et al. 10µM (Samarawickrema et al., 1997), but significantly lower than those used by Wassmann et al. 40µM (Wassmann et al., 1999), Tazreiter et al. 50µM (Tazreiter et al., 2008), and Leitsch et al. 50µM (Leitsch et al., 2007). Our strain adapted more quickly to 1µM MTZ compared to the strain used by Samarawickrema (7 vs. 38 days) but overall the length of time used to generate MTZR was comparable.

For reasons we do not understand, our attempts to further increase the level of resistance failed, and no resistance above the concentration used here could be established. We could

#### TABLE 3 | List of annotated genes that were downregulated in MTZR by > 3 fold.

#### TABLE 4 | qRT-PCR validation of selected genes from our microarray data.

not culture MTZR consistently at drug concentrations higher than 12µM. In contrast, strains of Trichomonas and Giardia can be readily adapted to grow at 584µM and 115µM MTZ, respectively (Kulda et al., 1984; Townson et al., 1992). Our results are therefore consistent with previous reports indicating that MTZ resistant strains are more difficult to generate in E. histolytica (Samarawickrema et al., 1997). While the concentration we used is several times lower than serum levels after MTZ treatment (Van Oosten et al., 1986), the changes we observed may still provide insights as to what occurs in the parasite in vivo. It was previously reported that in an abscess, parasites encounter significantly lower levels of drug than those found in the serum as drug penetration is limited by poor perfusion and mechanical barriers such as fibrin clots and the abscess wall (Sirinek, 2000).

#### Decrease in Growth and Reversibility of Resistance in MTZR

Formation of drug resistance is often accompanied by fitness costs (Andersson and Hughes, 2010). One particular cost,

reduction in growth rate, is the main parameter that determines the rate by which resistance develops and level of resistance reached. Similar to resistant strains previously generated (Samarawickrema et al., 1997; Wassmann et al., 1999), the doubling time of MTZR was longer compared to HM-1. Fitness cost in growth associated with drug resistance are well documented in mammalian cells, e.g., lymphoma and cancer cells (Lee, 1993; Bishop et al., 2001). It should be noted, however, that the growth kinetics of MTZR in the absence of drug pressure improved over time. This indicates that some of the drugselection-induced changes developed by MTZR were temporal and reversible. This also indicates that cells in the culture may have varying degrees of resistance. In addition, MTZR (−) that was re-exposed to MTZ showed significantly lower growth compared to MTZR, but slightly higher compared to HM-1, which may indicate that some drug-selection-induced changes in MTZR were still operative even after the removal of drug pressure.

#### Cross-Resistance in MTZR

The resistance level displayed by MTZR is significantly lower compared to previous reports: the IC50 value of 12µM in this study compared to 40µM in the previous study (Samuelson, 1999; Wassmann et al., 1999). However, its survival in otherwise lethal concentration of MTZ for long periods of incubation, does indicate reduced sensitivity and resistance. The cross-resistance of MTZR to ornidazole and tinidazole is not surprising, because both are 5-nitroimidazoles like MTZ. It is therefore likely that their mode of action and mechanism of resistance were similar (Pasupuleti et al., 2014). T. vaginalis isolates that were characterized as having very high resistance to MTZ were similarly insensitive to tinidazole (Narcisi and Secor, 1996). In the case of paromomycin, emetine, chloroquine, and hydrogen peroxide, it is possible that the reduced sensitivity was nonspecific and could be due to an increased capacity of MTZR to deal with stress. Studies have shown that cell lines resistant to one drug are often cross-resistant to closely related drugs or to unrelated compounds when their mode of action is the same or when they carry membrane alterations (Upcroft and Upcroft, 1993).

#### Decreased Adhesion, Phagocytosis, Cytolysis, and Virulence in MTZR

In E. histolytica, establishment and outcome of infection depend heavily on adhesive and invasive capacity (Flores-Romo et al., 1997). Recently, drug resistance has been shown to modulate these factors and vice versa (Giha et al., 2006). The decreased rate of adhesion observed in MTZR indicated that MTZ selection negatively affected the synthesis or expression of surface adhesion molecules. While other studies have shown that cell adhesion is a key determinant in drug resistance (Shain and Dalton, 2001), this apparently was not the case in MTZ resistance. Inhibition of adherence in this parasite has previously been

shown to decrease host cell cytotoxicity (Ravdin et al., 1985) and experimental evidence strongly suggests the involvement of surface adhesins in phagocytosis (Heron et al., 2011). As shown in the results, MTZR also had decreased cytopathy and phagocytosis (**Figures 2A,C**). The decreased band intensity for CP1 and CP2 in the zymogram may partially explain this phenotype. CP1 has been shown to be upregulated in a mouse model of amebic colitis following invasion (Gilchrist et al., 2006), while CP2 has been reported to contribute to intestinal damage and liver abscess formation (Hellberg et al., 2001). Our microarray data, however, did not show any significant repression of these genes, except for two uncharacterized CPs that were repressed by more than 7 folds. These CPs had high similarity (81%) to CP7 (also called EhCP-B1) based on amino acid sequence (Tillack et al.,

transformants were cultured with 25–1600µM hydrogen peroxide for 4 h.

2007). No significant change was observed in the expression of the genes known to be involved in the inactivation and intracellular trafficking of CPs such as intrinsic inhibitors of CPs (Sato et al., 2006) and cysteine protease binding protein family 1 (Nakada-Tsukui et al., 2012) and other lysosomal hydrolase carriers (Furukawa et al., 2012, 2013; Marumo et al., 2014). It is also possible, however, that resistance to MTZ may not necessarily result to decreased virulence in vivo.

#### Implications from Transcriptomic Profiling of MTZR

Our transcriptomic profiling suggests that the transcriptomic changes we observed in MTZR strain cultured with MTZ were a combination of the changes responsible for MTZ resistance and those associated with the adaptation to stresses caused by the exposure to MTZ. However, we believe that among the observed changes, the changes that were specific to MTZR strain with MTZ and absent in HM-1 with MTZ were responsible, at least in part, for resistance, rather than adaptation. This is based on the following observations: the transcriptomic profile of E. histolytica HM-1 cultured with MTZ was remarkably different from that of MTZR strain cultured with MTZ, while MTZR strain cultured with or without MTZ showed similar transcriptomic profiles. In other words, a larger number of common changes in gene expression were identified between MTZR strain cultured with MTZ and those without MTZ when compared between MTZR strain cultured with MTZ and HM-1 cultured with MTZ.

GO enrichment of MTZR-associated transcriptomic changes revealed regulation of genes encoding for proteins potentially involved in nucleotide binding, metabolism, oxidative stress response, and signal transduction. These are apparently the processes most influenced by MTZ resistance. However, since most of the genes are without GO annotations, it is likely that additional processes are involved. When we examined the genomic locations of these genes, we found that 30% of them formed dyads or triads that occupied the same contig. While some genes forming dyads and triads are apart by more than 10 kb, the distance between some of the adjacent genes in dyads and triads was less than 1 kb. Thus, co-expression of some genes in dyads and triads in MTZR could be attributed to a shared regulatory system.

Consistent with previous findings, no significant difference in PFOR mRNA levels was observed between MTZR and HM-1 (Samarawickrema et al., 1997; Wassmann et al., 1999; Tazreiter et al., 2008). This observation could mean 3 things. First, it's likely that the level of resistance achieved in this study was not sufficient to downregulate PFOR, although a similar finding was reported in strains resistant to 40µM MTZ (Wassmann et al., 1999). Second, since E. histolytica lacks pyruvate dehydrogenase (Clark et al., 2007; Tazreiter et al., 2008) or pyruvate decarboxylase (Lo and Reeves, 1978), the significance of PFOR cannot be overstated. The lack of an alternative enzyme to compensate for its downregulation makes this enzyme difficult to repress without severe consequences to the ameba. Third, in contrast to Giardia and Trichomonas, lack of PFOR downregulation may indicate the existence of an alternative mechanism for MTZ resistance in this parasite. It should be noted, however, that clinically resistant isolates of Trichomonas have been reported with no decrease in PFOR activity (Müller and Gorrell, 1983).

Two previous studies have reported the upregulation of Fe-SOD and downregulation of Fdx in the MTZ resistant ameba (Samarawickrema et al., 1997; Wassmann et al., 1999). In the previous work, fold changes were determined based on enzyme activity assay or northern blot (Samarawickrema et al., 1997; Wassmann et al., 1999). Disparity, however, between changes in mRNA abundance and enzyme activity have been reported, and in some cases, increases in enzyme activity can exceed those of mRNA abundance (Glanemann et al., 2003). Interestingly, the lack of Fe-SOD modulation in cells exposed to MTZ was also reported by Tazreiter et al. (2008). On the contrary, in this work, neither gene was modulated significantly. It is possible that similar to PFOR, differences in resistance levels could explain the lack of modulation in the levels of Fe-SOD and Fdx. Other studies also indicated that Fe-SOD and Fdx are not tightly associated with MTZ resistance. It was shown that parasites in which Fdx was repressed did not display full resistance to MTZ (Rasoloson et al., 2002) and in bacteria, increased SOD activity was not always associated with MTZ resistance (Smith and Edwards, 1995).

In this study, genes involved in nucleic acid synthesis and binding, stress response, metabolism, and vesicular trafficking were mostly affected in MTZR. The significance of these genes relative to phenotypic changes and drug resistance will be discussed here through their known roles in E. histolytica or in other organisms, in regulating key cell functions. One should also note that since only a single MTZ resistant line was analyzed in this study, it is possible that MTZ resistance can be also conferred by the mechanisms that are independent of those described in this study.

#### Nucleic Acid Synthesis and Binding

In prokaryotes, upregulation of DNA polymerases imparts plasticity to their genome, allowing them to adapt in unfavorable environmental conditions (Joseph et al., 2006). It has been suggested that pathogenic bacteria utilize this adaptation to develop resistance against therapeutic agents (Karpinets et al., 2006). Since it has been shown that primary effect of MTZ is rapid inhibition of DNA replication (Ludlum et al., 1988; Sisson et al., 2000; Leitsch et al., 2007), the upregulation of a DNA polymerase (EHI\_164190) in this study may indicate that this mode of action is operative in MTZ resistance. One hypothetical protein identified in this study (HP1, EHI\_006850) has some similarity to zinc finger proteins, which are known to be involved in DNA recognition, RNA packaging, and transcriptional activation (Laity et al., 2001). It has an NAD+ binding pocket and a tetrachlorodibenzo-p-dioxin (TCDD) inducible poly(ADP-ribose) polymerase (PARP)-like domain that is involved in the attachment of ADP-ribose units to DNA-binding proteins (Ma et al., 2001). It's also known to be a regulatory component induced by DNA damage and an enzyme involved in DNA repair (Nguewa et al., 2006; Marchler-Bauer et al., 2011). The dUTP nucleotidohydrolase (EHI\_072960) upregulated in MTZR may be involved in the removal of dUTP from the dNTP pool, de novo biosynthesis of dTTP, and DNA replication as reported (Richards et al., 1984; Gadsden et al., 1993). In cancer cells, high level of dUTPase is implicated as a mechanism of resistance to chemotherapy (Ladner et al., 2000).

#### Stress Response

ISFs that were modulated in MTZR are part of family of redoxactive proteins mainly found in anaerobic prokaryotes (Andrade et al., 2005). In NCBI, 18 ISF genes are annotated in the E. histolytica genome, although 50% of these are identical proteins (http://www.ncbi.nlm.nih.gov/). In this study 7 ISF probe sets were upregulated by at least 4 folds in MTZR. The overexpression of ISF1 (EHI\_138480) was also observed in HM-1 (+), while ISF2 was upregulated in both MTZR (−) and HM-1 (+). These 2 ISFs were upregulated in HM-1 (+) by least 20 folds, compared to 6 to 11 folds in MTZR, hinting that these genes are involved in stress response. These two genes were also induced in E. histolytica cultured under L-cysteine deprived and oxidative stress conditions (Vicente et al., 2009; Husain et al., 2011), but were downregulated in a mouse model of intestinal amebiasis (Gilchrist et al., 2006). Our findings together with these reports suggest that induction of these ISFs, particularly ISF1, is an adaptation that may enhance survival when cells experience environmental stress.

