Babesia divergens egress from host cells is orchestrated by essential and druggable kinases and proteases

doi:10.21203/rs.3.rs-2553721/v1

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Babesia divergens egress from host cells is orchestrated by essential and druggable kinases and proteases

https://doi.org/10.21203/rs.3.rs-2553721/v1

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Apicomplexan egress from host cells is fundamental to the spread of infection and is poorly characterized in Babesia spp., parasites of veterinary importance and emerging zoonoses. Through the use of video microscopy, transcriptomics and chemical genetics, we have implicated signaling, proteases and gliding motility as key drivers of egress by Babesia divergens. We developed reverse genetics to perform a knockdown screen of putative mediators of egress, identifying kinases and proteases involved in distinct steps of egress (ASP3, PKG and CDPK4) and invasion (ASP2, ASP3 and PKG). Inhibition of egress leads to continued intracellular replication, indicating exit from the replication cycle is uncoupled from egress. Chemical genetics validated PKG, ASP2 and ASP3 as druggable targets in Babesia spp. All taken together, egress in B. divergens more closely resembles T. gondii than the more evolutionarily-related Plasmodium spp. We have established a molecular framework for biological and translational studies of B. divergens egress.

Biological sciences/Microbiology/Parasitology/Parasite biology

Biological sciences/Microbiology/Parasitology/Parasite genetics

Biological sciences/Microbiology/Antimicrobials/Antiparasitic agents

Babesia spp. are tick-borne pathogens that infect, grow in, and eventually destroy the red blood cells (RBCs) of their vertebrate host. Several Babesia spp., including B. microti, B. divergens and B. duncani, are emerging zoonotic pathogens that can cause severe and even fatal disease¹. Babesia spp. also cause significant economic losses globally due to disease in livestock and companion animals, especially B. divergens, B. bovis and B. bigemina in cattle². Current treatment and prevention options for human and veterinary babesiosis are limited and suffer from poor efficacy, spontaneous resistance, severe side effects or render the animal products unsuitable for human consumption^1,3.

The Babesia genus is within the Apicomplexa phylum, which consists of single-celled obligate intracellular parasites. Apicomplexa include the human and veterinary pathogens, Babesia spp., Plasmodium spp., Toxoplasma spp., Cryptosporidium spp., Theileria spp. and Eimeria spp., amongst others. Babesia spp. are most closely related to Theileria spp. and Plasmodium spp., which all share the same vertebrate RBC niche for part of their lifecycle. The vast majority of apicomplexan cellular and molecular biology research has been performed in Plasmodium spp. and Toxoplasma gondii. Knowledge of a wider range of apicomplexan biology will hone in on conserved and essential functions that could be key targets to develop broad spectrum anti-apicomplexan drugs. Babesia spp. represent a minimalized apicomplexan genome with B. divergens having 4187 genes in comparison to P. falciparum (5720 genes) or T. gondii (8920 genes) (VEuPathDB v61). There are many similarities between Plasmodium spp., Toxoplasma spp. and Babesia spp., such as the presence of an apicoplast and apical invasion organelles. However, there are notable differences, such as Babesia spp. degrading their parasitophorous vacuole immediately after invasion and replicating in the host cell cytoplasm. The mechanisms of cell division also vary between apicomplexan parasites (reviewed in Gubbels, et al.⁴). In this study, a replication cycle refers to one cell undergoing division to form two (Babesia spp. and T. gondii) or more (Plasmodium spp.) daughter cells. A lytic cycle refers to the time from when a host cell is first invaded to when parasites egress from the same host cell, which may include multiple replication cycles in the case of Babesia spp. and T. gondii, but only one for Plasmodium spp. (Figure S1A)⁵.

Parasite egress from the host cells remains poorly characterized in Babesia spp. Egress in the two previously studied apicomplexans, Plasmodium spp. and T. gondii (reviewed in^6,7), begins when either intrinsic or extrinsic signals activate signaling pathways in the parasite that ultimately result in the release of parasite secretory organelles, the micronemes/exonemes, into the host cell cytoplasm. The contents of these organelles include proteases, phospholipases and perforin-like proteins (PLPs), which help permeabilize and eventually rupture the parasitophorous vacuole membrane (PVM) and host cell membrane, allowing the parasite to egress from the host cell. Many of the molecular players in the egress program are shared between Plasmodium spp. and T. gondii, including cGMP-dependent kinase (PKG), calcium-dependent protein kinases (CDPKs), aspartyl proteases, lipid and calcium signaling^8,9. However, there are key differences, such as protein kinase A (PKA) repressing egress in T. gondii but is instead required for invasion in P. falciparum^10–12. Significant effort has been utilized to develop inhibitors of egress in Plasmodium spp. and T. gondii, with the potential to develop pan-apicomplexan drugs (reviewed in^13,14).

Here, we have employed a range of cellular, genetic and genomic approaches to produce a foundational framework defining the key cellular features of B. divergens egress and identify the molecular mediators of these processes through functional analysis. We have developed stable transfection, a CRISPR/Cas9 system and inducible knockdown systems for B. divergens. These genetic systems were used in combination with small molecule inhibitors, to determine that the B. divergens kinases, PKG and CDPK4, and the aspartyl proteases, ASP2 and ASP3, are required for separate sequential steps in egress and invasion of the host RBC. Strikingly, inhibition of egress resulted in continued intracellular replication within a single RBC, suggesting the default pathway in these parasites is to continue replicating in the absence of either an egress signal or the downstream host lytic effector molecules. Moreover, chemical genetic approaches demonstrate that these conserved essential molecules present validated druggable targets in Babesia spp.

An induced egress assay to study B. divergens host cell egress and invasion

To make the study of egress accessible in B. divergens, we developed a method to induce egress, which otherwise occurs asynchronously in in vitro culture. A flow cytometry-based assay was used to screen for egress induction in B. divergens using known T. gondii egress-inducing compounds (Figs. 1A, S1B and S1D). 8-Br-cGMP, BIPPO (phosphodiesterase (PDE) inhibitor) and to a lesser extent, H89 (PKA inhibitor), induced egress and inhibited parasite replication (Figs. 1A, S1D-H)^12,15. A summary of the putative B. divergens egress signaling pathway can be found in S1C.

