Dissection of the in vitro developmental program of Hammondia hammondi reveals a link between stress sensitivity and life cycle flexibility in Toxoplasma gondii
Toxoplasma gondii , the causative agent of toxoplasmosis , is a globally ubiquitous Apicomplexan parasite capable of infecting all warm-blooded animals , including approximately one-third of the human population ( Dubey and Beattie , 1988; Dubey and Sreekumar , 2003; Pappas et al . , 2009; Jones et al . , 2001 ) . Although generally asymptomatic in immunocompetent individuals , T . gondii is capable of causing disease in the immunocompromised ( including HIV/AIDS patients ) , developing neonates , and occasionally healthy individuals ( Luft et al . , 1993; Remington and Klein , 2001; Carme et al . , 2002; Grigg et al . , 2001 ) . The global ubiquity of T . gondii is due , at least in part , to its broad host range and ability to persist undetected in an immunocompetent host where it is either capable of being transmitted to a new intermediate host or recrudescing in the original host ( Marty et al . , 2002; Takashima et al . , 2008 ) . These characteristics are lacking in the nearest extant relative of T . gondii , Hammondia hammondi . Despite sharing the vast majority of their genes in near perfect synteny ( Walzer et al . , 2013 ) and the same definitive felid host ( Dubey and Sreekumar , 2003 ) , H . hammondi is known to infect rodents , goats , and roe deer ( Dubey and Sreekumar , 2003; Lorenzi et al . , 2016 ) , and at least in mice it is naturally avirulent compared to T . gondii . Unlike T . gondii , H . hammondi has an obligately heteroxenous life cycle ( Dubey and Sreekumar , 2003 ) , where intermediate host stages ( i . e . , tissue cysts ) are only capable of infecting the definitive host , and definitive host stages ( i . e . , oocysts ) are limited to infection of an intermediate host . In addition to these life cycle differences , these two parasite species have in vitro behaviors that mirror their in vivo differences . Specifically , when T . gondii sporozoites ( VEG strain [Jerome et al . , 1998] ) are used to initiate infections in human host cells in vitro , they undergo a period of rapid replication , after which their growth rate slows and a subpopulation of parasites spontaneously convert to bradyzoite ( e . g . , cyst-like ) stages ( Conde de Felipe et al . , 2008 ) . However , T . gondii parasites cultivated in this manner still undergo multiple cycles of replication , egress and reinvasion , and indefinite propagation ( Jerome et al . , 1998 ) . In contrast , when H . hammondi sporozoites are put into tissue culture they replicate slower than T . gondii ( Jerome et al . , 1998; Sheffield et al . , 1976 ) and do not appear to be capable of subculture ( either via passage of culture supernatants or after scraping and trypsinization; ( Sheffield et al . , 1976; Riahi et al . , 1995 ) . Ultimately in vitro cultivated H . hammondi form bradyzoite cyst-like stages that are infectious only to the definitive host ( Sheffield et al . , 1976; Riahi et al . , 1995 ) . While the molecular determinants that control these dramatic phenotypic differences are unknown , the fact that life cycle restriction in H . hammondi occurs in vitro provides a unique opportunity to identify key aspects of H . hammondi biology that underlie its restrictive life cycle . By contrast , the life cycle restrictions in H . hammondi will allow for elucidation of the cellular and molecular differences in T . gondii that determine its comparatively flexible life cycle . It is important to note that with respect to other Apicomplexans ( including N . caninum and Plasmodium spp . ) , it is the T . gondii life cycle that is atypical; the majority of coccidian Apicomplexans are obligately heteroxenous ( Dubey and Sreekumar , 2003; Dubey , 1998; Muller and Baker , 1982 ) . The most parsimonious explanation is that the obligately heteroxenous life cycle of H . hammondi is the ancestral state , and that during speciation T . gondii acquired novel traits linked to any number of key parasite processes ( regulation of development , stress sensitivity , interactions with the host environment ) that underlie its life cycle flexibility . The obligately heteroxenous life cycle of H . hammondi makes experimentally characterizing and manipulating this parasite species difficult in comparison to other closely related apicomplexan parasites . We have characterized the invasion and replication rates of T . gondii ( VEG strain; [Dubey , 2001; Speer et al . , 1997] ) and H . hammondi ( HhCatEth1 and HhCatAmer [Lorenzi et al . , 2016; Dubey et al . , 2013] ) from sporozoite-initiated infections , representing the most thorough head-to-head comparison of the intermediate host life stages of these distinct parasite species . Using sporozoite derived infections , we have also compared the timing and frequency of in vitro differentiation and report the first ever transcriptome from replicating H . hammondi . Together , these data reveal previously unknown features of H . hammondi development . Using information gleaned from these experiments , we were able to successfully subculture H . hammondi both in vitro and in a murine model during a highly predictable window of infectivity and replicative capacity . Additionally , we generated the first ever transgenic H . hammondi , opening up the door to future molecular and genetic studies in this poorly understood parasite species . These data provide fundamental insight for understanding how virulence and pathogenicity have evolved in T . gondii through interspecies comparisons with its closest extant relative . Finally , we found that H . hammondi is resistant to alkaline pH-induced tissue cyst formation and determined that the H . hammondi transcriptome is refractory to alkaline pH exposure . Our data indicate that H . hammondi exists as a unique zoite life stage and follows a developmental path that is distinct from the canonical tachyzoite and bradyzoite life stages described for T . gondii . Moreover our work has identified sensitivity to external stressors as a likely culprit contributing to the intriguing developmental and virulence differences between T . gondii and H . hammondi , a finding that we speculate may have a direct impact on the ability of T . gondii to cause disease in immunocompromised humans and may also underlie its extensive host and geographic range .
While T . gondii oocysts and sporozoites have been studied extensively in vitro and in multiple hosts ( Jerome et al . , 1998; Dubey , 2001; Speer et al . , 1997 ) , H . hammondi and T . gondii have been directly compared in only a handful of studies ( e . g . , [Dubey and Sreekumar , 2003] ) . Therefore we performed a head-to-head characterization of sporozoite derived infections from both species during in vitro infections . As expected from previous work , we found that HhCatEth1 replicated significantly slower than TgVEG in multiple replicate assays ( Figure 1A , B; ** P=<0 . 01 , **** P=<0 . 0001 ) . Based on median vacuole size ( Table 1 , Figure 1A , B ) , TgVEG replicated ~2–3X faster than HhCatEth1 , with a maximum division time of 9 . 6 hr compared to 18 hr for HhCathEth1 ( Table 1 ) . Despite these differences in replication , TgVEG and HhCatEth1 sporozoites had similar infectivity rates in human foreskin fibroblasts ( HFFs ) , defined as the number of vacuoles counted as a fraction of the number of input sporozoites ( HhCatEth1 0 . 12 ± 0 . 02 N = 2; TgVEG; 0 . 10 ± 0 . 04 N = 2 p=0 . 64; Figure 1C–D ) . These data highlight parasite replication as a potentially important component in determining differences in virulence between these species , since pathogenesis in vivo is often ( but not always ) linked to parasite burden ( Saeij et al . , 2005 ) . All T . gondii strains studied to date can be cultured indefinitely by serial passage in rodent intermediate hosts or in tissue/cell culture ( Croken et al . , 2014a ) In contrast , when grown in tissue culture H . hammondi ultimately differentiates into a cyst form that is only orally infectious to the definitive feline host ( Dubey and Sreekumar , 2003 ) . Interestingly , some strains of T . gondii ( e . g . , strains VEG and EGS; [Jerome et al . , 1998; Dubey , 2001; Paredes-Santos et al . , 2013] ) are known to spontaneously form cysts in tissue culture . To elucidate the temporal dynamics of differentiation and tissue cyst formation in H . hammondi and compare them to T . gondii , we infected HFFs with TgVEG or HhCatEth1 sporozoites and stained them with Dolichos biflorus Agglutinin ( DBA ) at 4 and 15 days post-infection ( DPI ) to identify tissue cysts ( Figure 2A ) . While all TgVEG and HhCatEth1 vacuoles were DBA-negative at 4 DPI , at 15 DPI ~80% ( 152/182 ) of H . hammondi vacuoles were DBA-positive , compared to 15% ( 34/355 ) of TgVEG vacuoles ( p<0 . 