#### Metabolism

Several metabolic genes were upregulated in MTZR. NADspecific glutamate dehydrogenase (EHI\_075150) catalyzes the oxidative deamination of glutamate to α-ketoglutarate using NAD as cofactor (Plaitakis and Zaganas, 2001; Girinathan et al., 2014). Most likely, its upregulation is a part of stress response. In humans, its activity is regulated through ADP-ribosylation, and interestingly, Arf1 (EHI\_189960) was also upregulated in this study (Herrero-Yraola et al., 2001). Chitinase (EHI\_092100) (Escueta- De Cadiz et al., 2013) was also upregulated in MTZR and MTZR (−) by at least 3 folds, although its precise role in drug resistance is not known. In plants, its upregulation plays a defensive role against toxins and antibiotics (Gooday, 1999). Aldose reductase (EHI\_029620), on the other hand, can reduce a broad spectrum of substrates including cytotoxic aldehydes (Ramana, 2011). Its upregulation in human liver cancer cells was associated with resistance to daunorubicin and was confirmed by sensitizing cells to the drug by the addition of aldose reductase inhibitors (Lee et al., 2001).

The decrease in cell growth observed in MTZR may be partially explained by the repression of phosphoserine aminotransferase (EHI\_026360) (Ali and Nozaki, 2006; Mishra et al., 2010), which catalyzes the conversion of 3-phosphohydroxypyruvate to L-phosphoserine, the second step of phosphorylated serine biosynthetic pathway (Ali and Nozaki, 2006). L-serine has a well-recognized role in cell proliferation, providing precursors for amino acids, protein synthesis, and nucleotide synthesis (Nozaki et al., 2005; Tabatabaie et al., 2010). The downregulation of this enzyme, therefore, may contribute to the decreased growth rate. The repression of lecithin: cholesterol acyltransferase (EHI\_020250) may also be associated with decreased growth rate, because the enzyme is crucial in the maturation, remodeling, and metabolism of high density lipoprotein, HDL (Calabresi et al., 2011).

#### BspA1 Gene Family

Several genes encoding for leucine-rich repeat (LRR) proteins of the BspA family were also modulated in MTZR, and were the most downregulated genes with fold changes of more than 150. LRR is known to provide a structural framework for the formation of protein-protein interactions and serve as recognition motifs for surface proteins (Kobe and Kajava, 2001). BspA family protein was originally identified in Bacteroides forsythus and the LRR motifs of the BspA protein have been linked to cell binding to components of the extracellular matrix (Sharma et al., 1998). It is therefore possible that their downregulation may partially explain the reduced adhesion seen in MTZR. It was previously shown that an LRR BspA family protein in E. histolytica was located primarily on the plasma membrane, although it did not show whether the protein was involved in surface interactions (Davis et al., 2006).

#### AIG1 Gene Family

The E. histolytica AIG1 gene family consists of 47 members (Biller et al., 2010), and in this study 10 genes were found to be differentially expressed by 3 to 11 folds. Members of this protein family contain a domain found in Arabidopsis protein AIG1, which is likely involved in plant recognition and resistance to bacterial pathogens (Reuber and Ausubel, 1996). In E. histolytica, however, some AIG proteins appear to be linked with virulence. Comparative genomic analysis of Japanese clinical isolates indicated that an AIG family protein (EHI\_176590) and the region around it were uniquely present in KU50, a strain isolated from a diarrheic patient, but absent in a non-pathogenic isolate called KU27 (Nakada-Tsukui et al., unpublished data). Interestingly, the same gene was downregulated by 2.7 folds in MTZR, and two adjacent genes encoding for AIG family proteins (EHI\_176580 and EHI\_176700) were also repressed by 3 to 4 folds. A previous study also reported the upregulation of an AIG family protein (EHI\_180390) in a pathogenic strain of E. histolytica (Biller et al., 2010), although here the same gene was downregulated by 5 folds. It is therefore possible that the downregulation of these AIG family proteins might be related to the decrease in MTZR's cytopathogenicity.

Overall, we believe that these transcriptional changes participate in a global regulatory response resulting to the fitness costs and adaptive responses we observed in MTZR. The fact that more genes were upregulated than downregulated also suggests that increasing the amount of cellular components that play roles in stress response, DNA repair and others, is more important in developing low levels of resistance to MTZ than repression of proteins involved in activating the drug. Indeed, the most profound difference we observed between MTZR and HM-1 was the upregulation of genes for several ISF proteins. It should be noted, however, that more than half of the genes reported in this study are hypothetical. It is also likely that the effects of the repressed genes, although fewer in number, are more important in explaining the growth defect.

#### Functional Analysis of Potential Resistance Genes

In this study, we examined possible role of genes encoding HP1 and several ISF proteins in MTZ resistance. However, their episomal transfection and overexpression did not confer MTZ resistance to the extent observed in MTZR. Thus, at present, direct causal connection between HP1 and ISF overexpression and MTZ resistance has not been demonstrated. It is likely that in E. histolytica, resistance to the drug is multi-factorial in nature and requires the global regulation of genes involved in several key cell processes and not just DNA repair and stress response.

In conclusion, here we described the biological state of E. histolytica that was selected for MTZ resistance in vitro. Phenotypic and transcriptional profiling revealed fitness costs and adaptive responses that we could associate with drug resistance. Some of the consequences of resistance included decreased cell growth and virulence, which have significant implications on host-pathogen interaction and infection outcome. Comparative transcriptional analysis, on the other hand, revealed genes not previously associated with MTZ resistance in this parasite. The list of differentially transcribed genes also did not indicate in MTZR a reduced capacity to activate MTZ, but hinted on adaptations to tolerate the lethal effects of MTZ. Identifying the signaling mediators for the proteins that these genes encode, however, requires further investigation. Finally, while our data showed that high levels of MTZ resistance cannot be readily induced in vitro, the data presented here may still help researchers in better understanding the mechanisms involved should clinical cases of high levels of resistance are reported in the future.

# Acknowledgments

We thank Takashi Makiuchi, Ghulam Jeelani, Yumiko Saito-Nakano, and Joseph Marzo for valuable discussions. We thank Dr. Kentaro Hanada, Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases for providing CHO cells. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (23117001, 23117005, 23390099, 14506236), a grant for research on emerging and re-emerging infectious diseases from the Ministry of Health, Labour and Welfare of Japan (H23-Shinkosaiko-ippan-014, H26- Shinkosaiko-ippan-009), a grant for research to promote the development of anti-AIDS pharmaceuticals from the Japan Health Sciences Foundation (KHA1101).

# Supplementary Material

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb. 2015.00354/abstract

## References

relationship in Plasmodium falciparum causing severe malaria in an area of seasonal and unstable transmission. Acta Trop. 97, 181–187. doi: 10.1016/j.actatropica.2005.10.004

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2015 Penuliar, Nakada-Tsukui and Nozaki. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Quantitative RT-PCR assay for high-throughput screening (HTS) of drugs against the growth of Cryptosporidium parvum in vitro

#### Haili Zhang and Guan Zhu\*

*Department of Veterinary Pathobiology, College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, College Station, TX, USA*

#### Edited by:

*Anjan Debnath, University of California, San Diego, USA*

Reviewed by:

*Xianming Chen, Creighton University, USA Nigel Yarlett, Pace University, USA Momar Ndao, McGill University, Canada*

#### \*Correspondence:

*Guan Zhu, Department of Veterinary Pathobiology, College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, 4467 TAMU, College Station, TX 77843-4467, USA<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology*

Received: *09 May 2015* Accepted: *04 September 2015* Published: *22 September 2015*

#### Citation:

*Zhang H and Zhu G (2015) Quantitative RT-PCR assay for high-throughput screening (HTS) of drugs against the growth of Cryptosporidium parvum in vitro. Front. Microbiol. 6:991. doi: 10.3389/fmicb.2015.00991* Our laboratory has previously developed a qRT-PCR assay to assess drug efficacy on the growth of *Cryptosporidium parvum in vitro* by detecting the levels of parasite 18S rRNA. This approach displayed up to four orders of magnitude of linear dynamic range and was much less labor-intensive than the traditional microscopic methods. However, conventional qRT-PCR protocol is not very amendable to high-throughput analysis when total RNA needs to be purified by lengthy, multi-step procedures. Recently, several commercial reagents are available for preparing cell lysates that could be directly used in downstream qRT-PCR analysis (e.g., Ambion Cell-to-cDNA kit and Bio-Rad iScript sample preparation reagent). Using these reagents, we are able to adapt the qRT-PCR assay into high-throughput screening of drugs *in vitro* (i.e., 96-well and 384-well formats for the cultivation of parasites and qRT-PCR detection, respectively). This qRT-PCR protocol is able to give a >150-fold linear dynamic range using samples isolated from cells infected with various numbers of parasites. The new assay is also validated by the NIH-recommended intra-plate, inter-plate, and inter-day uniformity tests. The robustness and effectiveness of the assay are also confirmed by evaluating the anti-cryptosporidial efficacy of paromomycin and by a small scale screening of compounds.

Keywords: Cryptosporidium parvum, in vitro, qRT-PCR, high-throughput screening, assay development, paromomycin

# Introduction

Cryptosporidium is a genus of protozoan pathogens belonging to the Phylum Apicomplexa that infect humans and/or animals. Among them, C. parvum and C. hominis are the two major species causing water-borne and food-borne diarrheal illness in humans (Baldursson and Karanis, 2011; Budu-Amoako et al., 2011). These two and some other species, including C. meleagridis, C. muris, C. canis, and C. felis, also cause opportunistic infections in AIDS patients (AIDS-OIs) that can be chronic and fatal (Thompson et al., 2005; Feasey et al., 2011; O'connor et al., 2011; Shirley et al., 2012). A more recent multi-country study also revealed that Cryptosporidium is one of the top five diarrheal-causing pathogens in children in developing countries, in which the infection increased death rate and negatively affected the growth of children (Kotloff et al., 2013). However, options to treat cryptosporidiosis are limited. In fact, only a single drug, nitazoxanide (NTZ) is approved by the U.S. Food and Drug Administration (FDA) to treat human cryptosporidial infection in immunocompetent patients. Moreover, NTZ is ineffective in treating cryptosporidiosis in AIDS patients, and its efficacy in immunocompetent patients is also debatable (Cabada and White, 2010; Feasey et al., 2011; O'connor et al., 2011; Checkley et al., 2015).

Two approaches are commonly employed in early stages of drug discovery against infectious diseases. Hits and leads may be identified from new chemical entities by directly testing their efficacies against an in vitro or in vivo model of disease (traditional methods), or by screening compounds against drug targets from a pathogen such as essential enzymes or receptors (target-based methods). In the later case, identified hits/leads will still be evaluated on their in vitro efficacy before being advanced to in vivo drug testing. Therefore, the availability of a good in vitro disease model is critical in drug discovery against diseases including cryptosporidiosis. In vitro models of cryptosporidial infection are available, including the cultivation of C. parvum in HCT-8 or Caco-2 cells (Arrowood, 2002; Cai et al., 2005; Karanis and Aldeyarbi, 2011). However, there is a need for developing new methods that can efficiently evaluate the parasite growth in response to drug treatment in highthroughput or high-volume format. Conventional microscopic examination to count the numbers of parasites stained chemically or by immunofluorescence labeled antibodies is highly laborintensive and sometimes subjective, making it unsuitable for testing a large number of drugs. ELISA and chemiluminescence immunoassay methods were reported, but they appeared to have relatively narrow linear dynamic ranges of detection (i.e., the ratio between the largest and smallest signal values of detection is proportional to the difference between the two samples, such as the relationship between the OD values and concentrations of an antigen in an ELISA assay) and heavily relying on the quality and availability of specific antibodies.

