Nuclear Factor Y (NF-Y) Modulates Encystation in Entamoeba via Stage-Specific Expression of the NF-YB and NF-YC Subunits

The human parasite Entamoeba histolytica is an important pathogen with significant global impact and is a leading cause of parasitic death in humans. Since only the cyst form can be transmitted, blocking encystation would prevent new infections, making the encystation pathway an attractive target for the development of new drugs. Identification of the genetic signals and transcriptional regulatory networks that control encystation would be an important advance in understanding the developmental cascade. We show that the Entamoeba NF-Y complex plays a crucial role in regulating the encystation process in Entamoeba.

central factor in amebic biology, stage interconversion is extremely poorly understood at the molecular level.
The reptilian parasite Entamoeba invadens can be encysted efficiently in the laboratory and is used as a model system to understand the signaling mechanisms by which differentiation (from trophozoites to cysts) is triggered (3,4). Calorie restriction by glucose starvation and hypo-osmotic shock are the first and foremost environmental factors which trigger the encystation process under laboratory conditions. Two types of receptor-mediated signaling pathways have important roles in encystation: (i) pathways operating through the binding of galactosidase (Gal)-terminated ligands, provided by serum in the media, to Entamoeba Gal/Gal-NAc receptors (5,6); and (ii) adrenergic receptor (AR)-mediated signaling pathways operating through the binding of catecholamine compounds norepinephrine and epinephrine (Epi) (4). Other factors such as cyclic AMP (cAMP) (7), calcium signaling (8,9), and synthesis of cholesteryl sulfate (10) and phospholipase D (PLD), which are involved in lipid second messenger signaling (11), have important roles in Entamoeba encystation. Most recently, the metabolic cofactor NAD ϩ has been shown an important role in encystation (12). Overall, NAD ϩ /NADH levels were found to be elevated during encystation and the presence of extracellular NAD ϩ was found to enhance encystation in vitro (12).
These observations provide important insights into the conditions that may trigger developmental changes in Entamoeba, although the genetic factors regulating these responses are poorly understood. Therefore, identification of the genetic signals and transcriptional regulatory networks that control stage conversion would be an important advance in understanding stage conversion in this parasite. Only a small number of DNA motifs and transcription factors have been characterized in Entamoeba (13). Entamoeba possess an atypical TATA element (GTATTTAAA) located approximately 30 nucleotides (nt) upstream of the transcription initiation site (14), and E. histolytica putative TATA binding protein (TBP) has significant sequence divergence from the TATA binding protein of Drosophila melanogaster, Caenorhabditis elegans, and Plasmodium falciparum (15). A GAAC element (AATGAACT) and an initiator (Inr) element (AAAAATTCA) overlying the transcription initiation site were also reported earlier as representing a core promoter in Entamoeba (16,17). In Entamoeba, transcription factors regulate gene expression relevant to many important aspects of amebic biology, including virulence, oxidative stress response, and stage conversion (12,(18)(19)(20).
Some transcription factors have been identified which have important roles in stage conversion of Entamoeba (12,18). A developmentally regulated form of E. histolytica Myb (EhMyb-dr) belonging to the SHAQKY family of Myb proteins binds to a hexanucleotide CCCCCC motif, and overexpression of EhMyb-dr upregulates 117 genes in the encystation pathway (18). Another developmentally regulated transcription factor, encystation regulatory motif-binding protein (ERM-BP), is an NAD ϩ -dependent transcription factor which binds the CAACAAA motif and is found in the promoters of 131 cyst-specific genes (12). However, considering that 900 genes are upregulated during encystation, the entire transcriptional network operating during encystation is not well understood and there are likely other factors that regulate encystation.