To elucidate the cellular feature of B. divergens egress and invasion, we used video microscopy to follow 8-Br-cGMP mediated induced egress. As has been previously observed¹⁶, B. divergens egress is frequently initiated when the intracellular parasite contacts and deforms the host cell (observed in 76% (16/21) of egress events, Fig. 1B - red arrow). Soon after the initial deformation, the RBC ‘rounds up’ (mean ± S.D. = 2.6 s ± 3 s, n = 17) (Fig. 1B, yellow arrow). ‘Rounding’ is observed in P. falciparum immediately preceding PVM rupture and may be associated with increased PVM permeability, however, the mechanisms remains unclear^17–19. In B. divergens, which lack a PVM, rounding is likely due to permeabilization of the RBC. B. divergens parasites then become motile and escape the permeabilized RBC to invade new RBCs (mean ± S.D. = 1 s ± 2.4 s, n = 28, to escape after host cell permeabilization). Upon contact with a new RBC, the parasite strongly deforms the host cell around itself (Fig. 1C, green to purple arrows; mean ± S.D. = 6 s ± 2.7 s, n = 14), a process known as pre-invasion in P. falciparum, followed by the internalization phase when the parasite enters the RBC with relatively little deformation of the RBC (Fig. 1C, purple to blue arrows, mean ± S.D. = 4.5 s ± 1.1 s, n = 10; Video S1)²⁰. In contrast to P. falciparum egress that takes ~ 5–10 minutes (from initial PVM permeabilization to merozoite release)^18,21, B. divergens egress is rapid (mean ± S.D. = 10.8 s ± 3.0 s, n = 10).

To test the ability of B. divergens to egress throughout replication, parasites were synchronized to a 20-minute window and egress was induced every 1–2 hrs. The increased sensitivity to 8-Br-cGMP and increased number of parasites that egress as the parasites mature suggest that while B. divergens can egress throughout the replication cycle, it is more strongly primed to egress when parasites are fully mature (Fig. 1D). This feature of B. divergens egress is similar to T. gondii, which can be induced to egress throughout the replication cycle, albeit at reduced efficiency during S and M/C phase²², whereas P. falciparum egress is restricted to a narrow window at the end of the lytic cycle^15,23.

Loss of host cell integrity induces B. divergens motility and egress

To test if B. divergens motility is induced by exposure to extracellular conditions, as has been demonstrated in T. gondii, we observed parasites after the RBC was lysed using a low concentration of saponin in buffers mimicking either intracellular (IC, 140 mM K⁺, 5 mM Na⁺) or extracellular (EC, 5 mM K⁺, 140 Na⁺) concentrations of potassium and sodium, plus or minus calcium (2 mM). Upon saponin-induced RBC lysis in EC-Ca²⁺ or IC-Ca²⁺ buffer, parasites became motile and were able to escape from the permeabilized RBC (Fig. 1E, Video S3). In contrast, parasites in either IC or EC buffer without Ca²⁺ did not become motile (Fig. 1E, Video S4). PKG activation, through the addition of 8-Br-cGMP, bypassed the requirement for extracellular calcium in either buffer to initiate gliding motility (Fig. 1E). Together these results suggest that B. divergens motility is induced by exposure to extracellular concentrations of Ca²⁺, likely acting upstream or in parallel of PKG, but does not respond to Na⁺ or K⁺ concentrations. Similarly, T. gondii can sense exposure to the extracellular environment through serum albumin, a drop in potassium and an increase in extracellular calcium, which induces microneme secretion and motility^22,24−28. P. falciparum is also able to sense lipids and potassium which alter microneme secretion, egress or invasion, although their roles in the parasite are less clearly defined^29–31. Extracellular calcium is required for efficient invasion, but not egress, of P. falciparum^30,32,33.

Chemical inhibition of PKG, proteases and calcium release all impair egress

To identify the molecular processes required for B. divergens egress, we used the flow cytometry-based egress assay to screen for compounds that inhibit 8-Br-cGMP-induced egress. The compounds were selected based on known egress inhibition in P. falciparum or T. gondii. BAPTA-AM, which chelates intracellular calcium, and Compound 1 (C1), an inhibitor of apicomplexan PKG, both inhibited 8-Br-cGMP induced egress (Fig. 1F). Inhibitors of serine proteases, including TPCK, TLCK and PMSF, reduced induced egress at similar concentrations to those that inhibit P. falciparum gametocyte egress, whereas inhibitors of other protease classes exhibit modest inhibition (< 20%) (Figure S1H)^34–36. The short incubation time (15 min) may not identify inhibitors of certain proteases such as PfPMX/TgASP3 which act several hours prior to egress. Inhibitors of the lipid signaling pathway that inhibit egress in T. gondii and/or P. falciparum, including U73122 and propranolol which target PI-PLC and phosphatidic acid phosphatase, respectively, did not influence egress by B. divergens, whereas the diacylglycerol kinase inhibitor R59022 instead enhanced induced egress (Figure S1H)^27,29,37,38. In line with our flow cytometry assay, E64d treated parasites showed no obvious defect in egress, motility or invasion by video microscopy (Figure S1J). Taken together, these data demonstrate a requirement for cGMP signaling, calcium signaling (both intracellular and extracellular), serine proteases and gliding motility for efficient egress by B. divergens. Unlike in T. gondii, release of calcium with the ionophore A23187 does not induce egress or motility in B. divergens, suggesting calcium release is required, but not sufficient for egress. We note that a previous study found that A23187 could induce egress in B. bovis which could be due to differences between species, host cells, or technical such as the extracellular concentration of calcium³⁹.

Motility is required for B. divergens merozoites to egress from permeabilized RBCs but not for permeabilization

The initial deformation of the RBC in egress appears to be caused by direct contact with the parasite indicating the parasites actinomyosin motor may be used to physically disrupt the host membrane (Fig. 1B)¹⁶. To test if motility is required during egress, egress was induced with 8-Br-cGMP in the presence of phalloidin-Alexa Fluor 488, which selectively stains the cytoskeletons of permeabilized RBCs, and cytochalasin D, which inhibits actin polymerization and gliding motility. Cytochalasin D-treated parasites can be induced to lyse the host cell, but do not escape the permeabilized cell (Fig. 1G-H, Video S2). Immediately preceding egress, the host cell becomes ruffled, but no localized deformation is observed, followed by the RBC rapidly showing a reduced diameter, becoming round and being infiltrated by phalloidin, demonstrating the RBC membrane has been permeabilized (Fig. 1H, Video S2). T. gondii secreted lytic factors (e.g. PLP1) and host calpains damage the host cell, allowing the motile parasite to escape the permeabilized cell^27,40,41. The mechanisms of RBC lysis in asexual Plasmodium spp. remain unclear^42,43. B. bovis PLP1 is not strictly required for egress, however, its absence does lead to continued intracellular replication (4 parasites per cell instead of 2 which is typical of B. bovis), suggestive of a partial egress defect⁴⁴. In contrast to P. falciparum which fractures the RBC cytoskeleton during egress (Figure S1I)⁴⁵, the permeabilized RBC remains relatively intact throughout egress in B. divergens (Figs. 1B-C, 1G-H)¹⁶. P. falciparum secretes proteases (SERA6) which degrade the RBC cytoskeleton, leading to the to rupture of the RBC to release free merozoites, thus bypassing the requirement for gliding motility in egress^17,46,47. Consistent with this difference, Babesia spp. do not have an ortholog of any SERA protease nor an ortholog of the phospholipase TgLCAT/PbPL. Taken together, these data argue for a mechanism of host cell lysis in B. divergens relying centrally on secretion of lytic factors (e.g PLPs). While, we do not observe a requirement for parasite motility for host cell lysis, we cannot rule out that motility contributes towards cell lysis as has been observed in T. gondii⁴⁰.