More recent approaches include quantitating parasite loads by quantitative PCR (qPCR), qRT-PCR and automated fluorescence imaging analysis that are generally more advantageous than the earlier methods. Among them, the qRT-PCR method originally developed in our laboratory evaluates drug efficacy by detecting the relative levels of parasite 18S rRNA transcripts, which displays up to four orders of magnitude of linear dynamic range between the cycle threshold (CT) value and the logarithm of the number of oocyst inoculum (Cai et al., 2005; Ctrnáctá et al., 2010; Zhang et al., 2012). However, classic qRT-PCR protocol is not very amendable to the high-throughput or high-volume analysis when total RNA needs to be purified by lengthy, multi-step procedures.

Here we report an improved and simplified qRT-PCR protocol for evaluating of drug efficacies against the growth of C. parvum in vitro. This new protocol takes advantage of the commercially available lysis buffers to prepare cell lysates that could be directly used in downstream qRT-PCR analysis. We also re-optimized the primers and procedures, and adapted the in vitro cultivation of parasite in 96-well plates and real-time RT-PCR detection in 384 well plates, making the protocol suitable for high-throughput or high-volume evaluation of drug efficacy against the growth of C. parvum in vitro.

# Materials and Methods

#### Cultivation of Parasite In Vitro

Fresh oocysts of C. parvum (Iowa strain) were purchased from Bunch Grass Farm (Deary, ID). Oocysts were further purified by a Percoll-based gradient centrifugation method and surface sterilized with 10% bleach for 7 min on ice, followed by washes with phosphate-buffered saline (PBS). Only C. parvum oocysts less than 3 months old were used in all experiments. An ileocecal colorectal adenocarcinoma cell line (HCT-8, ATCC # CCL-244) was used to host the growth of C. parvum in vitro. One day before the inoculation, HCT-8 cells were seeded in 96-well plates (2.5 × 104 cells/well) containing RPMI 1640 medium supplied with 10% fetal bovine serum (200µL medium/well in all experiments) and allowed to grow overnight at 37◦C under 5% CO2 atmosphere until they reached ∼90% confluence. The infection and growth of C. parvum in vitro in 96-well plates were examined by differential interference contrast (DIC) microscopy and immunofluorescence (IF) labeling using a rabbit polyclonal antiserum raised against C. parvum sporozoite total membrane proteins (Zeng and Zhu, 2006; Zeng et al., 2006; Fritzler et al., 2007), and by qRT-PCR (as described below).

For generating standard curves, host cells were inoculated with five-fold serial dilutions of parasite oocysts (i.e., 4 × 104– 64 oocysts/well), responding to the ratios between parasite and host cells ranging from ∼1:1 to 1:625. For drug testing, host cells were infected with 2 × 104 oocysts per well (i.e., ratio 1:2). After inoculation, parasite oocysts were allowed to undergo excystation and invasion into host cells for 3 h at 37◦C. Free parasites and oocyst walls in the medium were removed from the plates by an exchange of the culture medium. Drugs at specified concentrations were added into the culture at this time point (i.e., immediately after the medium exchange). Parasite-infected cells were then incubated at 37◦C for additional 41 h (i.e., total 44 h infection time). For dose response of paromomycin (PRM), 3 × serial diluted PRM from 30 to 800µg/mL (i.e., 42–1120µM) was added to infected cells after 3 h post-infection (hpi) and the treatment lasted for 41 h. At least two independent experiments were conducted for every experimental condition, each including two replicates drugs and three replicates for negative and positive controls.

#### Preparation of Cell Lysates

Plates containing HCT-8 cells infected with C. parvum for 44 h and uninfected cells grown under the same condition were first centrifuged in a Sigma 2–5 plate centrifuge (Sigma Laborzentrifugen GmbH, Germany) for 10 min at 1000 × g to ensure that free merozoites in the medium were firmly settled on the bottom of the wells. Medium was removed by gentle flicking the plate over a container placed in the biosafety cabinet, followed by two gentle washes with PBS. After removal of PBS and blotting plates on paper towels to remove remaining drops, plates were placed on ice in a rectangle bucket. An eight-channel electronic pipette (Rainin Instrument, Oakland, CA) was used to add PBS and lysis buffer in subsequent procedures.

For extracting total RNA used in high-throughput screening (HTS), 200µL of ice-cold Bio-Rad iScript qRT-PCR sample preparation reagent (lysis buffer) (Bio-Rad Laboratories, Hercules, CA) was added into each well. Plates were sealed with adhesive and heat sealing films and the bucket containing racked plates on ice was secured in a multi-tube vortexer (VX-2500, VWR International, Radnor, PA) and subjected to vortex for 20 min at the speed set at 7. Plates were then incubated at 75◦C for 15 min, followed by centrifugation (5 min, 2000 × g) to settle down cell debris. Supernatants were used immediately in subsequent qRT-PCR reactions or the plates were stored at −80◦C until use.

#### Real-time qRT-PCR Assay

The levels of 18S rRNA transcripts from C. parvum and host cells (referred to as Cp18S and Hs18S) were detected by real-time qRT-PCR method using qScript™ one-step SYBR green qRT-PCR kit (Quanta Biosciences, Gaithersburg, MD). Cell lysates prepared as described above were diluted by 100 and 2000 folds for detecting Cp18S and Hs18S transcripts, respectively. Reactions were performed in hard-shell 384-well skirted PCR plates (Bio-Rad Laboratories, Hercules, CA) (10µL/well) containing 3µL diluted cell lysate, 5µL one-step SYBR green master mix, 0.2µl RT master mix and the following primers: Cp18S-1011F (5′ -TTG TTC CTT ACT CCT TCA GCA C-3′ ) and Cp18S-1185R (5′ - TCC TTC CTA TGT CTG GAC CTG-3′ ) primer pair for Cp18S rRNA (GenBank accession number: NC\_006986.1), and Hs18S-1F (5′ -GGC GCC CCC TCG ATG CTC TTA-3′ ) and Hs18S-1R (5′ -CCC CCG GCC GTC CCT CTT A-3′ ) primer pair for Hs18S rRNA (GenBank accession number: NR\_003286). Among them, Cp18S-1011F and Cp18S-1185R were described in our earlier studies reporting the original qRT-PCR assay and the efficacy of S-adenosylhomocysteine hydrolase inhibitors against the growth of C. parvum in vitro (Cai et al., 2005; Ctrnáctá et al., 2010), whereas Hs18S-1F and Hs18S-1R represented our newly optimized primers (that were also described in one of our more recent publications (Zhang et al., 2012). Hs18S levels were used as controls and for normalization. All reagents for the qRT-PCR were loaded using an epMotion 5070 automated pipetting system (Eppendorf, Hauppauge, NY).

Real-time qRT-PCR reactions were performed by a Bio-Rad CFX384 Touch Real-Time PCR Detection System. The reactions started with synthesizing cDNA at 50◦C for 20 min, followed by 5 min at 95◦C to denature RNA-cDNA hybrids and deactivate reverse transcriptase, and 40 two-temperature thermal cycles of PCR amplification at 95◦C, 10 s and 58◦C, 30 s. At the end of PCR amplification, melting curve analysis was performed between 65 and 95◦C. At least two technical replicates were included in qRT-PCR reactions for each sample.

After qRT-PCR reactions were completed, amplification curves and melting peaks were examined to assess the quality and specificity of the reactions, followed by the computation of relative parasite loads based on the cycle threshold (CT) values of Cp18S and Hs18S transcripts as previously described. Briefly, the means of CT values from technical replicates were first obtained for individual biological replicates, which were used to compute △CT values between Cp18S and Hs18S (i.e., △CT = CT[Cp18S]-CT[Hs18S]), and △△CT values between each experimental sample and control (i.e., △△CT = △CT[sample]-△CT[control]). Standard curves were generated by plotting △CT values against the logarithm of the oocyst numbers used to inoculate cells, followed by linear regression to obtain the slope value (parameter A in Equation below) to calculate the detection efficiency and parasite loads using the following equation as described (Cai et al., 2005):

$$Inhibtion\,(\%) = (1 - 10^{A \cdot \Delta \, \Delta C\_T}) \cdot \text{(100)}$$

Because all samples contained much lower levels of Cp18S rRNA than those of Hs18S rRNA, we used two different dilution factors (i.e., 100- and 2000-fold dilutions of lysates for detecting Cp18S and Hs18S, respectively) as described above to maintain the linearity of detections. These dilution factors needed to be considered in calculating the relative level between Cp18S and Hs18S in a specified sample:

$$Relative\left[Cp18\text{S}\right] = \frac{\left[Cp18\text{S}\right]}{\left[Hs18\text{S}\right]} = \left(\frac{100}{2000}\right) \cdot 10^{A\cdot\Delta C\_T\left[Cp18\text{S}\right] - H18\text{S}}\text{ [}\cdot\text{]}$$

However, the effect of the dilution factors was eliminated in subsequent computation of the relative growth of C. parvum (Cp) between a sample (s) and control (c) based on 11C values:

$$\begin{split} \text{Relative } & Cp \,\text{growth} = \frac{\left(\frac{[Cp18S]\_\text{s}}{[Hs18S]\_\text{s}}\right)}{\left(\frac{[Cp18S]\_\text{c}}{[Hs18S]\_\text{c}}\right)} = \frac{\left(\frac{100}{2000}\right) \cdot 10^{A \cdot \Delta C\_{T[s]}}}{\left(\frac{100}{[Bt18S]\_\text{c}}\right)} \\ & = 10^{A \cdot \Delta \Delta C\_{T[s-\iota]}} \end{split}$$

#### Evaluation of the Growth of Host Cells and Parasite in 96-well Plates

We also evaluated the growth of C. parvum in 96-well plates by qRT-PCR and microscopy, in which HCT-8 cells were cultured and infected with C. parvum as described above. For qRT-PCR analysis, total RNA was isolated from cells using an RNeasy RNA isolation kit (Qiagen), rather than directly using cell lysates, to ensure more precise measurements of the levels of Cp18S and Hs18S transcripts. RNA samples included those from cells infected with C. parvum for 3 and 44 h, respectively, and from uninfected cells cultured under the same condition and for the same time periods. For microscopic examination, sterilized glass coverslips were gently broken into pieces and placed into selected wells before seeding host cells. Cells infected with parasites for 3 and 44 h were fixed in 3.7% paraformaldehyde and stained with a rabbit polyclonal antibody against total C. parvum membrane proteins following standard protocols (Chen et al., 2003; Zeng et al., 2006). Samples were then mounted onto glass microscopic slides with a Anti-Fade mounting medium containing 4′ ,6 diamidino-2-phenylindole (DAPI) for counter-staining nuclei (Molecular Probes, Invitrogen), and examined under Olympus BX51 Research Microscope.

#### Assay Quality Assessment

In addition to determining the linear dynamic range from standard curve derived from samples infected with varied numbers parasites, the uniformity and signal variability of the assay were also validated according to the "Assay Guidance Manual" (see chapter "HTS Assay Validation" at http://www. ncbi.nlm.nih.gov/books/NBK53196/) (Iversen et al., 2012). This method is recommended by the National Center for Advancing Translational Sciences (NCATS), National Institutes of Health (NIH) (see "Pre-Clinical Research Toolbox" at http://www.ncats. nih.gov/expertise/preclinical). In this study, inter-day tests were performed by three independent experiments. Each experiment used three 96-well plates to assess inter-plate variations, and each plate contained three experimental groups, which included uninfected HCT-8 cells ("Min" background signal), cells infected with parasites but untreated ("Max" signal), and cells that were infected with parasites and treated with 120µg/mL (168µM) of PRM ("Mid" signal). These groups (32 wells each) were arranged in an interleaved-signal format according to the Assay Guidance Manual.