0001; Figure 2B ) . To more precisely determine the temporal dynamics of tissue cyst formation , we quantified spontaneous cyst formation with TgVEG ( Figure 2C ) or HhCatAmer ( Figure 2D ) over the course of 23 days . Both TgVEG and HhCatAmer spontaneously formed DBA-positive tissue cysts as early as 8 and 12 DPI , respectively . By 23 DPI 100% of all HhCatAmer vacuoles were DBA-positive , while the percentage of DBA-positive TgVEG vacuoles never exceeded 20% ( Figure 2C , D ) . TgVEG cultures had to be passed three times over the course of the experiment to prevent complete lysis of the host cell monolayers , which is consistent with their propagation as a mixed population of tachyzoites and bradyzoites . These data demonstrate that H . hammondi undergoes a much more rigid developmental program during in vitro cultivation compared to T . gondii , resulting in 100% tissue cyst conversion and what is likely a near complete arrest in growth . Our previous work showed that the H . hammondi oocyst transcriptome ( consisting of sporulated and unsporulated oocysts ) was significantly more ‘bradyzoite-like’ than T . gondii sporozoites ( Walzer et al . , 2014 ) , and we reasoned that this was consistent with the propensity of this species to spontaneously form cysts during in vitro cultivation . However , these transcriptome studies were from the quiescent oocyst life stage and provided no insight into the developmental dynamics of the H . hammondi transcriptome compared to T . gondii . To compare the dynamics of the replicating H . hammondi and T . gondii transcriptomes during in vitro development , we used RNAseq on day 4 ( D4 ) and day 15 ( D15 ) in vitro cultured H . hammondi and T . gondii . Using existing genome annotations ( Lorenzi et al . , 2016 ) , we identified 7372 putatively orthologous genes between T . gondii and H . hammondi ( ToxoDB . org; likely a conservative estimate of shared gene content between these species ) . As expected based on their in vitro growth rates ( Figure 1 ) , the number of reads mapping to the H . hammondi transcriptome was lower overall compared to those mapping to T . gondii ( Figure 3—figure supplement 1A ) , and this was particularly pronounced at 15 DPI . To mitigate the effect this could have on falsely categorizing transcripts as being of higher abundance in T . gondii compared to H . hammondi , we included a given gene in downstream analyses only if one H . hammondi D4 and one H . hammondi D15 sample had at least five reads . This reduced the overall size of the queried gene set to 4146 , and all analyses were performed with this subset . When log2 transformed and DESeq2-normalized RPM ( mapping Reads Per Million; all data in Supplementary Data File 1 ) values were plotted for D4 and D15 between species we observed a high correlation in transcript levels at both time points ( Figure 3—figure supplement 1B ) . While the majority of genes were expressed at similar levels between T . gondii and H . hammondi , we identified multiple transcripts of significantly different abundance ( Figure 3A and blue data points in Figure 3—figure supplement 1B ) . We used Gene Set Enrichment Analysis ( GSEA; [Subramanian et al . , 2005] ) with previously published ( Croken et al . , 2014b ) and our own curated gene sets ( Table 2 ) to identify gene classes with species-specific enrichment on D4 and D15 ( see Methods and Table 2 for Gene Sets ) . We identified seven gene sets that showed significant species-specific enrichment ( FDR q-value <0 . 01; NES scores shown in Figure 3B ) . H . hammondi profiles showed significant enrichment for in vitro ( D4 , D15 ) and in vivo ( D15 ) bradyzoite gene sets ( Figure 3B , C; heat maps shown in Figure 3—figure supplement 1D ) , showing that replicating H . hammondi zoites maintain the ‘bradyzoite-like’ transcriptional profile previously observed in sporozoites after at least 15 days in culture ( Walzer et al . , 2014 ) . Moreover , T . gondii showed significant enrichment compared to H . hammondi for in vitro tachyzoite genes at 15 DPI ( Figure 3B; heatmap shown in Figure 3—figure supplement 1C ) , a time point at which VEG is <10% DBA-positive while H . hammondi is between 40–80% DBA-positive ( Figure 2B , C ) . Overall these data are consistent with our general understanding of transcript abundance changes that occur during the tachyzoite to bradyzoite transition ( albeit entirely from studies using T . gondii ) . A more unexpected finding was that at both D4 and D15 , H . hammondi had a transcriptional profile that was significantly enriched for genes of high transcript abundance in T . gondii merozoites replicating in cat enteroepithelial cells but poorly expressed in other life stages ( [Croken et al . , 2014b; Behnke et al . , 2014]; CAT STAGE SPECIFIC 1 , 2; Figure 3C , D ) . Specifically , out of the 21 detectable members of the ‘CAT STAGE SPECIFIC 1’ gene set , 18 were ranked in the top 308/4146 ( 7 . 4% ) and 174/4146 ( 4 . 2% ) of genes of greater abundance in H . hammondi D4 and D15 zoites , respectively ( Figure 3C ) . To validate this finding we performed RNAseq on T . gondii and H . hammondi-infected THP-1 cells 24 hr post-infection and found a consistent and highly significant enrichment for these same gene sets ( Figure 3—figure supplement 2A–C ) , and in this case a similar number of reads from H . hammondi and T . gondii mapped to their respective parasite transcriptomes ( Figure 3—figure supplement 2D ) . These data suggest that H . hammondi has aspects of its transcriptional profile that are independent of its spontaneous conversion to bradyzoites and that a subset of genes that are uniquely expressed in T . gondii merozoites and sporozoites may not share these same profiles in H . hammondi . We also validated a subset of transcripts that were of greater abundance in D4 and D15 cultivated H . hammondi parasites using qPCR ( heatmap of selected genes in Figure 3—figure supplement 3A ) . We used ROP18 as a control for a gene that was of significantly higher abundance in H . hammondi compared to T . gondii ( TgVEG and other Type III strains express very little ROP18 transcript due to a promoter indel; [Walzer et al . , 2013; Walzer et al . , 2014; Boyle et al . , 2008] ) , and CDPK1 and AMA1 as control genes of similar abundance between the two species . GRA1 transcript levels were used as loading controls . We used validated primer sets for eight genes ( which had to be made separately for each species; see Supplementary file 3 for all primer sequences ) to quantify eight transcripts ( plus ROP18 ) using qPCR on RNA freshly isolated from D4 and D15 HhCatEth1 and TgVEG cultures . As expected ROP18 transcript was found to be >1000 fold greater in abundance in HhCatEth1 compared to TgVEG , while CDPK1 and AMA1 transcript levels were not significantly different between species ( Figure 3—figure supplement 3B ) . The remaining nine queried transcripts showed significantly higher abundance in H . hammondi compared to T . gondii ( Figure 3—figure supplement 3B ) confirming the validity of our approach to analyze , normalize and filter cross-species RNAseq data with significant differences in read counts . From a phylogenetic standpoint H . hammondi is a good model to understand the genetic basis for traits that are unique to T . gondii . However , unlike all T . gondii strains studied to date , sporozoite-initiated cultures of H . hammondi in host cells from multiple organisms including humans , cattle , and non-human primates lose their ability to be subcultured in vitro ( Sheffield et al . , 1976; Riahi et al . , 1995 ) . In order to precisely define when H . hammondi sporozoites lose the ability to infect new cells we attempted to subculture H . hammondi zoites after defined periods of cultivation ( Figure 4A–B ) . We found that H . hammondi zoites were capable of infecting and replicating within new host cells for up to 8 days post-excystation ( Figure 4C–F ) , while we did not observe any evidence of growth when subcultures were initiated on days 11 and 15 post-excystation ( Figure 4G , H ) . In agreement with previous studies ( Dubey and Sreekumar , 2003; Conde de Felipe et al . , 2008; Sheffield et al . , 1976 ) we also observed that the number of visible H . hammondi vacuoles disappear over time ( Figure 2C–E ) . We did not identify any vacuoles derived from lysing and re-invading parasites ( which would have comparatively smaller sizes ) after 10 DPI as we only observed a general increase in vacuole size prior to their disappearance from culture ( Figure 4—figure supplement 1 ) . Together , our data support previous work showing that H . hammondi cannot be subcultured with high efficiency after approximately one week or longer in culture ( Sheffield et al . , 1976; Riahi et al . , 1995 ) , but importantly we identified a previously unknown window prior to spontaneous tissue cyst formation