Identification of putative egress, motility and invasion genes through transcriptomic analyses

The lytic cycle of B. divergens has been morphologically defined but remains poorly characterized at the molecular level in any Babesia spp.⁵ There are several Babesia spp. transcriptomes at different stages of the life cycle, however, there are no synchronous intraerythrocytic stage time course transcriptomes^48–52. We generated a synchronous bulk transcriptome of one replication cycle (0–12 hrs) for B. divergens. As a parallel strategy, we utilized a recent asynchronous single-cell transcriptome where expression profiles of individual genes were generated using a pseudo-time analysis with the start of gene expression identified by cross-correlation analysis between the bulk and single-cell expression curves⁵³. Genes that showed expression changes over time display a transcriptional cascade associated with “just-in-time” gene expression that has been observed in Plasmodium spp. and T. gondii (Figs. 2A and S2A)^54,55. The number of genes detected and that display significant changes over time for each method can be found in Fig. 2B. The timing of peak expression between the bulk and single-cell transcriptomes was well correlated for the majority of genes, supporting the use of the pseudo-time data for downstream analysis (Fig. 2D).

93 B. divergens genes were identified as orthologs or family members of known egress, motility or invasion genes we have collated for apicomplexan parasites. 91 of these genes are expressed in in vitro culture and 67 showed expression changes over time (Table S1). Genes that are expected to co-localize within the same subcellular compartment (e.g. micronemes) based on orthology display similar expression profiles as has been observed in Plasmodium spp. and T. gondii (Figs. 2C and S2B)^54,56,57. Secondary messenger-responsive proteins, including PKG, PKAc1, PKAr, and CDPK4 (PfCDPK4/TgCDPK3) peak ~ 8–10 hours post invasion (hpi) and remain high until 12 hpi when parasites naturally egress (Figs. 2C). Other putative members of the egress signaling pathway did not display stage-specific expression indicative of a role in egress or invasion (Table S1). The cysteine and aspartyl proteases, DPAP1 (no direct ortholog) and ASP3 (ortholog of PfPMX/TgASP3), respectively, display expression profiles matching that of microneme proteins (Figs. 2C). The aspartyl protease ASP2 (closely related to BdASP3) displays an expression profile matching rhoptry proteins (Figs. 2C). All nine B. divergens PLP genes are expressed (Data not shown). We found that only PLP1 (TgPLP1/PfPLP3) and PLP4 (no direct ortholog) display an expression profile matching other microneme and rhoptry proteins, respectively, suggestive of a role in egress and invasion or post-invasion PV breakdown, respectively (Figs. 2C).

Multiple invasion ligands were identified by orthology to known apicomplexan ligands and display expression profiles matching that of rhoptry or microneme proteins (Figs. 2E). These included TRAP2/P18, RAP1 and AMA1, which all have a demonstrated role in Babesia spp. invasion (Fig. 2C)^58–60. The B. divergens orthologs of the Plasmodium spp. or T. gondii rhoptry protein, SRA, and microneme proteins, CLAMP, GAMA, MAEBL and CelTOS represent novel vaccine targets that have not been investigated in Babesia spp.^61–65 Inner membrane complex (IMC) proteins, which are involved in cell structure and motility, are transcribed in three groups (Figure S2B), suggesting that IMC assembly with distinct profiles warrants future study.

A comparative transcriptomic approach using data from P. falciparum and T. gondii was used to identify novel proteins putatively involved in egress, motility or invasion (methods outlined in Figure S2C). 104 genes were identified, 31 of which were identified by multiple methods and whose orthologs have no known function (Figs. 2E; Table S1). Of the 8 genes found in all three species, 5 have a growth phenotype in the T. gondii CRISPR screen and the P. falciparum piggyBac screen (Table S1).

A genetic screen of high priority candidates reveals the essentiality of PKG, CDPK4, ASP2 and ASP3 for parasite proliferation

We focused on 11 high priority candidates based on transcriptomics as well as orthology to apicomplexan egress genes, including the kinases, PKG, PKAc1, PKAc2, CDPK4, CDPK5 and CDPK7, the perforin-like proteins, PLP1 and PLP4, and the proteases, ASP2, ASP3 and DPAP1. PKG is conserved between B. divergens, P. falciparum and T. gondii and has a well-characterized role in egress and invasion in these latter species^66,67. PKAc1 suppresses egress in T. gondii but is instead required for invasion by P. falciparum^{10,12,68−71}. The CDPK family of proteins are also required for P. falciparum and T. gondii egress and invasion, although the direct orthologs (defined by reciprocal blast) do not always have the same function between species (Reviewed in^72,73). PLPs are involved in host cell permeabilization and egress in T. gondii, Plasmodium spp. sexual stages and B. bovis^{40,44,74−76}. PfDPAP family members and BdASP2/ASP3 orthologs (PfPMX, PfPMIX and TgASP3) are required for egress and/or invasion in P. falciparum or T. gondii^77–82.

To develop a stable transfection system for B. divergens, multiple transfection methods and selection drugs with established resistance markers were tested for stable transfection of a GFP reporter plasmid (Figure S3A and C) (summarized in Table S2). Nucleofection of isolated merozoites and blasticidin-S selection was the most efficient method and was used for all further transfections. A CRISPR/Cas9 system was generated to introduce a HA tag, as well as the glmS riboswitch with and without a destabilization domain (DD) inducible knockdown system in series to the 3’ end of each gene (Figs. 3A and S3B)^83,84. Parasites containing the correct integration reached > 1% parasitemia 12–16 days after transfection for all constructs, except PKAc1, PLP1 and PLP4 which we were unable to tag (Figure S3F and H). PKG-HA-DD-glmS parasites were initially used to determine the effectiveness of the knockdown systems. Induction of the DD system, which induces protein degradation, generated a more rapid (~ 6 hrs) and stronger reduction of protein levels than the glmS system (Fig. 3B). The glmS system, which destabilizes mRNA, resulted in slower knockdown that was first observable by 24 hrs, but generated strong knockdown by 48 hrs (Fig. 3B). Combining both systems resulted in a stronger knockdown than either alone (Fig. 3B). This pattern was reflected in the effect of these knockdown systems on parasite growth when targeting PKG, CDPK4, ASP2 or ASP3, with the double knockdown producing the strongest defect in all lines, whereas the DD or glmS alone were not sufficient for all genes (Fig. 3D). DPAP1, PKAc2, CDPK5 and CDPK7 knockdown did not affect proliferation (Fig. 3D, data not shown).