The cultivation of host cells and C. parvum in vitro, preparation of cell lysates, and qRT-PCR assay were performed as described above. PRM was added into specified wells after the invasion and the removal of uninfected parasites. At the same time, DMSO at 0.5% final concentration was added into all wells. Based on the qRT-PCR data, mean (AVG), standard deviation (SD), and coefficient of variation (CV) were calculated for each group in each plate. Signal window (SW) and Z′ factor for each plate were calculated based on the Assay Guidance Manual as follows (Iversen et al., 2012):

$$SW = \frac{\left(AVG\_{\max} - 3SD\_{\max}/\sqrt{n}\right) - \left(AVG\_{\min} + 3SD\_{\min}/\sqrt{n}\right)}{SD\_{\max}/\sqrt{n}}$$

$$Z' = \frac{\left(AVG\_{\max} - 3SD\_{\max}/\sqrt{n}\right) - \left(AVG\_{\min} + 3SD\_{\min}/\sqrt{n}\right)}{AVG - AVG}$$

AVGmax − AVGmin

#### Small-scale Drug Screening

The assay is currently used in our laboratory to test drug efficacy and screen compound libraries against C. parvum in vitro. Here we report the data on 48 small molecules designated as C001–C048 to provide a snapshot on the assay performance. HCT-8 cells were cultured in 96-well plates and infected with C. parvum using procedures described above. In primary screening, drugs dissolved in DMSO were added into the culture immediately after the medium exchange at 3 h post-infection (hpi) time (10µM final concentration). Each plate contained five negative controls without receiving drug treatment and three positive controls treated by 100µg/mL (140µM) of PRM. Each well, including negative and positive controls, contained 0.5% DMSO. At 44 hpi time, cell lysates were prepared using Bio-Rad iScript lysis buffer and the anti-cryptosporidial efficacies of drugs were evaluated by qRT-PCR as described above. Compounds that inhibited >65% of the parasite growth were re-tested. Two of the most effective drugs were then selected to generate dose-response curves to determine IC50 values.

# Results and Discussions

#### Growth of Host Cells and Parasites in 96-well Format in the 44-h Infection Assay

Similar to other apicomplexans, Cryptosporidium sporozoites invade host cells and undergo at least two generation of asexual cell cycles (merogony) before entering sexual development (gametogenesis and fertilization between macro- and microgametes) to form oocysts. We have observed that most C. parvum cultured in 24- or 48-well plates may complete the second generation of merogony and a large number of free merozoites may be released into the culture medium at 48 hpi. This complicates the quantification of parasite loads because free merozoites in the medium may be easily washed off during the sample preparation. To minimize this problem, our laboratory uses the 44 h infection assay, in which the majority of the intracellular parasites are in the late stage of meronts and only a minimal number of merozoites are present in the medium. To confirm that C. parvum cultured in 96-well plates followed similar invasion and intracellular developmental timeline, we compared the parasite growth at 3 and 44 hpi by qRT-PCR and fluorescence microscopy. We also evaluate the host cell growth at the time points correlated to the 3 and 44 hpi by qRT-PCR.

Using qRT-PCR, we have observed rapid intracellular parasite growth (i.e., 34-fold increase of Cp18S rRNA from 3 to 44 hpi) (**Figure 1A**), which gave us a good dynamic range to quantify the relative parasite growth. For comparison, there was only ∼1.6 fold increase of uninfected Hs18S cultured for the same time period (**Figure 1B**), indicating that parasite grew >20 times faster than the HCT-8 cells in this cultivation system. We also observed that parasite infection slightly reduced the HCT-8 cell growth (i.e., ∼10% decrease based on Hs18S rRNA levels) (**Figure 1B**), which might be attributed by the arrest of host cell growth upon infection and cell death upon the completion of merogony development and the release of parasite merozoites as reported by other investigators (Dobbelaere and Küenzi, 2004; Brunet et al., 2008).

DIC microscopy and IF labeling revealed the successful invasion of C. parvum sporozoites into the host cells after 3-h initial incubation of oocysts with host cells, in which the elongated sporozoites were transformed to small round trophozoites (**Figure 1C**, 3 hpi panel). However, it was possible that some sporozoites might have attached to, but failed to invade into host cells or undergo further development at the 3 hpi time point. The growth of parasite into late stage of meronts as evidenced by the presence of multiple nuclei in many meronts by DAPI staining (**Figure 1C**, 44 hpi panel). Intracellular parasites in these stages were also clearly visualized by IF staining using a rabbit antiserum against C. parvum sporozoite membrane proteins (**Figure 1C**, TRITC staining). These observations indicated that parasite cultured in 96-well plates exhibited an intracellular development timeline similar to those cultured in 24- and 48-well plates. Because majority of the parasites still remained intracellular at 44 hpi, they could be lysed together with host cell monolayers for RNA extraction. However, we still included a plate centrifugation step before washing cell monolayers and preparing cell lysates to ensure any free merozoites released into the medium were not excluded from subsequent analysis.

#### Optimization the Sample Preparation used for Simplified qRT-PCR

Several commercial lysis buffers are currently available for preparing cell lysates suitable for qRT-PCR without further

FIGURE 1 | Comparison of the growths between Cryptosporidium parvum and host cells (HCT-8 cell line) cultured in 96-well plates for 44 h post-infection (hpi) as determined by detecting their relative levels of 18S rRNA transcripts. (A) The growth rate of *C. parvum* in HCT-8 cells between 3 and 44 hpi. The levels of 18S rRNA transcripts from the parasite (Cp18S) and host cells (Hs18S) were determined by qRT-PCR, and those of Hs18S were used for normalization before calculating the fold changes in Cp18S transcripts. (B) The relative levels of Hs18S in HCT-8 cells infected with *C. parvum* for 3 and 44 hpi. Uninfected cells were grown in parallel under the same condition for the same time periods. Bars represent standard error of the mean (SEM, *n* = 9) from three independent experiments. (C) Micrographs showing the development of *C. parvum* cultured in HCT-8 cells at 3 and 44 h post-infection (hpi) time points, in which intracellular parasites were labeled with a rabbit antiserum against *C. parvum* sporozoite total membrane proteins and a TRITC-conjugated goat anti-rabbit IgG secondary antibody and counter-stained with DAPI for nuclei. DIC, differential inference contrast microscopy. DAPI, 4′ , 6-diamidino-2-phenylindole for counterstaining of nuclei. TRITC, Tetramethylrhodamine.

purification of RNA (Ho et al., 2013). At the early of the project, we briefly evaluated Ambion Cells-to-cDNA™ II kit (Life Technologies) and iScript™ RT-qPCR Sample Preparation Reagent (Bio-Rad), and observed slight better performance in releasing intracellular parasite RNA by the Bio-Rad's reagent (data not shown) that was then used in our subsequent assay development. We also observed that standard sample preparation procedures recommended by the manufacturer was insufficient to release parasite RNA, largely because intracellular parasites were contained in parasitophorous vacuoles and protected by extra layers of membranes. We hence performed experiments to determine the optimal conditions including the amounts of lysis buffer and the addition of vortex and heating steps.

We observed that heat treatment of cell lysates at 75◦C for 10– 30 min significantly improved the release of parasite RNA. When samples were lysed in l00, 150, or 200µL/well lysis buffer, heat treatment for 10, 20, or 30 min reduced the CT[Cp18S] values from 24.4, 24.0, and 23.9 to 22.4, 22.3, and 22.2 in the 100 µL/well group, from 25.3, 24.9, and 24.5 to 23.6, 23.4, and 23.3 in the 150µL/well group, or from 25.6, 25.5, and 25.2 to 23.9, 23.6, and 23.6 in the 200µL/well group, respectively (p < 0.005 by Student t-test in all samples in comparison to the unheated counterparts) (**Figure 2**). However, we also observed certain levels of heatinduced host cell RNA degradation (i.e., CT[Hs18S] values in heated samples were increased by ∼0.2–1.0) (**Figure 2**). One possible explanation was that the host cell membranes could be rapidly lysed to release RNA with ice-cold lysis buffer (as heating was not included in the manufacturer's protocol), and there was a small window of time at the beginning of the heating process for the host cell RNases to become active before being deactivated by heat. On the other hand, more parasite RNA was released after the temperature reached to 75◦C and RNases were deactivated. However, the final host cell RNA concentrations appeared to be consistent within each experimental group (i.e., CV values at 0.75–3.04% for all groups), indicating that the RNA degradation would not result in inconsistency in subsequent determination of relative Hs18S levels. Finally, based on the observation that heating between 10 and 20 min in wells containing 200µL lysis buffer resulted in the least degradation of host cell RNA (△CT at 0.20–0.25), but the most improvement in releasing parasite RNA (△CT at −1.8 to −2.0), we determined that the optimal condition was the use of 200µL iScript™ RT-qPCR Sample Preparation Reagent and heating for 15 min.

The subsequent qRT-PCR amplification protocol in 384-well format using cell lysates was effective and specific based on agarose gel analysis of amplicons (data not shown) and postrun analysis of amplification and melting curves (**Figure 3**). In the melting curves, Cp18S and Hs18S displayed two strong and distinguishable peaks, although there was a small minor peak in the Cp18S curves (**Figure 3B**).

#### Assay Validation on Plate Uniformity, Linear Dynamic Range, and Drug Testing

Three experimental cell culture groups were used in assay validation (i.e., a total of 864 uninfected, infected/untreated, and infected/PRM-treated samples in nine plates). For uninfected cell samples containing that should not contain any Cp18S transcripts, the determined Cp18S CT values were resulted from low level non-specific amplification due to the lack of specific templates and/or self-amplification of primers, which was similar to the background signals produced in negative controls without

FIGURE 2 | Effects of the amount of lysis buffer and heat treatment on the release of RNA from Cryptosporidium parvum and HCT-8 cells. Cell lysates were first diluted by 100 and 2000 times with nuclease-free water prior to qRT-PCR detection for Cp18S and Hs18S rRNA transcripts, respectively. The plotted CT values were not calibrated to equal volume of lysis buffer. Bars represent standard error of the mean (SEM, *n* = 6). Heat treatment vs. un-treatment control, *p* < 0.005 by Student's *t*-test in all samples.

enzyme in a biochemical assay. Under the conditions used in the assay, the overall mean of CT values for Cp18S from uninfected cells in all plates was 30.14 (between 29.34 and 31.07). These CT values were much higher than those derived from cells infected with C. parvum (mean = 22.15, between 21.40 and 22.90) (**Table 1**). Because of the negative correlation (inversely proportional) relationship between CT values and template concentrations, the higher CT values in uninfected cells indicated that the noise signals in this assay (i.e., background signals in negative controls in the absence of parasites) were roughly >250-fold lower than the signals from the infected cells (i.e., noise levels equivalent to 0.4% of the signals). For Hs18S transcripts, the overall mean of CT values from all groups was 13.46 (between 12.92 and 14.06). In the infected/untreated groups, the average 1CT value difference between those of Hs18S and Cp18S was 8.6 (**Table 1**). After considering the dilution factors (i.e., cell lysates used for detecting Hs18S and Cp18S transcripts were diluted by 100 and 2000 folds, respectively), these CT values suggested the presence of 7000–8000-fold more Hs18S than Cp18S in a typical sample.

Using the validation protocols recommended by NIH/NCATS, we observed high intra-plate, inter-plate, and inter-day uniformity for the assay (**Table 2**). All CV values were <5%, much better than the 20% threshold recommended by the Assay Guidance Manual. No significant inter-plate and inter-day data shifts were observed. The SW and Z′ values in the nine plates were ranging from 17.35 to 44.14 and 0.73 to 0.87, respectively. Both values were much higher than the recommended acceptance criterions (i.e., SW ≥ 2 and Z′ ≥ 0.4). The excellence of the assay is largely due to high specificity in the

*aAll mean values are CT values derived from each plate in three independent cell cultivation experiments (Exp) performed in different dates. PRM, paromomycin at 120* µ*g/mL (168*µ*M).*

*aAll mean values are* △*CT values between Cp18S and Hs18S derived from each plate in three independent cell cultivation experiments (Exp) performed in different dates. % Inh, % inhibition; PRM, paromomycin at 120*µ*g/mL (168*µ*M); SW, signal window; Z*′ *, Z*′ *score.*

RT-PCR amplification, resulting in a ∼256-fold signal difference between infected and uninfected samples as described above. In the PRM-treated groups, the classic anti-cryptosporidial drug inhibited the parasite growth by 66.07–71.46% in all plates except for the plate 2 in experiment 1 that was at a slightly lower level of 54.90% (**Table 2**). These values were consistent with our in vitro drug efficacy tests using total RNA samples isolated by traditional column-based isolation kits from cells cultured in 24 and 48-well plates (Cai et al., 2005; Zhang et al., 2012; Yu et al., 2014).