where this parasite can be effectively subcultured . When we performed similar experiments in vivo , we observed that the ability to be subcultured in vitro and infectivity in a mouse model of infection were temporally correlated . We cultivated H . hammondi sporozoites in HFFs for 5 days and used them to infect mice intraperitoneally ( 50 , 000 zoites ) . As early as 2 DPI , we measured interferon-gamma ( IFNγ ) production indicative of an active infection ( Figure 5A ) . We used PCR to detect H . hammondi DNA in spleens and resident peritoneal cells in these same mice 3 weeks post-infection ( PI ) ( Figure 5B ) . Histological examination of muscle from infected mice using anti-Toxoplasma antibodies that cross-react with H . hammondi showed the presence of the parasites in the muscle tissue of the mouse infected with in vitro-passed H . hammondi ( right panel , Figure 5C ) which were similar to those observed after oral inoculation with H . hammondi sporulated oocysts ( middle panel; Figure 5C ) . Overall these data show that H . hammondi parasites grown in vitro are capable of ( a ) inducing an immune response in mice during the acute phase of infection , and ( b ) disseminating to multiple mouse tissues and establishing a chronic infection that resembles an infection resulting from oral gavage with sporulated oocysts . To determine how long mouse infectivity was retained in in vitro cultivated H . hammondi , mice were injected intraperitoneally with 50 , 000 zoites grown in vitro for 6 , 8 , 10 , 13 , and 15 days , and monitored for signs of infection by measuring serum IFNγ levels . The ability to be subcultured in vitro correlated perfectly with the ability to initiate infections in mice in vivo . Specifically , we observed an increase in IFNγ levels in mice infected with 6 DPI or 8 DPI in vitro zoites ( Figure 5D ) , while we did not see an increase in IFNγ levels in those infected with parasites cultured for 10 , 13 , and 15 days ( Figure 5D ) . To our knowledge this is the first time that sporozoite-derived growth stages of H . hammondi have been used to initiate infections in an intermediate host , since all previous studies focused on parasite that had already terminally differentiated . Genetic manipulation of H . hammondi has not been reported in the literature . Given the genetic similarity between T . gondii and H . hammondi ( Walzer et al . , 2013; Lorenzi et al . , 2016 ) and the apparent interchangeability of promoter sequences between them ( Walzer et al . , 2013; Walzer et al . , 2014 ) , we transfected ~4 million HhCatAmer sporozoites with a UPRT-targeting CRISPR/CAS9-GFP plasmid ( Shen et al . , 2014 ) along with a PCR2 . 1 Topo-cloned dsRED cassette with 20 bp sequences flanking the CAS9 nuclease cut site within the H . hammondi UPRT gene . We used the Toxoplasma-specific plasmid to target H . hammondi UPRT as the gRNA sequence is identical between T . gondii and H . hammondi . Both plasmids were taken up and expressed by H . hammondi , as evidenced by GFP-fluorescence in the parasite nuclei and red fluorescence in the cytoplasm ( Figure 6A ) . Importantly , transgenic parasites replicated in vitro since we observed multicellular vacuoles in the infected monolayer ( Figure 6A ) . Transfection efficiency of viable parasites determined by counting the percentage of replicating parasites with CAS9-GFP and/or dsRED fluorescence was ~4% ( 15 out of 350 vacuoles at 48 hr post-transfection ) , which is consistent with typical transfection efficiencies observed with most strains of T . gondii ( Kim and Weiss , 2004 ) . To make a stable transgenic parasite line , we transfected ~4 million parasites with the HhUPRT:dsRED repair cassette and the HhUPRTgRNA:CAS9:GFP plasmid and cultured them in 10 µM FUDR for 5 days . After FACS sorting ~12 , 000 dsRED-expressing zoites ( ~3% of the total parasite population; Figure 6B ) , we injected two female Balb/c mice intraperitoneally with ~6000 dsRED-expressing zoites each . For the first week of the infection , mice were injected 1X daily with 200 µL of 1 mM FUDR , and mice were euthanized and fed to a cat 4 weeks PI . Fecal floats showed the presence of dsRED-expressing sporozoites within some of the oocysts ( Figure 6B ) , and ~32% of isolated sporozoites expressed dsRED based on FACS analysis ( Figure 6B , bottom FACS plot ) . PCR screening ( Figure 6—figure supplement 1 ) and the inability to grow parasites long term in vitro confirmed the identity of the fluorescent parasites as H . hammondi . To determine if our transgenic approach to disrupt the UPRT gene and insert a dsRED expression cassette was successful , we quantified the growth of dsRED+ and dsRED ( - ) parasites in the presence of FUDR and compared it to wild type HhCatAmer . Fixed and permeabilized parasites were stained with cross-reactive anti-Toxoplasma antibodies to identify all vacuoles . After 4 DPI , we quantified vacuole size and dsRED-expression status in >100 vacuoles for each strain ( WT and dsRED ) and condition ( Vehicle or 20 µM FUDR ) . As expected , HhCatAmer:WT grew poorly in FUDR ( Figure 6C ) , while HhCatAmer:dsRED parasites grew similarly to untreated parasites ( Figure 6C ) . When we separated parasite growth in FUDR based on dsRED expression , we found that dsRED ( - ) parasites in the H . hammondi population were just as susceptible to FUDR as were HhCatAmer:WT parasites ( Figure 6D , first four bars ) , while dsRED+ parasites grew equally well in FUDR compared to Vehicle ( Figure 6D , right two bars ) . These data suggest that most , if not all , of the dsRED-expressing parasites are also null for the UPRT gene , either by direct replacement of the UPRT gene with the dsRED cassette or by incorrect non-homologous end joining and insertion of the dsRED cassette at another genomic location . This represents the first stably transgenic H . hammondi line . Furthermore , PCR analysis of linearly amplified genomic DNA from the HhCatAmer population expressing dsRED demonstrated that the HhUPRT:dsRED repair cassette was incorporated into the genomic DNA of parasite within this population ( Figure 6—figure supplement 1 ) . While T . gondii can spontaneously form tissue cysts following an in vitro sporozoite infection , it can also be chemically induced to form bradyzoites using a variety of treatments ( Jerome et al . , 1998 ) . Given the dramatic developmental differences between T . gondii and H . hammondi in vitro and in vivo , we reasoned that these parasite species may respond differently to external stressors that induced cyst formation . We therefore exposed T . gondii and H . hammondi zoites to alkaline pH bradyzoite induction media and identified cysts using DBA staining as above . As expected we found that TgVEG vacuoles grown in bradyzoite induction media were ~55% DBA-positive after 2 days of exposure and ~90% positive after 4 days of alkaline pH treatment ( Figure 7A , B ) . However , we did not find a single DBA-positive H . hammondi vacuole at any time point ( out of 297 total vacuoles; Figure 7A–B ) . Given the importance of parasite replication events and bradyzoite conversion ( Radke et al . , 2003 ) , it is possible that the lack of cyst conversion in H . hammondi over a 4 day treatment was due to fewer replication cycles . While vacuole sizes for T . gondii and H . hammondi differed significantly after 2 days of growth in standard and bradyzoite induction media ( Figure 7C ) , pH treatment had no significant effect on vacuole size in either species ( Figure 7C; p=0 . 63 , 1 . 0 , respectively ) . This result suggests that bradyzoite conversion occurs independently of changes or differences in replication rate and provides strong support for the fact that H . hammondi are immune to cellular stresses induced by bradyzoite induction conditions . Given the well-established impact of alkaline pH media on T . gondii cyst formation in vitro and the propensity of H . hammondi to spontaneously form cysts in vitro , we found it remarkable that we did not identify a single DBA-positive H . hammondi cyst after even 4 days of alkaline pH exposure . An intriguing hypothesis emerging from this result is that T . gondii is more adept than H . hammondi at responding to stress signals in its environment . To test this hypothesis with respect to pH exposure , we treated T . gondii and H . hammondi sporozoites with bradyzoite induction media for 2 days and performed RNAseq analysis to identify changes in their respective transcriptomes ( Figure 8—figure supplement 1A ) . For T . gondii treated with alkaline pH , we observed significant enrichment in the IN VITRO BRADYZOITE and IN VIVO BRADYZOITE ( Croken et al . , 2014b ) gene sets ( NES = −2 . 4902 , –1 . 800 , respectively; FDR q =~0 . 00 for both ) for TgVEG treated with alkaline pH ( Figure 8A , B ) , and the IN VITRO TACHYZOITE and IN VIVO TACHYZOITE gene sets under control conditions ( NES = 1 . 