We analyzed developmentally regulated genes to identify transcription factors which may control stage conversion. Our analysis revealed that the nuclear factor Y (NF-Y) complex (composed of three subunits, NF-YA, NF-YB, and NF-YC) is developmentally regulated in Entamoeba (11). Using electrophoretic mobility shift assays (EMSA), we demonstrated that the NF-Y protein(s) from Entamoeba cyst nuclear extract binds a CCAAT motif. Silencing of NF-YC resulted in reduced stability of the complex, mislocalization of NF-YA during encystation, and a significant reduction in encystation efficiency. We demonstrated that the NF-Y complex follows ERM-BP and NAD ϩ induction, defining a temporal framework for transcriptional control of Entamoeba development.

RESULTS
The NF-Y complex was upregulated in amebic cysts. In order to identify transcription factors that may be regulated during development, we searched Entamoeba developmentally regulated genes for transcription factors (11,24). We identified homologues of nuclear factor Y (NF-Y) that are upregulated during E. invadens stage conversion. In eukaryotes, NF-Y is composed of three different subunits, NF-YA, NF-YB, and NF-YC, which form a complex and bind to CCAAT boxes in promoters of target genes (23). We found that the amino acid sequences of all three subunits (EIN_249270 NF-YA, EIN_057000 NF-YB, and EIN_380690 NF-YC) are present in E. invadens and are also well conserved in all Entamoeba species that form cysts (see Table S1 in the supplemental material). The Entamoeba NF-YA and NF-YC subunits are divergent from homologues in human and other eukaryotes (25,26); however, the domains essential for NF-Y subunit interactions and DNA binding are conserved in Entamoeba (Fig. 1), suggesting that the NF-Y transcription factor complex might be fully functional in Entamoeba. Previous work in higher eukaryotes has shown that the N-terminal region of human NF-YA (amino acids [aa] 31 to 140) contains a glutamine-rich domain, which is the minimal domain required for the interaction with Zinc-fingers and homeobox-1 protein (ZHX-1) (27). An amino acid sequence consisting of positions 141 to 269 and containing a serine/threonine-rich domain is the minimal domain required for the interaction with serum-responsive factor (SRF) (27). Amino acid sequence 1 to 212 of NF-YA corresponding to human is missing in Entamoeba; thus, the interaction with other proteins through this N-terminal region of NF-YA may not occur in Entamoeba.
On the other hand, Entamoeba NF-YC has a unique N-terminal sequence (89 aa, absent in human) and the C-terminal sequence of NF-YC (aa 121 to 335, corresponding to human NF-YC) is missing in Entamoeba. It is reported that the C-terminal sequence of NF-YC interacts with c-Myc in human and that deletion of amino acid sequence 101 to 335 in NF-YC produces a result that mimics an interaction with c-Myc (28). NF-YC is also reported to interact with TBP, and the interacting domains of NF-YC which interact with TBP are conserved in Entamoeba (29). The C terminus of NF-YC interacts with other proteins in plant systems. In Arabidopsis thaliana, the QQS protein interacts with the C terminus of NF-YC, plays an important role in the regulation of metabolic processes affecting carbon and nitrogen partitioning among proteins and carbohydrates, and modulates leaf and seed composition (30). Deletion of amino acid sequence 73 to 162 in AtNF-YC4 abolished the binding to QQS (30).
To further define stage-specific expression, we performed semiquantitative reverse transcriptase PCR (RT-PCR) in E. invadens trophozoites and cysts at different time points of encystation (24,48, and 72 h) (see Fig. S1A in the supplemental material). We found that NF-YA (EIN_249270) was constitutively expressed both in trophozoites and in cyst. However, EIN_057000 (NF-YB) and EIN_380690 (NF-YC) were stage specifically expressed during encystation of Entamoeba. NF-YB (EIN_057000) was undetectable in both trophozoites and was upregulated only at later time points (48 h and 72 h) of encystation. EIN_380690 (NF-YC) was undetectable in trophozoites and upregulated early (24 h) during encystation and continued to show high levels at later encystation time points (48 to 72 h) ( Fig. S1A and data not shown).