We also generated standard curves from cells infected with various numbers of parasites. We considered that this approach was better than the traditional method using serially diluted RNA templates, because standard curves derived from various numbers of infected parasites would correct systematic errors from infection, sample preparation to qRT-PCR procedures, rather than to only correct the errors introduced at the qRT-PCR step. In this assay, linear relationship was observed between the △CT values and the samples isolated from cells infected with 64–4 × 104 oocysts per well (**Figure 4A**). The △CT values from samples infected with more or less parasites frequently fell outside of the linear range (data not shown). The linear dynamic range for this new assay (∼156-fold) was smaller than our previously reported assay using conventional protocols (Cai et al., 2005). There were two possible explanations. Firstly, the 96-well format had lower limits on the number of parasites that could be cultured in 96-well without density effect. Secondly, in comparison to conventional RNA isolation methods, cell lysates contained much less RNA due to the less efficiency in releasing RNA by the lysis buffer and higher dilutions needed to minimize the inhibition of RT-PCR reactions caused by substances present in the lysis buffer. However, the 156-fold linear dynamic range was sufficient for testing drug efficacy in vitro, as it could theoretically resolve <1% changes of parasite loads in the assay system.

We further evaluated the assay by testing the dose-response of PRM on the parasite growth, in which the drug at concentrations from 30 to 800µg/mL reduced the growth of C. parvum by 19– 97%. Using a non-linear curve fit with a sigmoidal model, the IC50 value was determined at 88.1µg/mL (123µM) (**Figure 4B**). These inhibition values were comparable to those obtained in our previous report at 89.7µg/ml (Cai et al., 2005), suggesting that the assay was suitable for determining the inhibitory kinetics and IC50 values of anti-cryptosporidial compounds identified by HTS.

#### Testing the Assay by a Small Scale Drug Screening

In the primary screening of 48 small molecules (10µM each), two compounds (C012 and C020) displayed cytotoxicity on HCT-8 cells, while others displayed a wide range of effects on the C. parvum growth in vitro (i.e., from −86.6 to 98.8% inhibition) (**Figure 5**). The negative inhibition values suggested that a few compounds might actually promote the parasite growth and/or affected more on host cells than on parasites, which could be evaluated by looking at the Hs18S and Cp18S CT values. For example, for C023 with −86.6% inhibition, the mean CT values for Hs18S and Cp18S were 13.38 and 18.08 (vs. 13.63 and 19.17 in the DMSO control), suggesting that this compound likely promoted the parasite growth more than reducing the host cell growth. Because we were interested only in identifying anti-cryptosporidial inhibitors in this study, we repeated the primary testing on the seven most efficacious compounds, in which six compounds exhibited similar efficacies (**Figure 5A**, red circles). We also performed dose-response experiments on C004 and C028, in which both compounds at 10µM displayed the same efficacy as observed in the two primary testing experiments (**Figure 5B**). Their IC50 values were at 0.98 and 1.33µM, respectively, suggesting that these two compounds might be further explored for drug development or serve as leads. Experiments to further characterize these two compounds and other top hits from the 48 compounds are still ongoing as part of our larger effort in discovering new anti-cryptosporidial drugs, which will be reported separately. Nonetheless, these observations conformed the robustness and suitability of the assay in high-throughput screening of drugs by directly evaluating their anti-cryptosporidial efficacy in vitro.

# Conclusions

We have developed a simplified real-time qRT-PCR assay suitable for high-throughput or high-volume screening of drugs against

FIGURE 5 | In vitro anti-cryptosporidial activities of 48 small molecules (C001–C048). (A) Efficacies of all 48 compounds (10 µM each) obtained from primary screening (solid teal round dots). A second test was performed on seven top hits (red circles), in which six compounds displayed similar levels of efficacy between the two tests as shown by overlaps or near distances of their corresponding teal dots and red circles. Only one compound showed significantly different efficacies between the two tests (highlighted by a yellow line to link the corresponding teal dot and red circle). Compound codes are given on the seven top hits and one of the potential parasite growth enhancer as discussed in the text. (B) Dose-response curves on the anti-cryptosporidial activities of C004 and C028 compounds. Paromomycin at 100 µg/ml (140 µM) was used as a positive control in this assay. Bars represent standard errors of the mean (SEM) derived from at least two biological replicates.

the growth of C. parvum in vitro. This was achieved by directly using cell lysates in qRT-PCR amplification. The assay was adapted and optimized in 96-well format for culturing parasite and 384-well format for SYBR-green based one-step real-time RT-PCR amplification and detection. Intra-plate, interplate, and inter-day uniformities were validated by NIH/NCATS recommended tests (i.e., SW and Z′ values = 17.35–44.14 and 0.73–0.87, respectively). The assay exhibited >150-fold linear dynamic range using samples isolated from cells infected with serially diluted parasites. The robustness and effectiveness of the assay were also confirmed by evaluating the anti-cryptosporidial

# References

efficacy of paromomycin, and by a small scale screening of 48 compounds.

# Acknowledgments

Research reported in this study was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under award number R21AI099850 (to GZ). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

multidimensional analysis of the performance of reagents and workflows. PLoS ONE 8:e72463. doi: 10.1371/journal.pone.0072463

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2015 Zhang and Zhu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Oleylphosphocholine (OlPC) arrests *Cryptosporidium parvum* growth *in vitro* and prevents lethal infection in interferon gamma receptor knock-out mice

*Karine Sonzogni-Desautels1,2†, Axel E. Renteria1,3†, Fabio V. Camargo1, Thomas Z. Di Lenardo3, Alexandre Mikhail1, Michael J. Arrowood4, Anny Fortin5,6 and Momar Ndao1,2,3\**

*1 National Reference Centre for Parasitology, Research Institute of the McGill University Health Centre, Montreal, QC, Canada, 2 Institute of Parasitology, Macdonald Campus, McGill University, Montreal, QC, Canada, 3 Department of Experimental Medicine, McGill University, Montreal, QC, Canada, 4 Division of Foodborne, Waterborne, and Environmental Diseases, Center for Disease Control and Prevention, Atlanta, GA, USA, 5 Department of Biochemistry, McGill University, Montreal, QC, Canada, 6 Dafra Pharma R&D, Turnhout, Belgium*

*Cryptosporidium parvum* is a species of protozoa that causes cryptosporidiosis, an intestinal disease affecting many mammals including humans. Typically, in healthy individuals, cryptosporidiosis is a self-limiting disease. However, *C. parvum* can cause a severe and persistent infection that can be life-threatening for immunocompromised individuals, such as AIDS patients. As there are no available treatments for these patients that can cure the disease, there is an urgent need to identify treatment options. We tested the anti-parasitic activity of the alkylphosphocholine oleylphosphocholine (OlPC), an analog of miltefosine, against *C. parvum* in *in vitro* and *in vivo* studies. *In vitro* experiments using *C. parvum* infected human ileocecal adenocarcinoma cells (HCT-8 cells) showed that OlPC has an EC50 of 18.84 nM. Moreover, no cell toxicity has been seen at concentrations ≤50 μM. C57BL/6 interferon gamma receptor knock-out mice, were infected by gavage with 4000 *C. parvum* oocysts on Day 0. Oral treatments, with OlPC, miltefosine, paromomycin or PBS, began on Day 3 post-infection for 10 days. Treatment with OlPC, at 40 mg/kg/day resulted in 100% survival, complete clearance of parasite in stools and a 99.9% parasite burden reduction in the intestines at Day 30. Doses of 30 and 20 mg/kg/day also demonstrated an increased survival rate and a dose-dependent parasite burden reduction. Mice treated with 10 mg/kg/day of miltefosine resulted in 50% survival at Day 30. In contrast, control mice, treated with PBS or 100 mg/kg/day of paromomycin, died or had to be euthanized between Days 6 and 13 due to severe illness. Results of parasite burden were obtained by qPCR and cross-validated by both flow cytometry of stool oocysts and histological sections of the ileum. Together, our results strongly support that OlPC represents a potential candidate for the treatment of *C. parvum* infections in immunocompromised patients.

Keywords: *Cryptosporidium parvum*, cryptosporidiosis, oleylphosphocholine, OlPC, miltefosine, interferon gamma receptor knockout mice, oocysts, parasite burden

#### *Edited by:*

*Anjan Debnath, University of California, San Diego, USA*

#### *Reviewed by:*

*Dmitri Debabov, NovaBay Pharmaceuticals, USA Kayode K. Ojo, University of Washington, USA*

#### *\*Correspondence:*

*Momar Ndao, National Reference Centre for Parasitology, Research Institute of the McGill University Health Centre, 1001 Decarie Boulevard, Room EM3 3244, Montreal, QC H4A 3J1, Canada<EMAIL_ADDRESS>†These authors have contributed*

> *equally to this work. Specialty section:*

*This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology*

*Received: 12 May 2015 Accepted: 01 September 2015 Published: 23 September 2015*

#### *Citation:*

*Sonzogni-Desautels K, Renteria AE, Camargo FV, Di Lenardo TZ, Mikhail A, Arrowood MJ, Fortin A and Ndao M (2015) Oleylphosphocholine (OlPC) arrests Cryptosporidium parvum growth in vitro and prevents lethal infection in interferon gamma receptor knock-out mice. Front. Microbiol. 6:973. doi: 10.3389/fmicb.2015.00973*

# Introduction

*Cryptosporidium parvum* is an obligate parasite of the phylum Apicomplexa that infects the microvilli of the small intestine of many mammalian hosts including humans (Ryan and Xiao, 2014). Considered as a major waterborne pathogen (drinking and recreational water) that can also be transmitted through contaminated foods (Fayer et al., 2000; Cacciò and Putignani, 2014), *C. parvum* outbreaks have been widely reported in numerous countries from all continents (Cacciò et al., 2005; Putignani and Menichella, 2010; Cacciò and Putignani, 2014). This parasite can remain infectious for months in a humid environment (Robertson et al., 1992; King et al., 2005; Cacciò and Putignani, 2014). As a result, *C. parvum* has been categorized as a class B bioterrorist pathogen by the Center for Disease Control and Prevention (CDC; Feldmann et al., 2002; Rotz et al., 2002).

As one of the major causative agents of cryptosporidiosis in humans, along with *Cryptosporidium hominis* (Cacciò et al., 2005), *C. parvum* causes self-limiting watery diarrhea or persistent and severe diarrhea depending on the age and the immune status of the patient (Chalmers and Davies, 2010; Putignani and Menichella, 2010; Shikani and Weiss, 2014). Indeed, *C. parvum* infection has been reported to be lifethreatening in AIDS patients, among which the prevalence of cryptosporidiosis was determined to be 14% in developed countries and 24% in developing countries (Collinet-Adler and Ward, 2010; Putignani and Menichella, 2010). *Cryptosporidium* spp., along with *Giardia* spp., has also been identified as the leading cause of chronic or persistent diarrhea in children in a context of malnutrition or immunodeficiency (Thapar and Sanderson, 2004; Putignani and Menichella, 2010). The Global Enteric Multicenter Study identified Cryptosporidium as one of the top five pathogens causing mild to severe diarrhea in children under the age of 2 in developing countries (Kotloff et al., 2013). Studies have shown that *Cryptosporidium* infections in young children have often resulted in stunting and lead to poor cognitive functions later in childhood (Berkman et al., 2002). However, currently available treatments have demonstrated limited effect in these vulnerable populations (Mead and Arrowood, 2014).

Nitazoxanide, the only American Food and Drug Association (FDA) approved drug to treat cryptosporidiosis in immunocompetent patients, has shown little activity to fight against *C. parvum* infections in AIDS patients (Cabada and White, 2010; Rossignol, 2010; Mead and Arrowood, 2014). Similarly, paromomycin, the currently used drug to treat *C. parvum* infections in AIDS patients, has shown modest activity and limited results in different case studies (Hewitt et al., 2000; Rossignol, 2010). With no efficient way to treat immunocompromised patients, many drugs have been tested over the years, but very few have shown consistent activity (Mead and Arrowood, 2014).