94 , FDR q = 0 . 001; NES = 1 . 87 , FDR q = 0 . 002 , respectively; Figure 8A ) . This was in stark contrast to HhCatAmer in which members of the IN VITRO BRADYZOITE gene set showed no enrichment after alkaline pH exposure ( NES = −1 . 1681 , FDR q = 0 . 2078; Figure 8A–B ) . For the H . hammondi transcriptome , the IN VITRO BRADYZOITE gene set seemed to consist of two classes of genes: ( a ) those that were not induced upon pH exposure in H . hammondi but were in T . gondii ( top half of heatmap in Figure 8C ) and ( b ) those that were of high abundance in H . hammondi prior to exposure to high pH ( lower half of heatmap in Figure 8C ) . While we identified 90 transcripts that significantly differed in TgVEG following pH exposure ( fold change value ≥2 and Padj value <0 . 01 ) , we only identified 24 hr . hammondi transcripts with pH-altered abundance ( these are shown in Figure 8D ) . Importantly while the majority of pH-altered transcripts were of higher abundance in T . gondii , 21 out of 24 pH-altered transcripts in H . hammondi were of lower abundance , indicating that H . hammondi and T . gondii have distinct responses to high pH bradyzoite induction ( Figure 8C , D; See supplementary file 2 for normalized RNAseq data ) . Since transcripts for the canonical bradyzoite genes BAG1 , LDH2 , and ENO1 ( Weiss and Kim , 2000; Lyons et al . , 2002 ) were below the levels of detection defined for H . hammondi samples in our RNAseq analysis ( see Materials and methods ) , we conducted qPCR analysis of these genes in TgVEG and HhCatAmer treated with alkaline pH for 2 ( TgVEG only ) and 3 days . We found a significant increase in transcript abundance for all three genes in TgVEG exposed to pH 8 . 2 for 2 days ( BAG1 p=0 . 0023 , ENO1 p=0 . 0047 , LDH2 p=0 . 0079 ) . In HhCatAmer , we did observe a significant pH-dependent increase in transcript abundance for BAG1 ( 2 Days p=0 . 0261 and 3 Days p=0 . 0276 ) , while ENO1 and LDH2 transcript levels remained statistically unchanged ( Figure 8—figure supplement 1B ) . Taken together , our DBA staining , RNAseq and qPCR data strongly support the hypothesis that H . hammondi is refractory to alkaline pH induced stress at the transcriptional level , and this is correlates well with the complete lack of high pH-induced cyst wall formation in this species .
While spontaneous cyst formation during growth in vitro has been reported for a handful of T . gondii strains ( e . g . , [Jerome et al . , 1998; Paredes-Santos et al . , 2013] ) , T . gondii of most genetic backgrounds can be induced to form tissue cysts in vitro through the application of a variety of chemical modifications ( e . g . , alkaline pH , nitric oxide donors , serum starvation; [Jerome et al . , 1998; Buchholz et al . , 2013; Eaton et al . , 2006] ) and these cysts are infectious to both the definitive and intermediate host ( Dubey , 1998 ) . H . hammondi was resistant to high pH exposure at the transcriptome level , and 100% resistant with respect to tissue cyst formation . This highlights a key distinction between T . gondii and H . hammondi biology: the dynamic nature of the T . gondii tachyzoite permits movement in both directions along the tachyzoite-bradyzoite continuum which is at least partly driven by sensing changes in the external environment . The ability to dynamically alter ( and even delay ) stage conversion may be made possible by the existence of T . gondii effectors that block responses to host immune effectors like IFN-γ ( e . g . , TgIST; [Olias and Sibley , 2016] ) or otherwise manipulate the host response . If the progression from replicating H . hammondi zoite to tissue cyst is as predictable in vivo as it is in vitro ( which is unknown but likely given that even IFN-γ knockout mice survive infection with H . hammondi; [Dubey and Sreekumar , 2003] ) , and H . hammondi has non- or less-functional orthologs of genes like TgIST ( Hakimi et al . , 2017; Olias et al . , 2016; Gay et al . , 2016 ) , this may prevent the fixation of any polymorphisms in the population that might result in the suppression of spontaneous bradyzoite conversion . Given that bradyzoites induce less inflammation ( Kim et al . , 2007 ) and are protected from immune assaults by the tissue cyst wall ( Tu et al . , 2017; Alday and Doggett , 2017 ) , the timing of these events may be under very strong selective pressure in H . hammondi . It will be interesting to see how the developmental dynamics of H . hammondi in vivo are timed with that of the intermediate host immune response , particularly in rodents ( Sheffield et al . , 1976; Frenkel and Dubey , 1975; Mason , 1978 ) , since this may have directly impacted the timing of cyst formation during the evolution of this parasite . It is likely that H . hammondi is capable of sensing the stresses that are induced upon high pH exposure of the host cell ( as evidenced by significantly reduced transcript abundance for 24 H . hammondi genes ) , but if so it is clear that this species responds quite differently than T . gondii . The link between stage conversion and parasite replication ( Radke et al . , 2003 ) makes it possible that the lack of cyst wall formation after pH stress in H . hammondi is because the parasite is arrested in a phase of the cell cycle that prevents conversion . However , in the present study there were no significant differences in vacuole and/or tissue cyst size between T . gondii and H . hammondi after pH exposure , indicating that parasite replication continued in both species at a similar ( albeit slightly slower ) rate . Moreover in T . gondii stress-induced ( Bohne et al . , 1994 ) or spontaneous ( Jerome et al . , 1998 ) cyst formation , slowed replication is a requirement for expression of bradyzoite antigens and ultimately stage conversion . When taken together with the dynamics of spontaneous differentiation in H . hammondi , these data further demonstrate that H . hammondi terminally differentiates into a unique life stage that is no longer infectious to anything but the definitive host . While we did not conduct any formal analyses of the cell cycle stage ( s ) found within replicating and tissue cyst-like forms of H . hammondi , we would predict that spontaneous H . hammondi in vitro-derived tissue cysts form after a set number of division and would be homogenous in terms of cell cycle stage ( likely G0 and 1N ) given their terminal differentiation phenotype . It is also not known if terminally differentiated parasites are capable of reinvading neighboring cells after egress , nor if they could be coaxed to infect cat gut epithelial cells in vitro , although the existence of cat intestinal organoids ( e . g . , [Powell and Behnke , 2017] ) might allow for this to be tested directly in an accessible in vitro system . Some insight into the H . hammondi developmental processes underlying these unique life stages can be gleaned from the D4 and D15 transcriptional profiling data . H . hammondi consistently had higher transcript levels for genes previously defined as being uniquely expressed in T . gondii bradyzoites ( whether in vivo or in vitro ) as early as 4 DPI , indicating that there is a high degree of similarity between the spontaneous differentiation process that occurs in H . hammondi and the stress-induced process observed in T . gondii ( Jerome et al . , 1998; Croken et al . , 2014a; Croken et al . , 2014b; Weiss and Kim , 2000; Lyons et al . , 2002; Behnke et al . , 2008 ) . Importantly this enrichment for bradyzoite genes became more robust by 15 DPI compared to 4 DPI , indicating that the parasites are changing their transcriptional profiles throughout in vitro development ( rather than becoming less viable or dying ) . Less expected , however , was the higher expression of genes in H . hammondi ( observed in 24 hr PI , 4 DPI , and 15 DPI zoites ) that have been found previously to be expressed more abundantly in T . gondii cat enteroepithelial stages and sporozoites ( Behnke et al . , 2014; Hehl et al . , 2015 ) . While the exact role of these gene products in determining life cycle and growth differences between T . gondii and H . hammondi are unknown , this provides strong evidence that sporozoite-derived life stages of T . gondii and H . hammondi are distinct , at least when grown in vitro . Importantly this enrichment becomes more apparent at 15 DPI , indicating that this is not entirely due to residual transcript from the sporozoite stage . Assuming that the majority of these transcript expression differences are manifested at the protein level ( which is expected based on multiple studies in T . gondii; [Wang et al . , 2017; Xia et al . , 2008] ) , it is exciting to consider what impact these distinct expression profiles might have on parasite development and even the host response . Expression of these T . gondii merozoite-specific genes in H . hammondi zoites may reflect its terminally differentiated state , being infectious only the felid host ( Dubey and Sreekumar , 2003 ) . At a minimum our data may implicate only a subset of so-called ‘merozoite-specific’ genes ( Behnke et al . , 2014; Hehl et al . , 2015 ) as being truly key distinguishing features of the T . gondii merozoite , since a number of them are highly expressed in H . hammondi zoites growing in HFFs . In contrast to previously published work , we identified a short but very predictable replicative window during which H . hammondi could be used to infect new host cells and identified the precise timing of what appears to be an inevitable developmental program towards cyst formation . This finding is in contrast to previous studies where subculture was unsuccessful ( Sheffield et al . , 1976; Riahi et al . , 1995 ) , although in these studies subculture was attempted after 1 , 2 , 4 , and 6 weeks of growth and therefore most were outside of the window of infectivity we identified for H . hammondi . We exploited this window to genetically manipulate H . hammondi using transfection and gene editing strategies developed for T . gondii ( Shen et al . , 2014 ) . Our transgenic H . hammondi parasite line expresses dsRED ( 30–40% after the first round of selection ) and is >99% resistant to FUDR . This represents a new tool in the arsenal to track H . hammondi biology in vitro and in vivo , and opens up the door for the development of genetic screens to potentially disrupt spontaneous differentiation in H . hammondi to generate a mutant that can be cultured indefinitely , as well as utilized in gene-by-gene phenotypic screens . The nearly 3-fold difference in replication rate is likely a major determinant of reduced H . hammondi virulence ( Table 1 , Figure 2D , E ) , assuming this phenotype is recapitulated in vivo . Besides the impact that a slower replication rate would have on acute virulence , it may also impact infection outcome in the chronic phase . While H . hammondi parasites can at times be detected in the brains of mice after infection with oocysts ( Dubey and Sreekumar , 2003 ) , central nervous system ( CNS ) infection which is a hallmark of T . gondii infection ( Dubey and Sreekumar , 2003; Shen et al . , 2014 ) is much less common after exposure to H . hammondi ( although H . hammondi parasites have been detected in mouse brains [Dubey and Sreekumar , 2003] ) . A simple explanation for reduced CNS infection in H . hammondi is that it never reaches a high enough burden to infect the CNS at any significant level . It is also possible that H . hammondi has additional deficiencies that underlie differences in tropism ( such as poor trafficking by innate immune cells and/or inability to cross the blood-brain barrier; [Konradt et al . , 2016; Lambert et al . , 2006; Sanecka and Frickel , 2012] ) , although these have yet to be thoroughly explored . In contrast to differences in replication rate , we observed no substantial differences in the ability of T . gondii and H . hammondi to invade and form vacuoles within HFFs , suggesting that their invasion machinery is mostly conserved and intact . This idea finds some support in our transcriptome data , where we detected no enrichment for gene sets consisting of microneme , rhoptry or dense granule genes , and detected similar amounts of crucial invasion genes like AMA1 , SpAMA1 , as well as multiple rhoptry neck proteins ( see Supplementary file 1 for normalized RNAseq data ) . Moreover all genes known to be a part of the cellular invasion machinery appear to be well-conserved in both sequence and expression between these species ( [Lorenzi et al . , 2016]; Supplementary file 1 ) . T . gondii and H . hammondi oocysts co-occur in the natural environment . A study conducted in Germany and surrounding countries found that the number of feline fecal samples that contained either T . gondii or H . hammondi oocysts was strikingly similar ( Schares et al . , 2016 ) , meaning that humans would be just as likely to encounter H . hammondi oocysts as those of T . gondii . While more studies are needed to determine if co-occurrence is a widespread phenomenon , it raises important questions about the likelihood that H . hammondi is capable of infecting humans . Importantly , at present all serological tests would be expected to misdiagnose a H . hammondi infection as a T . gondii infection due to antibody cross-reactivity ( Dubey and Sreekumar , 2003 ) . While this is a very poorly explored line of research based on our data we would predict that an infection with H . hammondi should be less lethal than one with T . gondii since we would not expect a significant amount of recrudescence to occur . To further address the question of human infection and associated disease , mouse reactivation models and the development of differential diagnostic serological tests will be required in order to further determine human and animal infection rate differences between H . hammondi and T . gondii in the field .
Oocysts of Hammondia hammondi strain HhCatEth1 ( Dubey et al . , 2013 ) , HhCatAmer ( Frenkel and Dubey , 1975 ) and Toxoplasma gondii strain VEG ( Dubey , 2001 ) were isolated from experimentally infected cats as described previously ( Dubey , 2001 ) . Briefly , wild type ( for T . gondii ) or interferon-γ ( IFN-γ ) knockout ( for H . hammondi ) mice were orally infected with 104 sporulated oocysts and sacrificed 4–6 weeks post-infection , and leg muscle ( for H . hammondi ) or brain ( T . gondii ) tissue were fed to 10–20 week old specific pathogen-free cats . Feces were collected during days 7–11 post-infection , and unsporulated oocysts were isolated via sucrose flotation . Oocysts were placed at 4°C in 2% H2SO4 to allow for sporulation to occur and for long-term storage . Due to the limited availability and difficulty associated with the generation of oocysts , two strains ( HhCatEth1 and HhCatAmer ) of H . hammondi generated from multiple cat-derived oocysts preparations were used in experiments described below . Furthermore , various MOIs were utilized throughout the experiments described below to compensate for differences in replication between T . gondii and H . hammondi . Sporulated oocysts were washed 3X in Hanks’ balanced salt solution ( HBSS; Life Technologies , 14175145 ) and treated with 10% bleach in PBS for 30 min while shaking at room temperature . Washed pellets were resuspended in 3 mL of HBSS in a 15 mL falcon tube , and 4 g sterile glass beads ( 710–1 , 180 µM; Sigma-Aldrich , G1152 ) were added . Parasites were vortexed on high speed for 15 s on/15 s off , 4X . Supernatant was removed and pelleted by centrifugation . The pellet was resuspended and syringe-lysed using a 25 gauge needle in 5 mLs of pre-warmed ( 37°C ) and freshly made , sterile-filtered excystation buffer ( 40 mL PBS , 0 . 1 g Porcine Trypsin ( Sigma-Aldrich , T4799 ) , 2 g Taurocholic Acid ( Sigma-Aldrich , T4009 ) , pH 7 . 5 ) . After 45 min in a 37°C water bath , the suspension was syringe lysed again , and 7 mLs of cDMEM ( 100 U/mL penicillin , 100 µg/mL streptomycin , 2 mM L-glutamine , 10% FBS , 3 . 7 g NaH2CO3/L , pH 7 . 2 ) was added to quench the excystation media . The mixture was centrifuged , the pellet was resuspended in cDMEM , and used in downstream applications described in more detail below . Parasites were washed twice with PBS , fixed with 4% paraformaldehyde in PBS ( Affymetrix , 19943 ) , washed twice with PBS , and blocked/permeabilized in blocking buffer ( 50 mL PBS , 5% BSA , 0 . 1% Triton X-100 ) . After 10 min incubation on HFFs at 37°C in 5% CO2 , monolayers containing freshly excysted parasites were scraped , serially syringe lysed ( 25 and 27 gauge needles ) , and pelleted . The pelleted parasites were resuspended in cDMEM , filtered through a 5 μM syringe-driven filter ( Fisher Scientific , SLSV025LS ) and used to infect 4-chambered slides ( Lab-Tek II , 154526 ) containing a confluent monolayer of HFFs at an MOI of 0 . 5 for both H . hammondi CatEth1 and T . gondii VEG . After 1 , 2 , and 3 days of growth , cells and parasites were fixed as described above and stored in blocking buffer at 4°C until needed . Cells were immunostained with goat polyclonal Toxoplasma gondii Antibody ( ThermoFisher Scientific , PA1-7256 , RRID: AB_561769 ) at 1:500 . The secondary antibody , Alexa Fluor 594 Donkey anti-Goat IgG ( H + L ) ( ThermoFisher Scientific , A-11037 , RRID: AB_2534105 ) was used at 1:1000 . Coverslips were mounted to 4-chambered slides using VECTASHIELD Antifade Mounting Medium with or without DAPI , depending on the application ( Vector labs , H-1000 ) . Vacuole formation was used as a proxy for sporozoite invasive capacity . To quantify this , the number of vacuoles with at least one parasite was determined in 100 random FOVs ( 1000x magnification ) for three technical replicates for each parasite species at 1 , 2 , and 3 DPI . The entire experiment was repeated twice with different cat-derived oocyst preparations . For replication rate quantification , images of parasite-containing vacuoles were taken with an Axiovert 100 inverted fluorescent microscope with Zen lite 2012 software . The number of parasites per vacuole was determined for each image ( biological replicates 1 and 2 ) , and vacuole size ( Biological replicate one only ) was determined for each image using ImageJ software ( NIH ) . For Dolichos Biflorus Agglutinin ( DBA ) staining , monolayers containing freshly excysted parasites were scraped , syringe lysed , and pelleted 24 hr after growth . The pellet was resuspended in cDMEM , filtered through a 5 µM syringe-driven filter ( Fisher Scientific , SLSV025LS ) , and passed at MOIs of 0 . 