PKG and CDPK4 are essential for egress and their depletion results in continued intracellular replication

We analyzed knockdowns of PKG and CDPK4 following synchronized invasion to understand when the block in proliferation occurs. PKG or CDPK4 knockdown parasites continued to replicate within a single iRBC, to form up to 16 parasites per RBC by 40 hpi (Figs. 3C-E and S3I). The fraction of parasites that replicate to > 4 per iRBC is dependent on the degree of knockdown (Fig. 3E). Knockdown of PKG prevented induced egress by 8-Br-cGMP, further supporting that 8-Br-cGMP induces egress through activation of PKG (Figure S3D). The reduced susceptibility to 8-Br-cGMP mediated induced egress in PKG-HA-DD-glmS parasites without inducing knockdown compared to WT parasites is likely due to a partial knockdown in the absence of induction, due to tagging as observed in other parasite recombinants (Figure S3D)^85,86. A model of the molecular pathways involved in B. divergens egress can be found in Fig. 3F.

The proteases ASP2 and ASP3 are required for egress and invasion

The orthologs of B. divergens ASP2 and ASP3 in P. falciparum (PfPMIX/PMX) and T. gondii (ASP3) are the most upstream proteases known to date in the protease cascade that matures many proteins in the micronemes and rhoptries that are required for egress and invasion^80,81. ASP2 and ASP3 knockdown resulted in an increased number of free merozoites in culture (Fig. 3D). In the ASP3 knockdown culture, clusters of parasites were also observed in lightly stained RBCs (Fig. 3C, ASP3 left panel). To further define these phenotypes, induced egress in knockdown parasites was observed by video microscopy. The time to egress and rate of escape from the lysed cells was the same between ASP2 knockdown and WT parasites (Fig. 4A, B and E). After egress, ASP2 parasites typically bound to a single RBC and were able to strongly deform it (pre-invasion) similar to WT, however, were unable to complete invasion and eventually detached from the cell (Figs. 4C-E; Video S5). The time from initial deformation of the RBC by the intracellular ASP3 knockdown parasite to the time of lysis of the RBC was significantly longer than in WT parasites and fewer parasites were able to escape the lysed RBC (Figs. 4A-B, F-G). Fewer ASP3 knockdown parasites were able to escape the lysed cell, and parasites that successfully egressed were able to bind to and deform the RBC, however, unlike WT or ASP2 knockdown parasites that typically bind strongly to a single host cell, ASP3 parasites maintained gliding motility over the surface of the cell, often contacting multiple host cells, but were rarely able to complete invasion (Fig. 4B-D, 4F-G; Video S6). Together, these results are consistent with the transcriptomic data suggesting ASP2 functions in the rhoptries and knockdown parasites are able to reorient but are not able to undergo the final step of invasion which requires rhoptry proteins^87–89. The ASP3 phenotype suggests that it is required for maturation of microneme proteins that are required for RBC lysis and reorientation and/or anchoring of the apical end of the parasite to the RBC prior to rhoptry release^21,23. In comparison to T. gondii, which requires only TgASP3 to process proteins in the rhoptries and micronemes, our data suggest that BdASP2 and BdASP3 are functionally orthologous to PfPMIX and PfPMX, respectively, and likely responsible for processing proteins required for invasion in the rhoptries, and egress and invasion in the micronemes, respectively.

PKG, ASP2 and ASP3 are druggable targets required for egress and/or invasion

Significant effort has been invested to develop specific inhibitors of apicomplexan PKG, CDPKs and aspartyl proteases (PfPMIX, PfPMX and TgASP3) (reviewed in^90–94). To demonstrate that PKG is a druggable target in B. divergens we used the apicomplexan PKG inhibitors compound 1 (C1), and ML10 a potent and highly specific P. falciparum PKG inhibitor with in vivo activity^95,96. To determine the specificity of C1 and ML10 for BdPKG, CRISPR/Cas9 was used to introduce a putative resistance mutation (T651Q) equivalent to the gatekeeper mutations that confer resistance in P. falciparum and T. gondii (Fig. 5A)^67,97. A 500 bp region within a plasmid was used as a repair template, achieving ~ 50% editing, with decreasing efficiency further from the cut site (Figure S3E and F). We utilized the ability to perform sequential transfection with the same resistance marker to generate double mutants, containing the T651Q mutation and the glmS knockdown system. Treatment of WT parasites with C1 or ML10 resulted in the same continued intracellular replication as was observed with PKG knockdown (Fig. 5B). The potency of C1 and ML10 against B. divergens is ~ 25-fold and 281-fold less than against P. falciparum, and 4.4- and 8.4-fold less than against B. bovis ^66,96,98,99. A 3- and 10-fold for C1, and 15- and 8-fold for ML10, reduction in the IC₅₀ was observed between WT compared to PKG-glmS parasites with or without knockdown, respectively (Figs. 5C-D). A trending, but not statistically significant shift in the IC₅₀ was observed between WT and the PKG-T651Q parasites (10.5 vs 14.1 µM for C1, and 591 nM vs 1209 nM for ML10), whereas the same mutation in knockdown parasites restores susceptibility to near WT levels (Figs. 5C-D). Together these results support a model in which C1 and ML10 are able to target PKG, however, at concentrations required for killing B. divergens there are secondary targets contributing to growth inhibition, which is likely to be CDPK4 that is the only other CDPK to share the same small gatekeeper residue as PKG (Figure S3G). The ability to rapidly and specifically inhibit PKG with C1 when combined with partial knockdown was used to investigate the role of PKG during RBC invasion. PKG knockdown increased the sensitivity to C1 invasion inhibition by 20-fold (Fig. 5E), suggesting PKG signaling is also required for host cell invasion.

To determine the druggability of ASP2 and ASP3, we screened 22 aspartyl protease inhibitors, including compounds known to be active against P. falciparum PfPMIX and PfPMX as well as clinical inhibitors of human beta-secretase, against WT and knockdown parasite lines (Figure S4)^81,100−103. 14 compounds displayed an IC₅₀ < 25 µM, with the most potent compound (TCMDC-134675) having an IC₅₀ of 891 nM against WT parasites (Fig. 5G and Table 1)^81,103. We observe a 2.7-fold and 3.6-fold increase in sensitivity to TCMDC-134675 in ASP2 and ASP3 knockdown parasites, respectively. CWHM-0000166 was less active against WT parasites (8.8 µM), but displayed the largest increase in sensitivity of 7.7-fold and 26-fold against ASP2 and ASP3 knockdown parasites, respectively (Fig. 5F). In P. falciparum, incomplete PMX inhibition results in parasites lysing the PVM, whereas complete inhibition blocked PVM rupture^80,104. To determine if the phenotype observed in ASP3 knockdown parasites was incomplete, we combined knockdown with CWHM-0000166 treatment. Under these conditions we observed that parasites continued to replicate intracellularly, as was observed with inhibition of PKG and CDPK4, further supporting a role of ASP3 in egress (Fig. 5B). We also observed clusters of parasites within a lysed RBC, in line with the live microscopy results that parasites that do lyse the host cell escape at a reduced rate. The similar phenotype seen following inhibition of ASP3 to the protein kinases CDPK4 and PKG, suggests that failure to egress by multiple mechanisms results in a default for continuation of the intracellular lytic cycle. No synergy was observed between ASP2 or ASP3 knockdown and ML10, suggesting the kinase and protease egress pathways act independently. Together these data demonstrate that PKG, ASP2 and ASP3 are essential and druggable proteins in B. divergens that are required for egress and/or invasion.