Localization and enrichment of NF-YA and NF-YC in the nuclei of amebic cysts. In order to analyze the expression of NF-Y subsets at the protein level, we identified commercial polyclonal antibodies to human NF-YA and NF-YC that correspond to regions with high homology to the amebic proteins; a commercial antibody that would recognize the amebic NF-YB was not identified. Western blot analysis performed with human NF-YA antibody in amebic lysates detected a band at below 25 kDa, in both trophozoites and cysts, which was consistent with the predicted molecular mass of Entamoeba NF-YA (19 kDa) and consistent with its mRNA expression in both amebic trophozoites and cysts (Fig. S2A). As expected, the NF-YA antibody recognized a protein of around 40 kDa in human cells (Fig. S2A). Cellular fractionation revealed that NF-YA was present at similar levels in nuclear extract (NE) and cytosolic extract (CE) in trophozoites; however, an enrichment of NF-YA in nuclear extract in cysts was observed (data not shown). Western blot analysis performed with human NF-YC antibody resulted in detection of a single band in amebic lysate at ϳ25 kDa, which is consistent with the predicted molecular mass of Entamoeba NF-YC (23 kDa); the blot demonstrated stage-specific expression of NF-YC in cysts consistent with the earlier mRNA expression data (Fig. S2B). As expected, the NF-YC antibody recognized a protein at 59 kDa in human cells (Fig. S2B).
To determine the subcellular localization, an immunofluorescence assay (IFA) was performed. Immunostaining with NF-YA antibody in E. invadens demonstrated localization in the cytosol and a faint signal in the nucleus of trophozoites ( Fig. 2A). In cysts, however, NF-YA localized exclusively to the nucleus ( Fig. 2A). Immunostaining with antibody to NF-YC revealed no staining in trophozoites and localization to the nucleus only in cysts (Fig. 2B). In addition to the nuclear localization, we also observed localization of NF-YC in the form of dense patches which resembled chromatoid bodies (CB) within cysts ( Fig. 2B; white arrowhead). Chromatoid bodies are rod-shaped or bar-shaped cellular inclusions that have been reported in Entamoeba cysts (31). In order to determine if the staining was in chromatoid bodies, we performed coimmunostaining using anti-NF-YC antibody and anti-DEAD box RNA helicase (DDX4) antibody, which was used as a marker for the presence of chromatoid bodies in mouse testes (32). Western blot analysis performed with human anti-DDX4 antibody in amebic lysates detected a band at around 37 kDa and a band at around 75 kDa in human cell lysate, as expected (Fig. S2C). Staining with anti-DDX4 antibody (in red) colocalizes with NF-YC (in green) (shown with white arrowheads in Fig. 2C) provided substantial evidence of localization of NF-YC to the chromatoid body (Fig. 2C). The chromatoid body localization is very specific for NF-YC, however, as NF-YA does not show localization into the chromatoid body (Fig. 2D). Our quantitative analysis in E. invadens cysts identified localization of NF-YC in the nucleus only (63% Ϯ 11%) and in both the chromatoid body and the nucleus (27% Ϯ 8%), as well as in the chromatoid body only (11% Ϯ 3%) (Fig. S3).
Entamoeba cyst nuclear protein(s) bound specifically to the CCAAT motif. In other systems, the NF-Y complex binds to a CCAAT motif (21,23). We analyzed the promoter of 900 cyst-specific genes and identified 354 genes with a CCAAT motif in their promoter regions, which is a significant enrichment relative to the number of occurrences of this motif in the entire promoter set of E. invadens (as determined using the hypergeometric distribution) (P Ͻ 0.0004), implying that this motif may have an important role in encystation. All the cyst-specific genes with a CCAAT motif are listed in Table S2. To determine where the CCAAT motif resides in the promoters, we analyzed its distribution within 500 nucleotides (nt) upstream from the start codon and found that it was distributed throughout the promoter regions but was not enriched in any specific promoter region (Fig. S1B). In order to determine whether the CCAAT motif binds an amebic nuclear protein(s), we performed an EMSA with radiolabeled CCAAT probe. Our analysis demonstrated that the CCAAT motif showed strong and specific binding to a protein(s) from cyst nuclear extracts whereas nuclear extracts from trophozoites showed a very weak band (Fig. 3A). We performed gel supershift (SS) assays using radiolabeled CCAAT probe, crude nuclear extract from cysts, and antibodies to NF-YA and NF-YC, which resulted in the presence of a supershift (SS) band; EMSA performed with the control actin antibody did not result in a supershift (Fig. 3B). Taken together, the data suggest that the NF-Y complex from cyst nuclear extracts binds to the CCAAT motif in Entamoeba.