3 ( HhCatEth1 ) and 0 . 001 for ( TgVEG ) onto coverslips containing a confluent monolayer of HFFs . Three coverslips were infected per strain for each time point analyzed . At 4 and 15 DPI , cells and parasites were fixed as described above and stored in blocking buffer at 4°C until immunostaining was conducted . In addition to being stained with rabbit Toxoplasma gondii Polyclonal Antibody ( Invitrogen , PA1-7252 RRID: AB_561769 ) at a dilution of 1:500 and a 1:1000 dilution of Alexa Fluor 594 goat anti-rabbit IgG ( H + L ) ( ThermoFisher Scientific , A-11037 , RRID: AB_2534095 ) , coverslips were stained with Fluorescein labeled Dolichos biflorus Agglutinin ( DBA; Vector Labs , FL-1031 ) at a dilution of 1:250 . Coverslips were then mounted to microscope slides using ProLong Diamond Antifade Mountant with DAPI ( ThermoFisher Scientific , P36962 ) . The number of DBA positive vacuoles and total vacuoles were quantified in 20 FOVs in three coverslips for each parasite at 4 and 15 DPI . Images were obtained with an Axiovert 100 inverted fluorescent microscope with Zen lite 2012 software and edited using ImageJ software ( NIH ) . For time course tissue cyst formation analysis , freshly excysted and filtered HhCatAmer and TgVEG sporozoites were used to infect confluent monolayers of HFFs grown on glass coverslips at an MOI of 0 . 5 . After three days of growth , the media was changed every 2 to 3 days . At designated time points , coverslips were was twice with PBS , fixed with 4% formaldehyde in PBS , washed twice with PBS , and stored in blocking buffer 4°C until immunostaining was conducted using Rhodamine labeled DBA . The number of DBA positive vacuoles and number of total vacuoles were quantified in 15 parasite containing FOVs for each time point . Monolayers containing freshly excysted parasites were scraped , syringe lysed , and filtered through a 5 µM syringe-driven filter ( Fisher Scientific , SLSV025LS ) after 24 hr of growth . Filtered parasites were pelleted and passed at MOIs of 0 . 5 ( H . hammondi ) and 0 . 1 for ( T . gondii ) onto coverslips containing a monolayer of HFFs . Three coverslips were infected per parasite per treatment group . After 2 days , media was changed to pH 8 . 2 bradyzoite switch media ( DMEM with 100 U/mL penicillin , 100 µg/mL streptomycin , 2 mM L-glutamine 10 mM HEPES , 2 g/L NaHCO3 , and 1% FBS [Gay et al . , 2016] ) or cDMEM . Coverslips with pH 8 . 2 media were grown at 37°C in the absence of CO2 . Media was changed again at 3 DPI . After 4 DPI , cells and parasites were fixed as described above and stored in blocking buffer at 4°C until needed . DBA and counter-staining was conducted using Fluorescein or Rhodamine labeled DBA and rabbit Toxoplasma gondii Polyclonal Antibody as described above . The number of DBA-positive vacuoles was quantified in 15 FOVs in three coverslips for each strain grown at either pH 7 . 2 or pH 8 . 2 and the percentage of DBA positive vacuoles was determined . Images were obtained and analyzed as described above . Sporozoites were obtained using the excystation protocol described above with the exception of syringe lysis and previously described host cell incubation . Six confluent monolayers of HFFs grown in T-25’s were infected with H . hammondi American ( HhCatAmer ) sporozoites at an MOI of ~2 ( 2 . 8 million sporozoites ) . After a three-hour incubation at 37°C , the Day 0 Transfer flask was scraped , syringed lysed 5X with 25 gauge needle , filtered through a 5 µM syringe-driven filter ( Fisher Scientific , SLSV025LS ) , and passed to a new confluent monolayer of HFFs grown in a T-25 . This process was repeated after 2 , 5 , 8 , 11 , and 15 days post-excystation . After 3 days of growth following passage , 4 . 17 cm2 of the T-25 was monitored daily . Images of vacuoles were obtained with an Axiovert 100 inverted fluorescent microscope with Zen lite 2012 software with a 40X objective , and vacuole sizes were quantified using ImageJ software ( NIH ) . Similar methods were used to generate parasite samples for RNAseq analysis , except cells were processed for RNA harvest using methods described below . Sporozoites were obtained using the excystation protocol described above . After 24 hr , HFF monolayers infected with excysted sporozoites were scraped , syringe lysed 5X with a 25 gauge needle , and pelleted . The pellet was resuspended in cDMEM , filtered through a 5 µM syringe-driven filter ( Fisher Scientific , SLSV025LS ) and passed onto confluent monolayers of HFFs grown in T-25s . Infected host cells were incubated at 37°C 5% CO2 from 6 , 8 , 10 , 13 , and 15 days . The media was replenished after 5–7 days . At each time point , infected host cells were scraped , syringe lysed 3X with a 25 gauge needle and 3X with a 27 gauge needle , and pelleted . Parasites were counted and diluted in PBS , and a dose of 50 , 000 parasites was injected intraperitoneally in 2 BALB/C mice for each time point . Serum samples were obtained from infected mice daily for 9 days post-infection and the mass of the mice was also monitored daily . After 9 days of infection , the mice were sacrificed and dissected . Tissue samples were preserved in 10% neutral buffered formalin ( Sigma HT501128 ) until immunohistochemistry analysis was performed . Fixed tissue samples were embedded in paraffin , sectioned , and stained using rabbit anti-Toxoplasma antibody ( ThermoScientific Cat# RB-9423-R7 ) by Research Histology Services at the University of Pittsburgh . For antigen retrieval , deparaffinized slides were steamed at pH = 6 . 0 in 10 mM Citrate buffer . After exposure to 3% H2O2 , slides were washed in Tris-buffered saline with 2 . 5% Tween-20 ( TBST ) , and blocked for 20 min each with Avidin and Biotin blocking reagents ( Vector labs; SP-2001 ) with TBST washes in between . Slides were blocked in 0 . 25% casein:PBS for 15 min , and incubated overnight in primary antibody ( 1:100 dilution in 3% goat serum in PBS ) . Slides were washed in PBST and incubated for 30 min with biotinylated goat anti-rabbit ( Vector laboratories; BA-1000; 1:200 dilution in 3% goat serum in PBS ) . Following three washes with PBST , slides were incubated for 30 min with streptavidin-HRP ( Vector laboratories; PK-6100 ) , washed 3X with PBST , and incubated with AEC substrate ( Skytec; ACE-500/ACD-015 ) for 15 min . Following rinses in water , slides were counterstained with aqueous hematoxylin and blued using Scott’s tapwater substitute . Slides were mounted in Crystal Mount . Blood samples were obtained from mice daily via submandibular bleed , allowed to clot for 4–24 hr at 4°C , and centrifuged at 100 x g . Serum was stored at −20°C until ELISAs were performed . IFN-γ levels were determined using the BD OptEIA Set Mouse IFN-γ kit ( Cat . # 555138 ) according to manufacturer's instructions . Serum samples were diluted 1:20 . Excysted sporozoites were prepared as described above and incubated overnight in a T-25 flask with confluent monolayer of HFFs . After 24 hr , the monolayer was scraped , syringe lysed 3X with a 25 gauge and 27 gauge needle , and filtered using a 5 μM syringe-driven filter ( ThermoFisher Scientific , Pittsburgh , Pennsylvania , USA; SLSV025LS ) . The filtered contents were pelleted by centrifugation at 800 x g for 10 min , and the pellet was resuspended in 450 µl of cytomix with 2 mM ATP and 5 mM glutathione . Resuspended parasites were transferred to a cuvette , electroporated at 1 . 6 KV and a capacitance of 25 µF , and used to infect confluent HFF monolayers on coverslips . The coverslips were fixed 5 DPI as described above and mounted using ProLong Diamond Antifade Mountant with DAPI ( ThermoFisher Scientific , P36962 ) . To create stable transgenic H . hammondi parasite lines , the CRISPR/CAS9 plasmid expressing a gRNA sequence targeting the uracil phosphoribosyl transferase gene ( UPRT; [Shen et al . , 2014]; kindly provided by David Sibley , Washington University ) and a repair template consisting of 20 bp UPRT-targeting sequences flanking a PCR-amplified a dsRED expression cassette driven by the T . gondii GRA1 promoter with a GRA2 3’ UTR ( amplified from the dsRED:LUC:BLEO plasmid described in ( Jeffers et al . , 2017; Liao et al . , 2013 ) were used for transfection . Following centrifugation ( 800 x g , 10 min ) , 24 h H . hammondi zoites were resuspended in 450 μl of cytomix with ATP and glutathione containing 20 μg of the CRISPR/CAS9:UPRT:gRNA plasmid and 20 μg of PCR2 . 