Table 1

IC₅₀ values of Aspartyl protease inhibitors against *B. divergens*. IC₅₀ (± S.D.)
	Wild-type	ASP2 (High Shld1)	ASP2 (Low Shld1 = Knockdown)	ASP3 (High Shld1)	ASP3- (Low Shld1 = knockdown)
TCMDC-134675	0.891 (± 0.307)	0.320 (± 0.212)	0.478 (± 0.288)	0.362 (± 0.129)	0.246 (± 0.095)
TCMDC-136879	4.03 (± 1.90)	1.57 (± 0.17)	1.63 (± 0.94)	1.39 (± 0.26)	0.539 (± 0.358)
CWHM-0000099	> 25	11.8 (± 0.8)	3.99 (± 3.15)	16.3 (± 3.7)	2.85 (± 1.52)
CWHM-0000117	6.81 (± 3.08)	2.66 (± 0.55)	2.56 (± 1.32)	3.17 (± 0.61)	1.13 (± 0.50)
CWHM-0000166	8.80 (± 3.18)	2.89 (± 1.27)	1.15 (± 0.70)	2.76 (± 0.96)	0.337 (± 0.157)
CWHM-0000047	19.5 (± 5.3)	18.5 (± 12.9)	6.32 (± 5.84)	16.9 (± 9.0)	8.47 (± 5.35)
CWHM-0000579	12.6 (± 6.6)	7.49 (± 4.73)	11.1 (± 6.5)	9.12 (± 6.12)	7.32 (± 6.48)
CWHM-0000162	13.3 (± 9.4)	7.15 (± 6.45)	6.47 (± 5.35)	8.00 (± 5.06)	6.41 (± 6.74)
lanabecestat	19.8 (± 5.3)	10.1 (± 3.4)	4.67 (± 3.06)	14.1 (± 2.6)	10.0 (± 5.4)
verubecestat	10.9 (± 3.3)	6.38 (± 3.27)	1.89 (± 2.78)	8.04 (± 3.36)	5.49 (± 4.29)
CWHM-0000068	16.6 (± 7.6)	15.7 (± 12.6)	8.02 (± 0.89)	13.3 (± 2.9)	9.88 (± 4.84)
CWHM-0000116	> 25	> 25	18.8 (± 8.2)	20.3 (± 11.3)	21.4 (± 10.2)
CWHM-0000123	9.46 (± 3.66)	8.39 (± 4.39)	10.8 (± 2.8)	11.2 (± 8.7)	9.22 (± 1.76)
CWHM-0000293	> 25	> 25	> 25	> 25	> 25
CWHM-0000299	> 25	> 25	> 25	> 25	> 25
CWHM-0000460	> 25	12.3 (± 10.2)	12.2 (± 1.5)	12.4 (± 5.6)	12.6 (± 4.0)
CWHM-0000580	> 25	> 25	> 25	> 25	> 25
CWHM-0000583	> 25	14.2 (± 9.5)	14.5 (± 8.9)	13.8 (± 5.1)	14.1 (± 6.9)
CWHM-0000658	16.2 (± 9.8)	12.6 (± 11.9)	6.47 (± 3.82)	9.35 (± 3.77)	7.82 (± 5.45)
AZD3839	25.7 (± 12.3)	15.8 (± 9.6)	8.64 (± 4.98)	15.8 (± 3.6)	18.6 (± 13.4)
LY2811376	24.2 (± 10.7)	6.37 (± 3.46)	2.57 (± 2.67)	13.4 (± 7.2)	12.1 (± 8.2)
LY2886721	18.5 (± 5.7)	6.38 (± 3.72)	5.76 (± 6.77)	9.62 (± 4.22)	9.30 (± 6.24)
ML10	0.864 (± 0.319)	0.794 (± 0.072)	1.56 (± 1.24)	0.682 (± 0.256)	0.628 (± 0.219)
Atovaquone	0.021 (± 0.007)	0.020 (± 0.005)	0.020 (± 0.008)	0.021 (± 0.006)	0.025 (± 0.011)

Here, we establish B. divergens as a genetically tractable in vitro model to study Babesia spp. cell biology. The synchronous B. divergens bulk transcriptome and single-cell transcriptome will serve as a resource for the study of Babesia spp. biology as well as for further comparative studies of apicomplexan biology. The majority of molecular research in apicomplexan parasites has been limited to P. falciparum, P. berghei and T. gondii. Transient or stable transfection systems exist for Babesia spp., Theileria spp., Cryptosporidum spp., Eimeria spp., Sarcocystis spp. and Neospora spp. (reviewed in Suarez, et al.¹⁰⁵). However, the number of studies and the range of available tools and resources remains comparatively limited in these organisms. Additional studies of a wider range of apicomplexan parasites will expand our knowledge of conserved and unique biological mechanisms of apicomplexan parasitism, that are often pathogens of metazoans.

Egress is a unique aspect of apicomplexan parasitism compared to their hosts that requires specialized processes and offers novel druggable targets. The overall process of egress is similar between Plasmodium spp. and T. gondii, however, there are notable differences in their cell biology (e.g. differences in division mechanisms) and in the host cell niche that place unique pressures on the parasite (T. gondii resides in nucleated cells, while the asexual stages of Babesia spp. and Plasmodium spp. reside in enucleated RBCs). While the signaling and molecular mechanisms differ in some aspects, we have found that egress of B. divergens at the cellular level closely resembles that of T. gondii and has several notable differences to Plasmodium spp. egress despite being evolutionarily closer and sharing the same RBC niche. B. divergens egress begins with localized deformation of the RBC by the intracellular parasite. The deformation requires the parasites actinomyosin motor but is not necessary for disruption of the RBC membrane, although it may still contribute. Once the parasite has permeabilized the RBC membrane, it utilizes its actinomyosin motor to pass through the RBC cytoskeleton which remains relatively intact throughout egress. This is similar to T. gondii but in sharp contrast to Plasmodium spp. that fracture the RBC cytoskeleton to release parasites without requiring motility.

T. gondii is able to sense both intrinsic signals of parasite density and extrinsic signals of host cell damage to induce egress^{22,24–27,37,106}. Similarly, P. falciparum can sense lipids and potassium, although their role in egress and invasion are less clearly defined^29,30. Since Plasmodium spp. egress is strictly required at the end of each replication cycle, the initial egress signal is likely to be linked to the cell cycle, however, this remains to be demonstrated. The only egress signal identified in B. divergens is extracellular calcium, but not potassium or sodium, which acts as an extrinsic signal of host cell damage (Fig. 1E, Video S2). Whether B. divergens responds to its environment to determine if parasites will egress after one or two replication cycles, or whether this is regulated stochastically remains unclear.