Oleylphosphocholine (OlPC, C23H48NO4P) belongs to the family of alkylphosphocholines (Hernandez et al., 2014) and is structurally related to miltefosine (van Blitterswijk and Verheij, 2008), with the chemical formula C21H46NO4P · xH2O. Both compounds have shown *in vitro* and *in vivo* anti-leishmanial activity (Fortin et al., 2014), and *in vitro* activity of miltefosine against *C. parvum* has also been reported (Shahiduzzaman et al., 2009). This study reports the strong activity of OlPC, both *in vitro* and *in vivo* in an immunocompromised C57BL/6 IFNγR-KO mouse model of *C. parvum* infection.

# Materials and Methods

#### Parasites

*Cryptosporidium parvum* (Iowa strain) oocysts, generously provided by Dr. Michael J. Arrowood (CDC, Atlanta, GA, USA), were maintained through a C57BL/6 IFNγR-KO mouse infection model previously described by our laboratory (von Oettingen et al., 2008). Oocysts were purified following the reported technique (von Oettingen et al., 2008) and were stored in 2.5% (w/v) aqueous potassium dichromate (K2Cr2O7, Sigma-Aldrich, Oakville, ON, Canada) at 4◦C.

#### Test Compound

Oleylphosphocholine was generously provided by Dafra Pharma Research and Development (Turnhout, Belgium) and stored at 4◦C, away from light. For *in vitro* studies, OlPC was diluted to a stock solution of 11.5 mM in ddH2O. Miltefosine (Sigma-Aldrich, Oakville, ON, Canada) was used as a control in the *in vitro* assays because of its previously reported activity against *C. parvum* (Shahiduzzaman et al., 2009). Paromomycin (Sigma-Aldrich) was also used as a control in the *in vitro* assays. The drugs were prepared fresh daily in phosphate-buffered saline (PBS) for animal studies.

#### Host Cells

For *in vitro* studies, human colonic tumor cells (ileocecal adenocarcinoma; HCT-8: ATCC CCL-244) were used. Cells were maintained in T225 flasks (BD Bioscience, Mississauga, ON, Canada) using DMEM media (Wisent, St-Bruno, QC, Canada). For OlPC *in vitro* studies, media was supplemented with 10 μg/ml gentamycin, 10 μg/ml streptomycin, 100 U/ml penicillin, 10% heat-inactivated fetal bovine serum (FBS), and non-essential amino acids (NEAA; Wisent). For the cytotoxicity assays, cells were grown in supplemented DMEM without phenol red and further supplemented with 4.5 g/L D-glucose (Sigma-Aldrich, Oakville, ON, Canada) and 110 mg/L sodium pyruvate (Sigma-Aldrich). For miltefosine and paromomycin *in vitro* studies, media was supplemented with 10 μg/ml gentamicin, 50 μg/ml streptomycin, 50 U/ml penicillin, and 10% heatinactivated FBS.

### Cytotoxicity of OlPC in HCT-8 Cells

XTT assay (Sigma-Aldrich) was used (Roehm et al., 1991), which relies on the ability of live cells to cleave XTT causing a colorimetric change, to quantify the possible cytotoxicity of OlPC in HCT-8 cells. For this assay, DMEM without phenol red was used. Initially, 5 × 104 HCT-8 cells in 200 μL of supplemented DMEM were seeded in a 96-well flat-bottom plates (BD Falcon, Franklin Lakes, NJ, USA) and incubated overnight (or until they reached ∼80% confluency) at 37◦C with 5% CO2. Freshly prepared serial dilutions of OlPC diluted in supplemented DMEM were used and 50 μL of these solutions were added to the wells to obtain final OlPC concentrations ranging from 10 to 1000 μM (4.33 to 433 μg/mL). Samples were run in quadruplicate and plates were incubated at 37◦C with 5% CO2 for 48 h. Then, 50 μL of XTT (dissolved in DMEM) was added to each well and plates were re-incubated for 4 h. Absorbance was measured at 450 nm using a plate reader (EL800 BioTek; Fisher, Nepean, ON, Canada).

#### *C. parvum* Infection Model in HCT-8 Cells

We tested the effect of OlPC on *C. parvum* sporozoites in HCT-8 cells. Briefly, 24-well flat-bottom plates (BD Falcon) were seeded with 3 × 105 cells in 1.4 mL of supplemented DMEM and incubated overnight (or until they reached ∼80% confluency) at 37◦C with 5% CO2. Stock oocysts in 2.5% K2Cr2O7 were washed three times with 0.1 M acetate-NaCl buffer, pH 5.5, and subsequently incubated in 10 mM sodium periodate on ice for 20 min. Oocysts were then washed three times with PBS containing 0.1% bovine serum albumin (BSA; Sigma-Aldrich). To release the sporozoites from the oocysts (excystation), parasites were incubated in DMEM containing 0.75% sodium taurocholate (Sigma-Aldrich) at 37◦C for 30 min or until 50% excystation was determined by microscopy. Then, sporozoites from 1 × 105 oocysts were inoculated in each well. Non-infected cells incubated with supplemented DMEM served as negative controls as well as infected cells incubated with supplemented DMEM, as positive controls. OlPC was serially diluted in supplemented DMEM and was subsequently added to each well at concentrations ranging from 10 pM to 100 μM (4.33 pg/mL to 43.3 μg/mL). Miltefosine and paromomycin, diluted in supplemented DMEM, were also tested at concentrations ranging from 10 pM to 10 μM (4.08 pg/mL to 4.08 μg/mL) and 10 pM to 100 μM (7.14 pg/mL to 71.4 μg/mL) respectively, to serve as controls. Plates were further incubated for 48 h at 37◦C with 5% CO2. *In vitro* assays were run two independent times and each condition was run in triplicate for the miltefosine assay and in duplicate for the OlPC and the paromomycin assays. At the completion of the incubation, supernatants were removed and cells were lifted using 0.25% trypsin in EDTA (Wisent). To assess parasite burden in each well and determine the efficacy of OlPC, miltefosine or paromomycin against *C. parvum*, DNA was extracted from samples using QIAamp DNA Mini Kit (Qiagen, Toronto, ON, Canada). Purified DNA was stored at −20◦C until use.

#### Quantitative Polymerase Chain Reaction Primers, Probe, and Standard Curve

Parasite burden from DNA samples from *in vitro* assays as well as from mouse fecal and intestinal samples were assessed by quantitative polymerase chain reaction (qPCR) targeting the hsp70 gene of *C. parvum* (GenBank: U69698.2) using the LightCycler 2.0 system (Roche, Laval, QC, Canada). Primers and probe sequences (TaqMan) were used according to previously published sequences (Shahiduzzaman et al., 2009). Briefly, a master mix per sample was prepared by adding 5 μL of PCR-grade water (Roche), 2 μL of 10 μM of forward primer (CP\_hsp70\_fwd: 5 -AACTTTAgCTCCAgTTgAgAAA gTACTC-3 ; Tib MolBiol, Adelphia, NJ, USA), 2 μL of 10 μM reverse primer (CP\_hsp70\_rvs: 5 -CATggCTCTTTACCgTTA AAgAATTCC-3 ; MolBiol), 2 μL of 2 μM 5 6-carboxy fluorescein reporter dye (FAM) probe with a 3 tetramethylrh odamine quencher dye (TMR; hsp-taq: 5 -AATACgTgTAgAAC CACCAACCAATACAACATC-3 ; MolBiol), and 4 μL of buffer mix containing dNTP, MgCl2, and Taq-polymerase (Roche). Prior to the polymerase chain reaction, 5 μL of sample and 15 μL of master mix were added to LightCycler capillaries (Roche). The initial denaturation step was conducted at 95◦C for 15 min followed by 45 cycles of denaturation at 94◦C for 15 s and annealing at 60◦C for 1 min (Shahiduzzaman et al., 2009).

In order to quantify parasite burden, a standard curve was necessary. First, *C. parvum* oocysts were diluted to concentrations of 1 × 101 to 1 × 106 in 200 μL and then, they were disrupted by five freeze-thaw cycles of 2 min incubation in liquid nitrogen followed by 2 min incubation in a 56◦C water bath. Second, DNA from the oocyst extracts was purified using QIAamp DNA Blood Mini Kit (Qiagen) and these samples were run (qPCR) in the LightCycler 2.0; qPCR was used to correlate threshold cycle values (Ct values) to parasite burden (*R*2 = 0.9989). This standard curve was used to extrapolate parasite burden from DNA samples from *in vitro* assays as well as from mouse stool and intestine samples.

### Immunocompromised Mouse Model of *C. parvum* Infection

*Cryptosporidium parvum* oocysts in 2.5% K2Cr2O7 were washed three times with PBS; parasites were then counted using a hemocytometer and diluted in PBS to a concentration of 4 × 104/mL. Six to eight week old C57BL/6 IFNγR-KO female and male mice (Jackson Laboratories, Bar Harbor, ME, USA) were infected with a lethal dose of 4000 oocysts in 100 μL of PBS on Day 0 using a 22-gage gavage needle (CDVM, St-Hyacinthe, QC, Canada). Mice were separated in eight groups (13 mice/group for treated groups and 4–8 mice/group for control groups). Groups 1 and 2 were non-infected controls, treated per os (oral gavage) with PBS or with 40 mg/kg/day of OlPC, respectively. Group 3 was infected and treated with PBS (positive controls). Groups 4–6 were infected and treated per os with 40, 30, and 20 mg/kg/day of OlPC, respectively. Groups 7 and 8 were infected controls treated per os with 10 mg/kg/day of miltefosine and 100 mg/kg/day of paromomycin, respectively. Treatment started at Day 3 and continued for 10 days. OlPC and control drugs were prepared fresh on a daily basis for the length of the treatment. Mice were weighted daily. Starting on Day 4, individual stool samples were collected every 3–4 days until study termination.

Five mice from each group were sacrificed at Day 10 (before daily treatment) to assess and compare disease progression. Other mice were maintained until study termination (Day 30) unless they were found dead or had to be sacrificed due to severe illness. Because of the lethality of this model (von Oettingen et al., 2008; Ndao et al., 2013) an objective mouse rating system based on symptom severity and behavioral changes was developed in accordance with the Animal Care Committee and the Canadian Council on Animal Care (CCAC) guidelines to determine when mice should be sacrificed due to illness. Upon death or euthanasia, the mouse ileum was preserved in formalin for histological purposes and the rest of the intestine (duodenum to rectum) was collected individually and chopped into 0.5 cm pieces into a 60 mL specimen container (ThermoFisher Scientific, Ottawa, ON, Canada) with 10 mL of 0.02% (v/v) Tween20 in PBS (one container per mouse). During necropsy, stool was also collected for oocyst purification.

After completion of the *in vivo* study, a constant volume of purified oocysts from intestinal samples of mice from each treatment groups was inoculated to naïve C57BL/6 IFNγR-KO mice to determine if the concentration of oocysts remaining in the intestines of treated mice was able to induce clinical signs of cryptosporidiosis in naïve mice.

All mouse studies were conducted at the Montreal General Hospital rodent animal facility site of the Research Institute of McGill University Health Center. All studies performed were approved and in accordance with the MGH Facility Animal Care Committee and also in accordance with the CCAC guidelines.