Silencing of NF-YC decreased encystation efficiency and altered nuclear localization of the NF-Y subunits. In order to better understand the role of NF-YC in Entamoeba development, we used a trigger-mediated RNA interference gene silencing Representative EMSA results determined in the presence and absence of different components (marked "ϩ" and "Ϫ" respectively) are shown. Radiolabeled CCAAT probe was used in each reaction with crude nuclear extracts (NE) from both trophozoites (Trophs) and cysts. Unlabeled CCAAT probe was used as a specific cold competitor at 10ϫ and 100ϫ as indicated. The arrow indicates the major specific band in the gel shift assay; the free probe is indicated at the bottom of the panel. (B) Gel supershift assay using radiolabeled CCAAT and crude nuclear extracts from cysts with anti-NF-YA, anti-NF-YC, and anti-actin antibodies (Ab) at 1 or 2 g. The arrow indicates a major band corresponding to specific binding to NF-Y. SS, supershifted bands whose presence was due to complex formation with anti-NF-YA and anti-NF-YC antibodies but not with control anti-actin antibodies.
approach to downregulate NF-YC (33,34). We were successfully able to silence NF-YC, and the NF-YC transcript level was undetectable in trigger-mediated NF-YC cell lines (Fig. 4A). In order to determine the impact of NF-YC silencing on developmental control, parasites with silenced NF-YC were encysted in 96-well plates and calcofluorstained cysts were imaged at different time points of encystation (33). Silencing of NF-YC significantly decreased the cyst number at 72 h of encystation, suggesting that the NF-Y complex has an important role in regulating encystation (Fig. 4B). We also calculated encystation efficiency by counting sarkosyl-resistant cysts and demonstrated that the silenced NF-YC parasites showed a significant reduction of encystation efficiency compared to the control (data not shown). Furthermore, we checked the levels of expression of NF-YA and NF-YC protein in parasites silenced for NF-YC. Our Western blot analysis showed no change in the level of NF-YA in trophozoites or cysts in parasites silenced for NF-YC; however, as expected the level of NF-YC protein was undetectable in silenced NF-YC parasites (Fig. 5A). Immunostaining with NF-YA in silenced NF-YC cells showed only cytosolic localization in trophozoites and cysts; the NF-YA localized to punctate focal areas in cysts but did not overlap the parasite nuclei (Fig. 5B). This suggests that although expression of NF-YA does not depend on NF-YC, correct localization into the nucleus during encystation is dependent on the expression of NF-YC. As expected, NF-YC protein was undetectable by IFA in silenced parasites (Fig. 5C).
NF-Y transcription factor induction followed induction of NAD ؉ and transcription factor ERM-BP. In order to determine the sequence and appearance of transcription factors during encystation, we next performed EMSA using cyst nuclear extracts from parasites silenced for NF-YC or silenced for ERM-BP and wild-type control cells using radiolabeled NF-Y motif (CCAAT) and the encystation regulatory motif binding protein (CAACAAA motif) (12). As expected, both motifs showed specific binding in cyst nuclear extracts (Fig. 6A). In parasites silenced for NF-YC, the CCAAT (NF-Y motif) binding was largely abolished compared to control cysts; however, binding of the ERM-BP protein CAACAAA motif still occurred. In parasites silenced for ERM-BP, binding of both the ERM-BP and NF-Y protein complexes was significantly reduced (Fig. 6A). We have previously reported that the intracellular NAD ϩ /NADH level is elevated during encystation (12). We found that the NAD ϩ /NADH level in parasites silenced for NF-YC NF-Y Is Essential for Entamoeba Development ® was increased at 48 h of encystation and was similar to that seen with control parasites (Fig. 6B).