1 TOPO vector containing the dsRED repair template . The parasites were electroporated as above and each transfection contained at least 4 million parasites . Transfected parasites were then transferred to HFFs and grown for 2 days in cDMEM , then selected for 3 days by incubation in cDMEM containing 10 μM FUDR ( Figure 5B ) . Parasites were again scraped , syringe lysed and filtered , and dsRED-expressing parasites were collected in PBS using flow cytometry . Sorted parasites were injected intraperitoneally into 2 BALB/c mice . After 3 weeks of infection , mice were euthanized , skinned , and the intestines were removed before feeding to specific pathogen-free cats . The oocysts were collected and purified as described above , and oocysts , sporozoites and replicating parasites were evaluated for dsRED fluorescence using microscopy , flow cytometry and FUDR resistance . To test for deletion of the UPRT gene we quantified transgenic H . hammondi resistance to 5-fluoro-2’-deoxyuridine ( FUDR ) . To do this , sporozoites of wild type and dsRED-expressing parasites were incubated overnight in a T25 flask with confluent monolayer of HFFs . After 24 hr , parasites were isolated by needle passage and filtration as above . We infected coverslips containing confluent HFFs with 50 , 000 parasites , and parasites were exposed to media alone or media containing or 20 μM FUDR . The parasites were allowed to grow for 4 days and then fixed using 4%PFA for 20 min . After blocking overnight in PBS/BSA/triton , coverslips were stained with rabbit anti-Toxoplasma antibody at a 1:500 dilution for an hour , and stained with Alexa-fluor 488-labeled goat anti Rabbit antibody ( RRID: AB_143165 ) . For each treatment/strain combination , we counted the number of parasites in at least 100 vacuoles , and for HhdsRED parasites we also quantified growth in both red and wild type vacuoles ( since the HhdsRED parasites were from a mixed population ) . To isolate genomic DNA from H . hammondi , freshly excysted parasites were used to infect HFFs . Following 7 days of growth , infected host cells were scraped , syringe lysed , pelleted and resuspended in 200 µL of PBS . Genomic DNA was isolated with the GeneJET genomic DNA Purification kit ( ThermoFisher , K0721 ) according to the manufacture . Alternatively , identical procedures were used on freshly excysted and filtered sporozoites . We used the Genomiphi Linear DNA amplification kit to linearly amplify DNA taken from our population of dsRED-expressing H . hammondi American parasites . We used ~5 ng of input DNA in a single reaction , and ethanol precipitated amplified DNA prior to quantification and use in PCR reactions . To verify that the dsRED cassette incorporated into the HhCatAmer genome , linearly amplified pure parasite genomic DNA ( HhamdsRED ) was amplified using primer sets targeting the UPRT homology arms of the dsRED cassette , the dsRED transgene , and a sequence specifically found in H . hammondi ( Sreekumar et al . , 2005 ) . ( See supplementary file 3 for primer sequences ) Controls included wild type HhCatAmer genomic DNA , purified from infected HFFs , and a water control . PCR reactions were conducted using 12 . 5 µL of 2X BioMix Red ( Bioline , BIO-25006 ) , 1 nM of both forward and reverse primers , 45 ng of template DNA , and water to 25 µL . Cycling conditions began with 5 min of 95°C denaturation , followed by 30 cycles of 95°C for 30 s , 50°C for 30 s , 72°C for 1 min and 30 s , followed by 72°C for 5 min and a 4°C hold . PCR products were visualized on a 1% agarose gel . Sporozoites were isolated from HhCatEth1 and TgVEG sporulated oocysts as described above and used to directly infect confluent HFF monolayers in 96 well tissue culture plates with MOIs of 3 , 1 or 0 . 5 purified sporozoites for each species . On day four post-infection , wells were observed and chosen for RNA analysis based on similar numbers of parasite-containing vacuoles lack of significant host cell lysis in both species . For the Day four samples , the MOI = 3 infected wells were chosen for H . hammondi , while the MOI = 0 . 5 infected wells were chosen for T . gondii . Wells were washed 3X with ~200 μL cDMEM , and RNA was harvested using Trizol ( Invitrogen ) . Remaining H . hammondi wells were washed with 3X with cDMEM on Day four and then again on Day nine prior to harvest on Day 15 post-infection . For T . gondii , remaining MOI = 3 and 1-infected wells were scraped and syringe lysed on Day four and Day nine and used to infect new monolayers in 96 well plates at an MOI of either 0 . 5 ( Day 4 ) or 0 . 3 ( Day 9 ) . On Day 15 , wells for both H . hammondi ( not subcultured ) and T . gondii ( subcultured 2x ) were washed 1X with cDMEM and then harvested for RNAseq analysis . Two samples were harvested for each species on Day 4 , and three samples were harvested from each species on Day 15 . Total RNA samples were processed for Illumina next generation sequencing using the Mobioo strand-specific RNAseq library construction kit . Samples were analyzed on an Illumina Nextseq and demultiplexed using NextSeq System Suite software . Reads were aligned to their respective species genome assembly ( H . hammondi v10 or T . gondii strain ME49 v10; toxodb . org; [Lorenzi et al . , 2016] ) using the Subread package for Linux ( v . 1 . 4 . 6; [Alday and Doggett , 2017] ) with subread-align using default parameters except for –u to keep only uniquely mapping reads . featureCounts from the subread package ( Alday and Doggett , 2017 ) was used to quantify the number of mapping reads per transcript , using default settings except for –s 2 ( for stranded read mapping ) , –t CDS , –g Parent ( for specific compatibility with the T . gondii and H . hammondi gff files ) , –Q 10 ( minimum mapping quality required ) . The –t CDS option was chosen for both T . gondii and H . hammondi because to date no 5’ or 3’ UTR sequences have been predicted for H . hammondi ( in contrast to T . gondii ) . Fastq files have been deposited in the NCBI short read archive ( SRX3734444-SRX3734449 ) . To validate genesets that were significantly enriched in D4 and D15 H . hammondi compared to T . gondii , we extracted sporozoites from oocysts and grew them overnight in HFFs as described above . Parasites were released by scraping and needle passage as above , and then passed through a 5 μm syringe-driven filter ( Fisher Scientific , SLSV025LS ) . Filtered parasites were used to infect monocytes ( THP-1 , RRID: CVCL_X588 ) at MOI of 4 . At 24 hr post-infection , RNA was harvested using RNeasy Mini Kit ( Qiagen; three samples for each species ) . Total RNA samples were processed for Illumina next generation strand-specific sequencing using the Illumina TruSeq stranded mRNA sequencing kit . Fastq files have been deposited in the NCBI short read archive ( SRX3734421-SRX3734428 ) . FASTQ reads were aligned to their respective species genome assembly ( H . hammondi release 34 or T . gondii strain ME49 release 30; toxodb . org ) with CLC Genomics Workbench v10 . 0 . 1 ( http://www . qiagenbioinformatics . com/ ) using default parameters for reverse strand mapping . Raw count data ( with at least one read in at all samples for HhCatAmer ) generated by CLC Genomics Workbench were loaded into R statistical software and analyzed using the DESeq2 package ( Varet et al . , 2016 ) . Read count data of HhCatAmer were compared to TgVEG to identify genes of different abundance and genes were deemed differentially expressed according to the threshold of log2 ( fold ) >1 and Padj <0 . 01 . Log2 transformed data were used for enrichment analysis using Gene Set Enrichment Analysis ( GSEA ) ( Subramanian et al . , 2005 ) . Data were compared to previously curated gene sets ( 434 total; as published in [Behnke et al . , 2014] ) as well as seven additional gene sets that we curated ourselves ( listed in Table 2 ) . All enrichment profiles were deemed significant if the FDR q-value was ≤0 . 01 . We also identified 1069 genes that were of different abundance between HhCatAmer and TgVEG at this 24 hr time point in THP-1 cells . Sporozoites were isolated from HhCatAmer and TgVEG sporulated oocysts as described above and used to directly infect confluent HFF monolayers in 24 well tissue culture plates with MOI of 1 . 