#### Oocyst Purification from Intestinal Samples and Oocyst Burden Analysis by qPCR

For each mouse intestine sample in 0.02% (v/v) Tween20 in PBS, 0.05 g of sputasol (dry mixture of 10% DTT, 76% NaCl, 2% KCl, 10% Na2HPO4, and 2% KHPO4) was added. Oocyst purification was performed using a previously described method reported in literature and by our lab (Meloni, 1996; von Oettingen et al., 2008). Briefly, each sample was homogenized for 2 min at 7000 rpm with the Polytron PT3000D homogenizer (Kinematica, Lucerne, Switzerland). Between each sample, the homogenizer was cleaned with bleach and ethanol to avoid crosscontamination. Samples were placed on a rotary mixer at low speeds for 90 min at room temperature. Samples were then transferred to 50 mL Falcon tubes (BD Falcon, Franklin Lakes, NJ, USA) and centrifuged at 2000 × *g* for 10 min at 4◦C. The supernatant was removed and the pellet was resuspended in 8 mL of 0.02% (v/v) Tween20 in ddH2O and 2 mL of diethyl ether (Sigma-Aldrich, Oakville, ON, Canada). Samples were thoroughly vortexed and centrifuged at 2000 × *g* for 10 min at 4◦C. The layers containing fatty cells and tissues as well as the supernatants were removed. The pellets were washed with 20 mL of cold ddH2O and centrifuged at 2000 × *g* for 10 min at 4◦C. Supernatants were discarded and the pellets resuspended in 20 mL of saturated NaCl. Samples were vortexed thoroughly, carefully overlayed with 5 mL of cold ddH20 and centrifuged at 2000 × *g* for 10 min at 4◦C. Five milliliters of the interphase were collected and centrifuged at 2000 × *g* for 10 min at 4◦C; supernatants were discarded and oocyst-containing pellets were resuspended in 1 mL PBS containing 10 μg/ml streptomycin and 100 U/ml penicillin. Purified oocyst samples were stored at 4◦C. For qPCR analysis, 200 μL were taken from each purified oocyst sample and five freeze/thaw cycles of 2 min incubation in liquid nitrogen followed by 2 min incubation in a 56◦C water bath were performed. DNA was then extracted using the QIAamp DNA Blood Mini Kit (Qiagen) and samples was used for qPCR analysis.

### Oocyst Purification from Stool Samples and Oocyst Shedding Analysis by qPCR

Individual stool samples were collected in 500 μL of distilled and deionized (18.2 M cm) water (ddH2O) and stored in microcentrifuge tubes at 4◦C until used. Samples were thoroughly vortexed until stools were homogenized to smaller particles, then centrifuged at 14000 × *g* and washed with cold ddH2O. Supernatants were discarded and the pellets resuspended in 1 mL of saturated NaCl. Samples were vortexed thoroughly, carefully overlayed with 250 μL of cold ddH2O and centrifuged at 1600 × *g* for 10 min at 4◦C. All of the supernatant was collected and the sample was exposed to another NaCl-ddH2O overlay. The supernatants collected from both overlays were centrifuged at 14000 × *g* for 3 min at 4◦C, combined into one tube and resuspended in 200 μL of ddH2O. Purified oocysts were exposed to five freeze/thaw cycles as described above. DNA was extracted using the QIAamp DNA Blood Mini Kit and stored at −20◦C until oocyst shedding was quantified by qPCR. Values were normalized by gram of stool.

#### Oocyst Burden Analysis by Flow Cytometry

All oocyst samples purified from mouse intestine were incubated in a final concentration of 1% paraformaldehyde for 15– 30 min prior to analysis. Briefly, 40 ul of sample and 50 ul of CountBrightTM absolute counting beads (Invitrogen, Burlington, ON, Canada) were added to a paraformaldehyde solution for a final volume of 500 ul. Oocysts within each sample were quantified by morphology (FSC-A and SSC-A) using BD LSRFortessaTM cell analyser (BD, Franklin Lakes, NJ, USA). Oocysts were initially identified with a FITC-labeled mouse IgG3 monoclonal antibody targeting a surface antigen (AbD Serotec, Raleigh, NC, USA). Results demonstrated that 98% of the FITC positive events occupied a distinct population by size and complexity in the FSC-A and SSC-A channels. Results generated by flow cytometry are based on unstained samples gating on this distinct oocyst population. Oocyst concentration per sample was calculated by determining the quantity of sample collected based on the number of CountBrightTM bead events collected. Final oocyst numbers were normalized by gram of mouse intestines.

#### Histopathology

*Cryptosporidium parvum* is known to preferentially infect the jejunum and ileum (Fayer and Xiao, 2008), thus the ileum was collected for histopathology. Liver and spleen were also fixed in formalin, paraffin-embedded and cut to 4 μm thick sections. Sections were stained with hematoxylin and eosin to further assess disease progression by light microscopy.

#### Data Analysis

All *in vitro* studies and qPCR data were analyzed using Microsoft Excel (Microsoft Corporation, Redmond, WA, USA). Graphs and *P*-values were obtained using GraphPad PrismR version 6.0 (GraphPad Software, Inc., La Jolla, CA, USA). Results represent mean ± standard error mean (SEM). For OlPC and miltefosine EC50 calculations, the same software, GraphPad PrismR version 6.0, the non-linear fit of EC50 shift and the "shared value for all data sets" constraint for Bottom and Top were used. Flow cytometry data was analyzed using FlowJo v10 analytical software (Treestar, Inc., San Carlos, CA, USA).

# Results

### OlPC and Miltefosine Inhibit *C. parvum* Infection in HCT-8 Cells *In Vitro*

To test the efficacy of OlPC to inhibit *C. parvum* infection *in vitro*, we used HCT-8 cells which allowed the parasite to replicate (Widmer et al., 2000). Miltefosine was used as a control as its efficiency to inhibit *C. parvum* infection *in vitro* has already been described (Shahiduzzaman et al., 2009). However, the dose of 100 μM was not tested for miltefosine as cell toxicity has been reported at a concentration of 24.5 μM when cells are incubated for 45 h (Shahiduzzaman et al., 2009). For the OlPC and the miltefosine assays, DNA was extracted after 48 h of incubation with the compound. A standard curve was used to correlate threshold cycle values (Ct values) obtained by qPCR to parasite burden. Then, for each well, the percentage of parasite burden reduction was assessed according to this formula:

=[1-(parasite burden from tested well/parasite burden of positive control)]∗100

Positive controls were infected cells incubated with supplemented media (no compound). **Figure 1** represents the mean of the parasite burden reduction of tested wells for each concentration of OlPC (A) and miltefosine (B) ± SEM.

HCT-8 cells treated with 100, 10, and 1 μM of OlPC showed the highest *C. parvum* inhibition with 87, 84, and 85% reduction in parasite burden, respectively (**Figure 1A**). Therefore, these concentrations of OlPC are not statistically different than uninfected controls showing the clear potency of these treatments to inhibit *C. parvum* infection *in vitro*. There is a statistical difference (*P* < 0.0001) between OlPC treatment and uninfected controls starting at the concentration of 100 nM due to the lack of efficiency of these doses to inhibit *C. parvum* infection. Results demonstrated a dose-dependent reduction of *C. parvum* burden when treated with increasing concentrations of OlPC and a calculated EC50 of 18.84 nM. Cell toxicity of OlPC was not observed at concentrations of ≤50 μM using an XTT assay (data not shown).

Miltefosine at concentrations of 10 and 1 μM showed the highest inhibitory effect with 85 and 69% reduction in parasite burden, respectively (**Figure 1B**). Similarly to the OlPC treatment, at these concentrations, there is no statistical difference between the miltefosine treatment and uninfected cells. So, OlPC is as potent as miltefosine at inhibiting *C. parvum* infection *in vitro* at a concentration of 10 μM (84 vs. 85%), but OlPC better inhibits infection at a concentration of 1 μM (85 vs. 69%). The calculated EC50 for miltefosine is 0.81 μM, which is slightly lower than the previously published value of 1.87 μM for this compound and this parasite (Shahiduzzaman et al., 2009), but within the same range. Batch differences in oocyst virulence due primarily to freshness are normal and can explain this type of variation. Concentrations of paromomycin ranging from 10 pM to 100 μM were tested to determine the ability of this compound to inhibit *C. parvum* infection *in vitro*. However, no significant effect was observed (data not shown), which is in agreement with what has been published previously (Ndao et al., 2013).

#### OlPC shows a Dose-Dependent Effect on *C. parvum*-Induced Mortality in an Immunodeficient Mouse Model

The C57BL/6 IFNγR-KO mouse model is an excellent model to assess the efficacy of compounds to treat acute *C. parvum* infection because of the high susceptibility of these knockout mice to this parasite (von Oettingen et al., 2008; Ndao et al., 2013). According to previous reports, mice infected with as low as 1500 oocysts had a mortality rate of 80% by Day 14 (Ndao et al., 2013). Therefore, in this study, mice were infected with 4000 oocysts, and survival, as well as parasite burden reduction, was used to differentiate an effective treatment from an inefficient treatment. To ensure the objectivity of the study, an external blinded examiner scored the mice reaching pre-defined critical clinical end-points according to a pre-established rating system.

As expected, infected controls started showing symptoms of illness as early as Day 5 and had a very high mortality rate at Day 14. For the PBS treated group, mice started showing signs of illness (such as hunched back, weight loss, dehydration, lethargy, and watery stools) at Day 5 and all mice from this group died or were euthanized between Days 6 and 10 (**Figure 2**). Mice treated with 100 mg/kg/day of paromomycin for 10 days displayed clinical symptoms of cryptosporidiosis at Day 7 and a 60% mortality rate was observed at Day 9. Some paromomycintreated mice did survive until Day 13 but, the Mantel-Cox or the Gehan–Breslow–Wilcoxon tests used to compare survival curves showed no significant difference with the PBS-treated group (**Figure 2**).

Mice treated with 10 mg/kg/day of miltefosine, the analog of OlPC, also showed signs of illness as early as Day 7 and a 50% mortality rate at Day 13 but, survivors remained alive until the end of the study (Day 30; **Figure 2**).This clear difference in survival rates with the PBS-treated group (*P* < 0.001) determined that a 10 mg/kg/day miltefosine treatment for 10 days significantly decreased mortality. Nevertheless, at Day 30, clinical signs of cryptosporidiosis were still visible in these mice such as 15% weight loss, hunched backs, dehydration, and soft stools.

Mice treated with 40 mg/kg/day of OlPC displayed a 100% survival rate by the experimental endpoint (Day 30; *P* < 0.001; **Figure 2**). Additionally, the onset of symptoms began later (Day 9) than the PBS-treated controls; clinical signs were less severe and did not last as long in comparison with other groups. Furthermore, at Day 10, which is the peak of infection (von Oettingen et al., 2008), weight loss (about 10% reduction from initial weight for the 40 mg/kg/day OlPC-treated group) was already significantly lower compared to any of the infected control groups (>15% of initial weight, *P* < 0.05, data not shown). By Day 30, 40 mg/kg/day OlPC-treated mice looked healthy as they displayed no visible signs of illness. Moreover, unlike the miltefosine-treated group, 40 mg/kg/day OlPC-treated mice had exceeded, by Day 30, their initial weights (*P* < 0.0001, data not shown). Mice treated with 30 mg/kg/day of OlPC for 10 days, had less severe clinical signs of cryptosporidiosis. For

these latter mice, on one hand, treatment was not able to rescue all mice which showed a severe weight loss of up to 15% of initial weight; on the other hand, the onset of symptoms was delayed to Days 8 or 9. Their survival rate was of 87.5% at Day 11, 75% at Day 30 (**Figure 2**) and, by Day 30, the survivors displayed no visible signs of illness and had returned to their initial weights (data not shown). Finally, the lowest dose of OlPC (20 mg/kg/day) was still able to rescue 75% of mice at Day 30 even if there were still visible signs of illness and mice had a 5% weight loss in comparison with initial weights. Non-infected mice treated with 40 mg/kg/day of OlPC presented a constant weight through the length of the study and no clinical sign were noted in this group (data not shown).

#### OlPC Eliminates Oocyst Shedding in a Dose-Dependent Manner

All infected mice began shedding oocysts between Days 5 and 7. Stool samples were collected from individual mice, processed and analyzed by qPCR. At Day 10, infected mice treated with PBS and with 100 mg/kg/day of paromomycin were shedding 8.52 × 107 and 1.84 × 108 oocysts per gram of stool (oo/g of stool) respectively (data not shown). Similarly, mice treated with 10 mg/kg/day of miltefosine were shedding 1.94 × 108 oo/g of stool at Day 10 (**Figure 3**). At Days 14 and 30, surviving mice of this group continued to shed considerable levels of oocysts; 1.49 × 107 and 1.5 × 107 oo/g of stool respectively (**Figure 3**).