Taken together, our data suggest that the pathways involved in expression of ERM-BP and increasing levels of NAD ϩ (12) were not affected in the parasites silenced for NF-YC; thus, the NF-Y function was later than the ERM-BP function in the encystation pathway (Fig. 7). We also observed that the cyst-specific genes with a NF-Y motif were largely distinct from cyst-specific genes with the ERM motif (Fig. S4). A total of 354 genes had the NF-Y motif in their promoter (Table S2), and a total of 131 genes had ERM (12), but there were only 19 genes with both motifs in their promoters (Fig. S4) (Table S4). Further studies are needed to understand the full transcriptional network regulated by NF-Y in Entamoeba.

DISCUSSION
In higher eukaryotes, the NF-Y transcription factor acts as an activator or repressor, depending on its interaction with HAT (histone acetyltransferase) or HDAC (histone deacetylase), and plays a critical role in development (35). In Drosophila, NF-Y functions as a transcriptional activator in the differentiation of R7 photoreceptor cells during development (23). In the plant Arabidopsis thaliana, transcript analysis revealed that all of the NF-Y genes display differential expression patterns during development and as a response to environmental stimuli (36)(37)(38). It has also been reported that the specific expression pattern of each NF-Y protein and interactions among the complementary subunits are important for the specific function of NF-Y complexes in transcriptional control (38). We found that the NF-Y complex is functional in Entamoeba cysts, provides important contributions to transcriptional control of stage conversion, and occurs downstream of ERM-BP transcriptional control. Our work begins to develop a framework for the network of transcriptional regulators affecting Entamoeba development.
All three NF-Y subunits are well conserved in Entamoeba, though the NF-YA and NF-YC subunits are shorter than those in human and other eukaryotes; however, the domains essential for subunit interactions and DNA binding are conserved in Entamoeba (25,26). In Entamoeba cysts, NF-YC localizes to the nucleus as well as to a dense rod-like structure, the chromatoid body. In many organisms, including Drosophila, germ cells are characterized by the accumulation of dense fibrous material into a cytoplasmic structure called the germplasm or nuage. In mammals, the chromatoid body is suggested to be a counterpart of nuage on the basis of its structural features and protein composition (39). Recent studies identified the CB as RNA-processing bodies in somatic cells (32,39,40). Dicer and components of microRNP complexes, including Argonaute proteins (Ago), RNA helicase (VASA homolog, Dead-Box RNA helicase) are highly concentrated into the CB (41). CBs are frequently observed in Entamoeba cysts, and may contain various amounts of DNA, RNA, and RNA binding proteins and play a role in cyst wall deposition during Entamoeba encystation (42). However, the exact function of EMSA results are shown in the presence and absence of different components marked "ϩ" and "Ϫ," respectively. Radiolabeled probes for the NF-Y motif (CCAAT probe) and ERM (CAACAA motif) were used as indicated with nuclear extracts from control, silenced NF-YC, and silenced ERM-BP cysts (48 h). Unlabeled probes for each were used at 100ϫ as a specific cold competitor as indicated. The arrow indicates the major specific band in the gel shift assay; the free probes are indicated at the bottom of the panel. In NF-YC-silenced cysts, ERM-BP still functionally bound to its motif. In ERM-BP-silenced parasites, the NF-Y complex did not bind to the CCAAT motif. (B) Measurement of intracellular NAD ϩ /NADH in trophozoites (Troph) and 48-h cysts in control and silenced NF-YC parasites. Data represent means Ϯ standard deviations (SD) (n ϭ 3) (Student's t test; *, P Ͻ 0.05; ns, not statistically significant). chromatoid bodies in Entamoeba encystation has remained elusive. The localization of the transcription factor NF-YC in Entamoeba chromatoid body links transcriptional control of development to other aspects of cellular RNA control and begins to define the molecular components of the CB.