In T. gondii and P. falciparum, the initial signals converge to either activate guanylate cyclase (GC) or inhibit PDE and thus raise cGMP levels, which in turn activates PKG. In T. gondii, PKAc1 suppresses cGMP levels to prevent premature egress, putatively by activating a PDE, whereas in P. falciparum PKAc1 is required for invasion^{10,12,68−71}. Chemical inhibition or activation of PKA suggests that PKA suppresses B. divergens egress and is also required for invasion (Fig. 1F, S1D and S1G). It is possible these functions are performed separately by the two PKAc orthologs, although only PKAc1 displays stage specific expression (Figs. 2C). The PKAc2 knockdown line did not display an obvious growth defect and we were unable to generate a PKAc1 knockdown line. The inability to generate several of the attempted knockdown lines could be due to the DD tag interfering with the proteins function, or the proteins being sensitive to partial knockdown induced by the DD or glmS even in the absence of induction. Alternative genetic systems will need to be developed for B. divergens to determine the function of these proteins.

In P. falciparum and T. gondii, PKG activates PI-PLC, which generates lipid second messengers. These eventually lead to the release of calcium from intracellular stores that are required for microneme/exoneme secretion. We have demonstrated that PKG is a central component of the B. divergens egress signaling pathway that is both necessary and sufficient to induce egress (Figs. 1A, 1F and S3D). Small molecules targeting the lipid signaling pathway, including propranolol and U73122, had little to no effect on B. divergens egress and will require further genetic studies to determine the role of lipid signaling. Downstream of PKG and calcium release, CDPKs are required in P. falciparum and T. gondii egress. Of the three CDPKs expressed in B. divergens only CDPK4 displayed late stage expression and an egress defect when knocked down.

Inhibition of the egress signaling pathway in B. divergens or B. bovis (PKG and CDPK4 inhibition or calcium chelation) result in continued intracellular replication (Figs. 3C and E)^107,108.

The egress signaling pathway leads to the release of the micronemes/exonemes. These organelles contain lytic factors, motility proteins and invasion ligands, many of which are proteolytically matured. In P. falciparum, proteolytic maturation in the rhoptries and micronemes is done in part by PMIX and PMX, respectively⁸¹. In contrast, T. gondii only contains one ortholog, TgASP3, which is localized in a post-Golgi compartment and matures a subset of microneme and rhoptry proteins^80,82. The putative localization of BdASP2 and BdASP3 based on transcriptomics and their knockdown phenotypes suggest they are functionally orthologous to PfPMIX and PfPMX, respectively. While further work will be required to determine the substrates of these proteases and overlap between apicomplexans, we hypothesize that the continued replication phenotype observed with ASP3 inhibition is due to a block in maturation of lytic factors and therefore host cell lysis. Inhibition of either the signaling pathway or the downstream lytic factors produces a similar continued intracellular replication phenotype in T. gondii^40,67. This implies that T. gondii and Babesia spp. lack a checkpoint to exit the cell cycle and that the default pathway is to continue replication. Inhibition of egress signaling in P. falciparum blocks replication, however, the transcriptional profile of stalled parasites continues to progress, suggesting that they may also lack a checkpoint at the transcriptional level¹⁰⁹.

Small molecules have been developed that target the kinases and proteases required for egress of Plasmodium spp. and T. gondii. Identification of shared parasite targets of these compounds in Babesia spp. and other apicomplexan parasites will help leverage drug development efforts for malaria that have significantly more resources. Here, we have developed transfection, alongside CRISPR/Cas9 and inducible knockdown systems to modify the B. divergens genome. Through knockdown and the introduction of resistance mutations, we identified C1 and ML10 as inhibitors of PKG, however, at concentrations required for killing it is likely that other parasite molecules are also inhibited, with CDPK4 being the most likely target as it also contains a small gatekeeper residue (Fig. 5C-E and S3G). We also identified several compounds that dually target ASP2 and ASP3, including compounds that have known activity against PfPMIX and PfPMX lines^81,100,101. The dual activity against these proteins may reduce the ability of the parasite to develop resistance to small molecule inhibitors. The same methods could be applied to these and other proteins to aid future drug development for Babesia spp. Notably, multiple new classes of potent inhibitors have been developed against PfPMX and PfPKG with drug-like properties and in vivo activity^104,110,111.

Here, for the first time we have established a molecular framework for the events surrounding Babesia spp. egress. Several major questions remain about Babesia spp. egress, such as how they selectively lyse the PVM after invasion, the role of lipid signaling, the initial signal to induce egress and how they regulate egress to occur after one or more replication cycles. With the cellular, transcriptomic and genetic tools developed here, future studies will be able to answer these questions and reveal the unique biology of Babesia spp. as well as conserved processes throughout Apicomplexa, providing a rational basis for the development of therapeutic interventions.

Acknowledgments

We would like to thank Dr. Simon A. Osborne (LifeArc) for kindly providing ML10 and Dr. Jeffrey D. Dvorin for providing Compound 1 and BIPPO. We would like to thank Dr. Sebastian Lourido for providing A23187 with demonstrated activity in T. gondii. This work was supported by grants 1R21AI153945 (MTD), 1R01AI167570-01 (MJG, KZ, MTD) and 1R21AI150090 (KZ, MJG) from the National Institutes of Health. BE was supported by an American Heart Association (AHA) postdoctoral fellowship (17POST33410556) and a Australian National Health and Medical Research Council postdoctoral fellowship (APP1148392). CDK was supported by an AHA predoctoral fellowship (19PRE34380106).

Author contributions

BE, CDK and YR conceived and performed experiments and wrote the manuscript. ASP, KAB, JAT, SAS and CKM performed experiments and wrote the manuscript. MJG, MJM, KZ and MTD conceived experiments and wrote the manuscript.

RESOURCE AVAILABILITY

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Manoj Duraisingh ([email protected]).

Materials availability

All unique reagents in this study are available from the lead contact. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Data and code availability

Single-cell RNAseq and bulk RNAseq data have been deposited at NCBI Sequence Read Archive (SRA) and are publicly available as of the date of publication. Accession numbers are listed in the key resources table. Microscopy data reported in this paper will be shared by the lead contact upon request. All code has been deposited at GitHub. DOIs are listed in the key resources table.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Parasite strains

Babesia divergens strain Rouen 1987, kindly provided by Kirk Deitsch and Laura Kirkman (Weill Cornell Medical College), was cloned by limiting dilution (BdC9) and was maintained in purified Caucasian male O+ human RBCs (Research Blood components). The Babesia bovis strain MO7 was kindly provided by David Allred of the University of Florida and maintained in purified bovine RBCs (hemostat). Parasites were cultured in RPMI-1640 media supplemented with 25 mM HEPES, 11.50 mg/l hypoxanthine, 2.42 mM sodium bicarbonate, and 4.31 mg/ml AlbuMAX II (Invitrogen), at 37^oC in a 1% oxygen, 5% carbon dioxide and 94% nitrogen environment.