3 for HhCatAmer and an MOI of 1 for TgVEG . Infected host cells were maintained as described the induction of bradyzoite section above . On day four post-infection , wells were washed twice with 1X PBS and RNA was harvested using the RNeasy Kit according to the manufacturer ( Qiagen , 74104 ) using QiaShredder spin columns ( Qiagen , 79654 ) to homogenize samples , and RNase-free DNase to degrade contaminating DNA ( Qiagen , 79254 ) . Total RNA samples were ethanol precipitated and processed for Illumina next generation strand specific sequencing as described for infected THP-1 RNAseq experiments . Library preparation and read alignment were performed as described in infected THP-1 RNAseq experiments . Raw count data per transcript ( generated by featureCounts or CLC genomics ) were loaded into R statistical software and analyzed using the DESeq2 package ( Love et al . , 2014 ) . Comparisons of D4 and D15 read count data were used to identify transcripts of different abundance at each time point , and differences were deemed significant at Padj <0 . 05 . Data were log2 transformed and normalized using the rlog ( with the blind = TRUE option ) function in DESeq2 for use in downstream analyses . Log2 transformed , normalized data from DESeq2 ( hereafter referred to as Log2 ( FPM ) ) were analyzed for enrichment using Gene Set Enrichment Analysis ( GSEA; [Subramanian et al . , 2005] ) . Since read count overall from the T . gondii libraries were much greater than those from the H . hammondi libraries , we took the 7372 genes matched based on the previously published gene-by-gene annotation ( Lorenzi et al . , 2016 ) and selected only those that had at least 1 day 4 sample and 1 day 15 sample with >5 reads for the D4 and D15 data and samples that had reads for each replicate and a total read average of 5 reads per three replicates for pH 8 . 2 analysis . In total , 4146 genes for the D4 and D15 analysis and 907 genes for the pH 8 . 2 analysis passed these benchmarks and were used in subsequent analyses . We used this approach to identify gene sets that were significantly different between days 4 and day 15 in culture in both species and those that were different between species at both day 4 and day 15 and in pH 8 . 2 treated and control samples . We compared these data using previously curated gene sets ( 434 total; as published in [Walzer et al . , 2014] ) as well as seven additional gene sets that we curated ourselves . These are listed in Table 2 . All enrichment profiles were deemed significant if the FDR q-value was ≤0 . 05 . For qPCR analysis , after 24 hr of growth , monolayers containing excysted sporozoites were scraped , syringe lysed , and pelleted . The pellet was resuspended in cDMEM , filtered through a 5 μM syringe-driven filter ( Fisher Scientific , SLSV025LS ) and passed at MOIs of 1 ( T . gondii ) and 7 ( H . hammondi ) into 96-well plates or 24-well plates containing HFF monolayers . Mock-infected controls consisted of filtering parasites through a 0 . 22 μM syringe-driven filter ( Fisher Scientific , SLGL0250S ) . Three replicates were made for each sample per isolation time point . RNA for D4 and D15 RNAseq validation was collected at day 4 and day 15 post-infection , ( equivalent to day 5 and day 16 post-excystation ) from parasites and mock infections grown in 96-well plates . RNA for pH induced bradyzoite transcript analysis was collected from day 4 and 5 post-infection samples grown in 24-well plates . RNA was collected using the RNeasy Kit according to the manufacturer ( Qiagen , 74104 ) using QiaShredder spin columns ( Qiagen , 79654 ) to homogenize samples , and RNase-free DNase to degrade contaminating DNA ( Qiagen , 79254 ) . Isolated RNA was ethanol precipitated , resuspended in RNase-free water , and these preparations were used to create cDNA using Superscript III Reverse Transcriptase Kit using Oligo ( dT ) primers according to the manufacturer ( ThermoFisher Scientific , 18080051 ) . All RNA samples were kept at −80°C , and all cDNA reactions were kept at −20°C . Prior to qPCR use , cDNA was diluted 1:10 ( for RNAseq validation ) or 1:5 ( for bradyzoite transcript analysis ) with H20 . qPCR assays were performed on a QuantStudio 3 Real-Time PCR System in a 10 μl reaction volume containing 5 μl 2x SYBR Green Master Mix ( VWR International , 95030–216 ) , 3 μl of cDNA template , 1 μl H20 , and 1 μl 5 μM primer . Controls included a reverse transcription negative control and a water-template control . The thermal cycling protocol was 95°C , 10 min; 40 cycles of ( 95°C , 15 s; 60°C , 1 min ) ; 4°C hold . The melt curve protocol was 95°C , 15 s; 60°C 1 min , 95°C 15 s . The control gene was dense granule 1 ( GRA1 ) , and samples were tested in duplicate or triplicate . Melt curves were performed on each plate ( with the exception of 2 plates ) . Data were analyzed using the 2-∆∆Ct method ( Livak and Schmittgen , 2001 ) , and statistical analyses were conducted on the ∆Ct values ( as in [Boyle et al . , 2003] ) . All mouse experiments were performed with 4- to 8-wk-old BALB/C and C57BL/6J mice . Animal procedures met the standards of the American Veterinary Association and were approved locally under Institutional Animal Care and Use Committee protocol no . 12010130 . RNAseq data has been deposited in the NCBI short read archive . Sequencing data generated and analyzed in this is available in supplemental files 1 and 2 . Gene set categorization of significant genes for all gene sets used in analyses in this study are available in supplemental files 4 and 5 . All additional data is available in the manuscript and supporting files .
Most eukaryotic parasites are obligately heteroxenous , requiring sequential infection of different host species in order to survive . Toxoplasma gondii is a rare exception to this rule , having a uniquely facultative heteroxenous life cycle . To understand the origins of this phenomenon , we compared development and stress responses in T . gondii to those of its its obligately heteroxenous relative , Hammondia hammondi and have identified multiple H . hammondi growth states that are distinct from those in T . gondii . Of these , the most dramatic difference was that H . hammondi was refractory to stressors that robustly induce cyst formation in T . gondii , and this was reflected most dramatically in its unchanging transcriptome after stress exposure . We also found that H . hammondi could be propagated in vitro for up to 8 days post-excystation , and we exploited this to generate the first ever transgenic H . hammondi line . Overall our data show that H . hammondi zoites grow as stringently regulated , unique life stages that are distinct from T . gondii tachyzoites , and implicate stress sensitivity as a potential developmental innovation that increased the flexibility of the T . gondii life cycle .
Over a billion people worldwide are infected , often for life , by a parasite known as Toxoplasma gondii . Most of the time , the parasites remain in a dormant state and cause no symptoms . However , the parasites can reactivate , which can be fatal for people with weaker immune systems . Currently no drugs can kill the dormant stages of T . gondii . One of the possible reasons T . gondii is so widespread is because it easily passes from host to host , and resists both detection and treatment . Like many parasites , T . gondii infects multiple hosts during its life cycle: it reproduces sexually in its ‘definitive' host , the cat , and reproduces asexually in its ‘intermediate’ hosts , which include virtually all warm-blooded animals . Unlike most parasites , all hosts can transmit T . gondii to other hosts , making it a very flexible parasite . However , its closest living relative , Hammondia hammondi , can only be transmitted from a definitive host to an intermediate host , or vice versa . The reason for this difference remains unclear , but comparisons between these species can hopefully help uncover how T . gondii came to be such a promiscuous parasite . Now , Sokol , Primack et al . report that a dramatic difference between H . hammondi and T . gondii is the ability to adapt to changes in the environment . While H . hammondi grows in a very strict and inflexible way , T . gondii can sense its environment and change its growth rate accordingly . This is important because it would allow T . gondii to grow until the host immune response is activated , at which time T . gondii can then quickly become dormant and hide from these host defences . Sokol , Primack et al . suggest that this unique adaptation may have helped T . gondii to become the globally successful parasite that it is today , and explain why H . hammondi is much less lethal . It is also possible that T . gondii reactivates after periods of dormancy because it senses changes in its environment . If so , and if scientists can identify the sensors required for this , it may also be possible to target them and effectively block reactivation in infected humans . Comparing T . gondii with H . hammondi may provide a good way to identify these sensors .