An earlier study in Entamoeba histolytica revealed that the CCAAT motif can act as cis-activator element and control the expression of multidrug-resistant P-glycoprotein gene EhPgp1 (43). Differential DNA-protein complex formation results were seen in the multidrug-resistant clone compared to the drug-sensitive clone involved in the regulation of the EhPgp1 gene expression (43). The nuclear factors that bind to these sites were semipurified by affinity chromatography but were not further characterized (44,45). Whether the protein complex that binds to the CCAAT motif in multidrug-resistant E. histolytica is the same as that which binds to the CCAAT motif in E. invadens cysts is not clear. However, our efforts using both biochemical (EMSA supershift) and genetic (silencing of NF-Y complex) approaches have definitively demonstrated that the NF-Y complex binds the CCAAT motif in E. invadens and is an important regulator of parasite development.
Silencing of NF-YC significantly reduces the encystation efficiency, directly implicating the NF-Y complex as an important regulator of Entamoeba development. However, silencing of NF-YC does not affect either intracellular NAD ϩ /NADH or ERM-BP binding, suggesting that NF-Y lies downstream of the ERM-BP and NAD ϩ biosynthesis pathway (Fig. 7). Using our data and published information, we outlined a temporal network of control mechanisms regulating Entamoeba development (Fig. 7). Further characterization of NF-Y target genes and interacting protein partners will help define the transcription machinery regulated by transcription factor NF-Y in Entamoeba.  (3). Among the signaling molecules, Gal-terminated ligands (5,6), catecholamine signaling (4), cyclic AMP (cAMP) (7), cholesteryl sulfate (10), Ca2 ϩ signaling (8,9), phospholipase D (PLD) (11) and NAD (NAD ϩ ) (12) have shown important roles in encystation. Among the transcriptional regulators of encystation, the roles of two previously described transcription factors (drMyb and ERM-BP) (12,18) and of nuclear factor Y (NF-Y) (in this present study) and their appearances at different time points of development are illustrated here. References are listed in parentheses in the figure.

MATERIALS AND METHODS
Parasite culture, transfection, and induction of stage conversion. E. invadens (strain IP-1) was axenically maintained (46). To make stable transgenic cell lines, parasites were transfected with plasmid DNA by electroporation (47). Stable cell lines were maintained at a G418 concentration of 80 g/ml unless otherwise stated. To induce encystation, E. invadens trophozoites were incubated in 47% LYI-LG (supplemented with 7% adult bovine serum) in a 96-well plate (48). Cyst numbers were determined by automated quantitative imaging as described in earlier studies (33,49). Briefly, calcofluor white, which specifically stains chitin in the cyst wall, was added to wells after 48 h or 72 h of encystation. The calcofluor-stained cysts were imaged at ϫ10 magnification using ImageXpress Micro (Molecular Devices) and quantified by using MetaXpress analysis software (Molecular Devices). The experiment was repeated at least three times with eight replicates for each sample. Data represent means and standard errors, and the t test was performed from a well-distributed data set (24 replicates) of each cell line.
Bioinformatics analysis to identify NF-Y promoter motif. Upstream promoter regions (500 nt) of 900 cyst-specific genes were analyzed to identify DNA motifs as described earlier (20,50). In brief, MEME was performed with the command line -dna -mod zoops -minw 5 -maxw 5 -minsites 5 -nmotfs 30. The MAST program was utilized to determine the total number of occurrences of each motif in the promoter sequence databases.