In mice treated with 40 mg/kg/day of OlPC, oocyst shedding reduction occurred as early as Day 10 with only 7.59 × 106 oo/g of stool which is a significant reduction in comparison with the 10 mg/kg miltefosine group (*P* < 0.01; **Figure 3**). At Day 14, after 10 days of OlPC-treatment, oocyst shedding in this group was even lower with 5.14 × 103 oo/g, representing a 99.96% reduction (4-log reduction) in oocyst shedding. At Day 14, the 40 mg/kg/day OlPC-treated group is also statistically different when compared with the 10 mg/kg miltefosine-treated mice (*P* < 0.05; **Figure 3**). Finally, at Day 30, there was a 100% elimination of oocyst shedding (>7-log reduction) in all mice of the 40 mg/kg/day OlPC-treated group (*P* < 0.0001; **Figure 3**). Light microscopy was used to confirm qPCR results. Indeed, all the samples of 40 mg/kg/day OlPC-treated mice at Day 30 were negative as no oocysts could be visualized under the microscope.

Mice treated with 30 mg/kg/day of OlPC showed a similar tendency in oocyst shedding as the 40 mg/kg/day OlPC-treated group. Effectively, all average oocyst counts were significantly

lower when compared with the 10 mg/kg/day miltefosine-treated group (**Figure 3**); at Day 10, the average oocyst shedding was 2.45 × 106 oo/g of stool (*P* < 0.0001), at Day 14, 4.9 × 102 oo/g of stool (*P* < 0.05) and, at Day 30, 13 oo/g of stool (*P* < 0.01). Mice treated with 20 mg/kg/day of OlPC had the least important reduction in oocyst shedding but remained significantly lower than the 10 mg/kg miltefosine-treated mice. In fact, 4.04 × 107, 3.68 × 105, and 5.95 × 103 oo/g of stool, was evaluated in these 20 mg/kg/day OlPC-treated mice at Day 10 (*P* < 0.01), Day 14 (*P* < 0.01), and Day 30 (*P* < 0.05) respectively (**Figure 3**).

Oocyst shedding was not statistically different between mice treated with 30 mg/kg/day and 40 mg/kg/day at Days 10 and 14. The fact that the numerical value of the mean for parasite burden/g of stool for the 40 mg/kg/day treated mice was slightly higher at Days 10 and 14 than the one for 30 mg/kg/day treated mice is most probably due to inter-individual variations in this mouse model.

#### OlPC Significantly Reduces Parasite Burden in the Intestines of C57BL/6 IFN**γ**R-KO Mice Infected with *C. parvum*

To quantitate parasite and oocyst burden in the intestines and assess disease progression of infected mice during and after their respective treatments, mice were euthanized and intestines were collected at two time points: Days 10 or 30. Intestinal samples from every mouse were processed individually to purify *C. parvum* parasites, analyzed using qPCR and normalized to parasites per gram of intestines (p/g of intestines). On Day 10, infected control groups revealed massive levels of *C. parvum* in the gut. Mice treated with PBS had the highest parasite burden with 4.77 × 107 p/g of intestines followed by mice treated with 100 mg/kg/day of paromomycin with 5.01 × 106 p/g of intestines (**Figure 4A**). Mice treated with 10 mg/kg/day of miltefosine also had very high parasite burden with 7.94 × 106 p/g of intestines (**Figure 4A**). In mice treated with OlPC, parasite burden was significantly lower compared to the infected PBS control group. After 7 days of treatment (Day 10), 20 mg/kg/day OlPC-treated mice showed a parasite burden of 3.44 × 106 p/g of intestines (*P* < 0.01; **Figure 4A**). In mice treated with 30 mg/kg/day of OlPC the parasite burden was even lower with 1.81 × 105 p/g of intestines (*P* < 0.001) corresponding to a 99.6% (2-log reduction) parasite burden reduction in comparison with the PBS control group (**Figure 4A**). Similarly, mice treated with 40 mg/kg/day of OlPC also revealed a 99.6% reduction in parasite burden with 2.25 × 105 p/g of intestines (*P* < 0.001) when compared to the infected PBS control mice (**Figure 4A**).

By Day 30, 17 days after the completion of the treatments, surviving mice treated with 10 mg/kg miltefosine exhibited a high parasite burden with 2.65 × 106 p/g of intestines (**Figure 4B**). Parasite burden decreased in mice treated with OlPC when compared to the 10 mg/kg miltefosine-treated mice following a dose-dependent response. Indeed, in mice treated with 20 mg/kg/day of OlPC, parasite burden obtained was 1.09 × 105 p/g of intestines (not significant, *P* > 0.05), whereas in mice treated with 30 mg/kg/day and 40 mg/kg/day, parasite burden was 4.66 × 103 p/g of intestines (99.8% parasite burden reduction, *P* < 0.05) and 692 p/g of intestines (99.97% parasite burden reduction, *P* < 0.01) respectively (**Figure 4B**). Additionally, by Day 30 there was an overall reduction in parasite burden in each group compared to their burden at Day 10.

To cross-validate results obtained by qPCR and to confirm the presence of *C. parvum* oocysts in the intestines, flow cytometry was used to quantify the number of oocysts per gram of intestines (oo/g of intestines). Flow cytometry data analysis allowed to

identify oocysts by their size and morphology (FSC-A and SSC-A) and to count them for each intestinal sample. In general, total parasite burdens calculated by flow cytometry were lower than those obtained by qPCR suggesting the presence of other stages of the parasite in the intestines that flow cytometry cannot distinguish. However, the results were supportive of those of the qPCR. Mice treated with PBS, 100 mg/kg paromomycin and 10 mg/kg miltefosine showed an oocyst burden of 1.46 × 106 oo/g of intestines, 2.3 × 105 oo/g of intestines and 4.53 × 105 oo/g of intestines respectively (**Figure 4A**). In mice treated with 20 mg/kg/day of OlPC, 9.4 × 104 oo/g of intestines was detected which consisted in a 93.5% decrease in oocyst burden compared to the PBS treated group (*P* < 0.01). The reductions in oocyst burden increase in mice treated with 30 or 40 mg/kg/day of OlPC were only 180 oo/g of intestines (99.98% reduction, *P* < 0.01) and 167 oo/g of intestines (99.98% reduction, *P* < 0.01) respectively (**Figure 4A**). In both cases an average 4-log reduction in oocyst burden was observed when compared to PBS treated mice. In surviving mice treated with 10 mg/kg miltefosine, oocyst burden at Day 30 was 1.4 × 105 oo/g of intestines which is comparable to their oocyst burden at Day 10 (**Figures 4A,B**). At Day 30, at 20 mg/kg/day of OlPC, oocyst levels were slightly lower (6.63 × 104 oo/g of intestines) than mice treated with 10 mg/kg/day of miltefosine, but this difference remained statistically non-significant (52.6% oocyst burden reduction, **Figure 4B**). At 30 mg/kg/day of OlPC, oocyst burden (2.81 × 103 oo/g of intestines) increased compared to the oocyst burden at Day 10 but still represented a 98% oocyst burden reduction compared to the oocyst burden at Day 30 for 10 mg/kg miltefosine-treated mice (**Figure 4B**). Finally, at 40 mg/kg/day of OlPC, mice showed the lowest oocyst burden in their intestines with 34 oo/g of intestine representing a 99.98% oocyst burden reduction when compared to the 10 mg/kg miltefosine-treated mice (*P* < 0.01, **Figure 4B**).

#### OlPC Eliminates the Presence of Oocysts in Histological Sections of the Ileum of C57BL/6 IFN**γ**R-KO Mice Infected with *C. parvum*

To visually validate the presence of *C. parvum* life cycle stages in the intestines of infected mice, the ileum was collected from all mice at time of death. Sections from the ileum were prepared and stained (H&E) for histopathology purposes. Ileum sections from mice treated with PBS and 100 mg/kg/day of paromomycin at Day 10 revealed an overwhelming presence of *C. parvum* life cycle stages and severe damage to the epithelium of the intestine (**Figures 5A,B**). Damage to intestinal mucosa includes blunting of the villi, acute inflammation and formation of fibrin (fibrinous hemorrhagic enteritis) resulting from local hemorrhage and clotting (**Figures 5A,B**). The ileum of mice treated with 10 mg/kg/day of miltefosine was as severely damaged and infected as the other controls (**Figure 5C**). At Day 10, *C. parvum* oocysts were also abundantly present in mice treated with 20 mg/kg/day of OlPC and intestinal damage was also present but to a lesser extent than in the infected controls (**Figure 6A**). At the dose of 30 mg/kg/day of OlPC, mild intestinal damage and inflammation were noticeable and *C. parvum* oocysts were scarcely visible (**Figure 6B**). Finally, at 40 mg/kg/day, mice displayed practically no signs of intestinal damage or inflammation and a rare presence of *C. parvum* oocysts at Day 10 (**Figure 6C**). Slides from the ileum of non-infected controls were also prepared to provide a negative reference for comparison (data not shown).

At Day 30, the surviving mice treated with 10 mg/kg/day of miltefosine revealed an abundant presence of *C. parvum* oocysts and severe damage to the gut epithelium (**Figure 7A**). At the lowest concentration of OlPC, mice displayed a moderate presence of *C. parvum* oocysts and only mild damage to the intestinal epithelium (**Figure 7B**). At 30 and 40 mg/kg/day of OlPC, mice showed a complete absence of *C. parvum* oocysts and a healthy ileum in comparison with controls (**Figures 8A,B**). Sections of ileum, liver and spleen (data not shown) were also processed in uninfected (control) mice treated with 40 mg/kg/day for 10 days and no inflammation or toxicity was noticeable.

#### Residual Infectious Oocyst Concentration from Mice Exposed to OlPC is not Sufficient to Cause a New Infection in Naïve C57BL/6 IFN**γ**R-KO Mice

Another experiment was conducted to determine whether there was a sufficient number of infectious oocysts (more than the minimal infectious dose) remaining in the intestines of surviving mice at Days 10 or 30 to transmit the parasite and cause a new infection. To do so, naïve C57BL/6 IFNγR-KO mice were

infected with a constant volume of the purified oocysts from mouse intestinal samples coming from the different treatment groups from both Days 10 and 30. In order to determine if naïve mice were infected, stool samples were taken once a week, processed and analyzed by qPCR and light microscopy (**Table 1**). Presence of *C. parvum* oocysts or DNA in the stool of these naïve mice suggests that a fully complete life cycle was achieved by the parasite. Therefore, the concentration of infectious oocysts in the purified intestinal samples of mice from the *in vivo* study would be considered to cause a new infection. Results demonstrated that only the purified intestinal samples from 40 mg/kg/day OlPCtreated mice at Day 30 were not able to successfully transmit a *C. parvum* infection in naïve C57BL/6 IFNγR-KO mice. For these inoculated naïve mice, no oocysts could be seen by light microscopy and no DNA could be detected by qPCR in the stool even by Day 35 (**Table 1**). Additionally, no sign of illness were observed and the histological sections of the ileum revealed a complete absence of oocysts (data not shown). This observation corroborates previous results as 40 mg/kg/day OlPC-treated mice have been shown to have practically cleared the infection at D30. However, naïve C57BL/6 IFNγR-KO mice infected with purified intestinal samples from other treatment groups developed signs

FIGURE 7 | Day 30 histological sections of the ileum of control and 20 mg/kg/day OlPC-treated C57BL/6 IFN**γ**R-KO mice infected with *C. parvum.* Representative transverse sections of the ileum stained with H&E showing disease progression. (A) Ileum of mice treated with 10 mg/kg/day of miltefosine shows severe epithelial damage and abundance of oocysts. (B) Ileum of mice treated with 20 mg/kg/day of OlPC shows some epithelial damage and moderate level of oocysts. Each group is represented at 100 and 400x magnifications. Red arrows indicate *C. parvum* oocysts.

FIGURE 8 | Day 30 histological sections of the ileum of 30 mg/kg/day OlPC-treated and 40 mg/kg/day OlPC-treated C57BL/6 IFN**γ**R-KO mice infected with *C. parvum.* Representative transverse sections of the ileum stained with H&E showing disease progression. (A) Ileum of mice treated with 30 mg/kg/day of OlPC. Section reveals no gut epithelial damage and an absence of oocysts. (B) Ileum of mice treated with 40 mg/kg/day of OlPC presents no sign of epithelial damage and an absence oocysts. Each group is represented at 100 and 400x magnifications.