METHOD DETAILS

Induced egress

To measure egress, mixed stage parasites at 2% final HCT, 10–15% parasitemia in a total volume of 40 µL in the presence of the stated drug, were incubated at 37^oC for one hour in a 96 well plate for 1 hr. Where not otherwise stated, 8-Br-cGMP was used at a final concentration of 500 µM. For inhibition of induced egress, parasites were first incubated with the stated compound at 1.33x concentration for 15 min at 37^oC, prior to induced egress with the addition of 8-Br-cGMP to a final concentration of 500 µM and 1x inhibitor concentration. A final concentration of 100 µg/ml of heparin was added to prevent reinvasion. After 1 hr, the parasites were washed 3x with PBS and stained with 1:5000 Syber green II. Parasitemia was determined by flow-cytometry (Macs quant, Miltenyi) and analyzed in FloJo. Induced egress was calculated as the drop in parasitemia of the treated parasites as a percentage of the RPMI only sample.

Live microscopy

All videos were taken on a Zeiss Axio Observer using a 60x oil immersion lens inside a chamber heated to 37^oC. Prior to imaging, parasites were allowed to settle on the bottom of a glass bottom slide (Ibidi, Cat#80827/81817) at 37^oC in a 5% CO₂ incubator for 10–15 min. Small molecule inhibitors were included in this incubation as necessary. Immediately prior to imaging, the media was removed and replaced with the RPMI/IC/EC with 500 µM 8-Br-cGMP, and any small molecules inhibitors being tested. For IC/EC experiments, the final buffer contained 0.0075% (w/v) saponin (Calbiochem, Cat#558255) to lyse the RBC. Alexa Fluor™ 488 Phalloidin (Invitrogen, Cat#A12379) was added to RPMI at a concentration of 1/150 where stated. All images were taken within 20 minutes of removal from the 5% CO₂ incubator. Plasmodium falciparum mature schizonts were isolated by magnetic affinity purification (MACs LS column, Miltenyi) and imaged as per the B. divergens protocol. Images were processed in Zen 2 (Zeiss) and ImageJ/Fiji.

Isolation of free merozoites for invasion assays, synchronization and transfection

~ 1–2 ml of packed iRBCs at 20–30% parasitemia was used to isolate free merozoites using a modified protocol from ⁵. Briefly, the iRBC was resuspended to 10% HCT in RPMI and passed through two 1.2 µM filters. The isolated merozoites and RBC debris were pelleted at 3000 x g for 3 min and the supernatant was removed. For invasion assays, the merozoites were resuspended in RPMI and added to a 96 well u-bottom plate containing 1.33x final drug concentration (30 µl total) and incubated at 37^oC for 10 min. Fresh RBCs were then added to a final of 2% HCT and 1x drug concentration (40 µl total). The plate was then incubated at 37^oC shaking at 600 rpm for 20 min. The assay was stopped by washing the parasites three times with 200 µL PBS, 400 x g for 2 min or by adding 200 µl 4% paraformaldehyde. Parasitemia was determined by flow cytometry as per induced egress section above. For synchronization, isolated merozoites were resuspended to a final volume of 1 ml of 20% HCT RBCs in RPMI and allowed to invade for 20 min shaking at 600 rpm and 37^oC. Parasites were then washed 3x with 10 ml RPMI at 400 x g to remove free merozoites and cell debris. 100 µg/ml heparin was added to prevent re-invasion throughout the time course when stated in the figure legend.

Plasmid construction

The sequences of all primers and synthesis products used in this study are found in table S3. The bi-directional promoter between the EF1alpha (Bdiv_030590) and LON peptidase (Bdiv_030580c) was amplified using primers BE-8 and BE-9, and cloned into XhoI/BamHI sites of pPfEF-GFP-BSD (EF1alpha side drives GFP/Cas9 expression). The DHFR (Bdiv_030660, primers BE-21/22) and HSP90 (Bdiv_037120c, primers BE-32/33) 3’UTRs were cloned into EcoRI/HindIII and SpeI/NotI sites, respectively, for the selection marker or GFP/Cas9, respectively. Cas9 was amplified from pDC2-Cas9 using primers BE-124/125 and cloned into the XhoI/SpeI sites in place of GFP. The U6 promoter, bbs1 sites (for the guide), guide tracer/scaffold, U6 terminator and PKG-T651Q repair template were synthesized by IDT (Synthesis 1) and cloned into the EcoRI site by gibson assembly to make the pEF-Cas9-PKG-T651Q plasmid. For the sgRNA, oligos were either phosphorylated, annealed and cloned into the BbsI sites, or the sgRNA was amplified using the corresponding ‘guide PCR’ primer (e.g. BE-551) and BE-550 (universal for all). For all inducible knockdown lines, the HA-glmS or HA-glmS-DD tags were amplified using primers BE-536/537. The 5’HR (HR1, 3’end of the gene) and 3’HR (HR2, 3’UTR of gene) were amplified using the corresponding HR1 and HR2 primers from Table S3 for each gene (e.g. BE_512–515 for CDPK4). The sgRNA, HR1, DD-glmS and HR2 PCR fragments were cloned into the Bbs1/PacI sites of the pEF-Cas9-PKG-T651Q plasmid in a single Gibson reaction. Correct integration of plasmids was confirmed with primers from Table S4 labelled “test integration” (e.g. BE_610/611 for CDPK4).

Transfection

For transfection of iRBCs using the Biorad Genepulser II, 100 µg DNA in 30 µl combined with 370 ul of cytomix (120 mM KCl, 0.15 M CaCl2, 2mM EGTA, 5mM MgCl2, 10mM K2HPO4/KH2PO4, 25mM HEPES, pH 7.6) and 200 µl iRBCs (~ 15% parasitemia). Transfection was carried out with Biorad genepulser II set to 0.31 kV, 950µF. For Amaxa nucleofection of free merozoites, merozoites were isolated from ~ 3x10⁹ iRBCs and all supernatant was removed. The pellet, containing free merozoites and cell debris, was resuspended in 100 µl of P3 solution (Lonza) plus 10 µl of water containing 2–10 µg of DNA. For Amaxa nucleofection of intact iRBCs, 10 µl iRBCs at 10% parasitemia was resuspended in 110 µl P3 + DNA solution, as per above. Transfection of either free merozoites or iRBCs was carried out in a 4D-Nucleofector System (Lonza), using the FP158 program. After electroporation of free merozoites, the parasite and buffer mixture was immediately transferred to 1 ml of RPMI containing 200 µl packed RBCs and pre-heated to 37^oC. Parasites were allowed to invade at 37^oC shaking at 600 rpm for 20 min before being washed with 10 ml RPMI to remove the P3 solution and returned to culture. Electroporated intact iRBCs were washed 1x with 10 ml RPMI and returned to culture. Cultures were selected with 15–20 µg of Blasticidin-S.