Electrophoretic mobility shift assays (EMSA). EMSA was performed as previously described (20). The oligonucleotides used in EMSA are listed in Table S3 in the supplemental material. Each motif had an additional 12 nt at the 5= end and 8 nt at the 3= end, which created a 5= overhang after annealing, and were utilized for radiolabeling using Klenow fragments (50). In brief, complementary overlapping probes were annealed and labeled using [␣-32 P]ATP and Klenow fragments (Invitrogen). The binding reaction mixture consisted of a total volume of 20 l, which included 2 l 10ϫ EMSA binding buffer (10 mM Tris-HCl [pH 7.9], 50 mM NaCl, 1 mM EDTA, 3% glycerol, 0.05% milk powder, 0.05 mg of bromophenol blue), 10 g of nuclear extract form trophozoites or 24-h cysts, 2 g of poly(dI-dC), and 50 fmol of labeled probe. For gel supershift assays, 10 g of nuclear extracts was preincubated for 3 h at 4°C in HEPES buffer with 1 and 2 g of each mouse monoclonal anti-NF-YA antibody (sc-17753), rabbit polyclonal anti-NF-YC antibody (ab232909), and rabbit polyclonal anti-beta-actin antibody (ab227387); followed by 30 min of incubation at room temperature with radiolabeled probe (50 fmol). The binding reaction mixtures were loaded onto a 9% nondenaturing polyacrylamide gel and run for 3 h. The gel was fixed, dried, and exposed to a phosphor screen. Gels were imaged using a Personal Molecular Imager (PMI) system with Quantity One software (Bio-Rad).
RNA extraction and RT-PCR. Total RNA was extracted from trophozoites and cysts using the TRIzol method (Life Technologies). RNA was subjected to DNase treatment (DNase kit; Invitrogen) and reverse transcribed using oligo(dT) primers (Invitrogen). The resultant cDNA (3 l) was used in subsequent PCRs (25 l total volume). The number of PCR cycles was set to 30, and 10 l of PCR products was run on a 1.5% agarose gel. The negative control (minus reverse transcriptase [RT]) was split away before the addition of Superscript RT (Invitrogen) but was otherwise treated like the other samples. The primers used in RT-PCR are listed in Table S3.
Plasmid construction. For gene silencing, the 152-trigger construct was used; a full-length coding region of the NF-YC gene was cloned downstream of the Trigger region at the AvrII and SacII sites as described earlier (33). The primers used in cloning are listed in Table S3. The construct was confirmed by sequencing before transfection into E. invadens.
Measurement of intracellular NAD ؉ /NADH levels. Intracellular NAD ϩ and NADH levels were determined per the manufacturer's protocol (NAD ϩ /NADH assay kit) (Abcam; catalog no. ab65348) and as described earlier (12). Briefly 2 ϫ 10 6 cells were lysed in NAD ϩ /NADH extraction buffer by sonication (five pulses at 15 A for 15 s). The lysate was centrifuged at 14,000 rpm, and the supernatant containing NAD ϩ /NADH was filtered through a 10-kDa spin column to get rid of enzymes, which may consume NADH rapidly. To detect the NADH in the sample, a decomposition step was performed by heating the samples at 60°C for 30 min; under such conditions, all the NAD ϩ is decomposed while the NADH is still NF-Y Is Essential for Entamoeba Development ® intact. A 100-l reaction mixture was prepared for each standard, and samples were processed in duplicate in a clear-bottom 96-well plate. The plate was incubated at room temperature for 5 min to convert NAD to NADH followed by addition of 10 l NADH developer into each well and was incubated at room temperature for 2 h. Optical density (OD) was measured at 450 nm using a plate reader (BioTek Cytation3).
Statistical analysis. Student's t test was performed for comparisons of two conditions. A P value of Ͻ0.05 in each independent experiment was considered significant.

ACKNOWLEDGMENTS
We thank all members of the Singh laboratory for helpful discussions and critical reading of the manuscript. We are especially grateful to Gretchen Ehrenkaufer for helpful technical and scientific advice.
A grant from NIAID AI119893-02 to U.S. supported the work.