Drug sensitivity and proliferation assays

For all compounds except the aspartyl protease inhibitors, parasites were diluted to 0.2% parasitemia in 2% hematocrit (100 µl total) in the presence of the stated compounds) in a 96-well U-bottom plate. Parasites were cultured for 72 hours. Parasitemia was measured by flow-cytometry. Testing of aspartyl protease inhibitors (Fig. 5F-G, Table 1), was performed using a whole-well SYBR assay¹¹⁶ in 384-well format, with initial parasitemia set to 0.4%, 2% HCT, 40 µl/well and 72 hrs of proliferation. SYBR lysis buffer contained (0.16% saponin; 20 mM Tris-HCl; 5 mM EDTA; 1.6% Triton X 100; adjust pH to 7.4). Where not otherwise stated, +Shld was 500 nM, +GlcN was 1 mM, -Shld/-GlcN was 0 nM, except for ASP2 drug assays (Fig. 5F-G, Table 1) where + Shld was 2 mM and -Shld was 250 nM.

RNAseq analysis

RNA was isolated from parasites using a hybrid protocol of organic extraction combined with column purification. Briefly, parasite pellets were resuspended in 500 µl TRIzol and then extracted with chloroform. The aqueous layer was then purified using Qiagen RNeasy Mini spin-columns following manufacturers protocols. RNA was quantified and normalized into individual wells, and libraries were prepared following the Smart-seq2 protocol ¹¹⁷. Libraries were sequenced on the illumina platform. Bulk synchronous RNAseq analysis workflow: The quality of reads was assessed using FastQC (Version 0.10.1). The reads were trimmed with Cutadapt from Trim Galore package (Version 0.3.7) (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/). The trimmed reads were mapped against the Bdivergens1802A reference genome (PiroplasmaDB release 46) and assembled with HISAT2 (Version 2.0.5 released)⁽https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4655817/). SAM files obtained from alignment results were processed using SAMtools (Version 1.4.1) and the relative abundance of transcripts were estimated using featureCounts (https://academic.oup.com/bioinformatics/article/30/7/923/232889). Normalization & Noise removal: Counts per million (cpm) values per gene was calculated using cpm() from edgeR R package (Version 3.24.3) (https://www.bioconductor.org/packages/release/bioc/vignettes/edgeR/inst/doc/edgeRUsersGuide.pdf). Genes with cpm value > 2 in at least 3 samples were maintained for further analysis. Gene counts were normalized and scaled to logarithmic form using edgeR’s TMM method (trimmed mean of M values) with DGEList(), calcNormFactors() and cpm() functions. The cpm() parameters were as following: y = DGEList.obj, log = TRUE, prior.count = 3, normalized.lib.sizes = TRUE. Batch effected samples were Identified through the analysis of hierarchical clustering and dissimilarities method using the R function hclust() with the default parameters and logCPM expression values. These samples were excluded from further analysis. The bad samples were the ones clustered in single batches and were not similar to the rest of samples based on Euclidean distance metric. PCA Analysis: Principal component analysis (PCA) was run on genes with FC > 1.5 with prcomp() from R. The first 2 principal components (PCs) were chosen for visualization. Ortholog Analysis: Orthologous genes between Babesia divergens 1802A, Plasmodium falciparum 3d7, Toxoplasma gondii ME49, Plasmodium berghei ANKA (all from VEuPathDB release 46) were assembled using blast (version 2.10.0)( https://academic.oup.com/nar/article/36/suppl_2/W5/2505810) and the R package Orthologr (version ‘0.4.0’) with implemented best reciprocal hit method. Smoothed expression curve analysis: Only genes with an average of ≥ 10 cpm across each timepoint were maintained. Curves were generated using loess.smooth function in R (span = 0.5, degree = 2, family = c(‘gasussian’)). Differentially expressed genes were those that displayed a ≥ 1.5-fold change over time. P. falciparum data was from otto et al 2010 (downloaded from PlasmoDB). Genes with an FPKM ≥ 5 were maintained. T. gondii data was from Behnke et al 2010 (Downloaded from ToxoDB). T. gondii data was manipulated to change the timepoints to match the other datasets (i.e. it starts at 6h from paper which is straight after invasion). Pearson correlation was calculated in R using the smoother curves for AMA1 and RON2. Genes were considered to be highly correlated if they displayed ≥ 2-fold change over time and had a Pearson correlation value of ≥ 0.9.

scRNA-Seq work flow

scRNA-Seq data was processed using the 10x cell-ranger pipeline and aligned to the Babesia divergens 1802A genome. Counts were subsequently normalized and processed using the R Seurat package. A total of 9450 cells and 3620 genes were retained after removing cells and features with low counts (Seurat parameters: min.cells = 10, min.features = 100, nFeature_RNA > 200 nFeature_RNA < 1200). Dimensionality reduction and clustering analysis were performed using PCA and graph-based KNN as implemented in the Seurat Package. A total of 4 clusters were identified by the KNN algorithm. To make the size of the data manageable, each cluster was downsampled to include 800 cells. Global differential expression performed on each cluster (log(FC) > 1 and adjusted p-value < 0.01) identified 544 differentially expressed genes. Pseudo-time analysis: Pseudo-time analysis was performed on the first two PCA components by fitting a principal curve to the data and orthogonally projecting the cells on the curve. Gene expression curves were then constructed using the pseudo-time and the start of gene expression was identified by cross correlation analysis between the bulk and single cell expression curves. Further details can be found in¹¹⁸.

QUANTIFICATION AND STATISTICAL ANALYSIS

All statistical analysis was performed in Graphpad PRISM 9. IC₅₀ values were calculated using the Non-linear regression function (variable slope – four parameters, least squares regression). Statistical significance of IC₅₀ changes were determined using ANOVA. All image analysis was performed in Zen 2 (Zeiss) and ImageJ/Fiji. Details of each analysis can be found in the corresponding figure legend.

KEY RESOURCES TABLE

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Anti-Histone H3 antibody	Abcam	ab1791; RRID:AB_302613
Anti-HA 3F10	Sigma (Roche)	12158167001; RRID:AB_390918

Bacterial and virus strains
E. coli XL10-Gold Ultracompetent cells (all cloning)	Agilent	200315

Chemicals, peptides, and recombinant proteins
8-Bromoguanosine 3′,5′-cyclic monophosphate sodium salt (8-Br-cGMP)	Sigma-Aldrich	B1381
SYBR™ Green I Nucleic Acid Gel Stain	Invitrogen	S7563
KT5720	Sigma-Aldrich	K3761
Heparin sodium salt from porcine intestinal mucosa	Sigma-Aldrich	H3393
8-Bromoadenosine 3′,5′-cyclic monophosphate sodium salt (8-Br-cAMP)	Sigma-Aldrich	B7880
1-[6-[((17β)-3-Methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione (U-73122)	Sigma-Aldrich	U6756
1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM)	Sigma-Aldrich	A1076
Cytochalasin D	Sigma-Aldrich	C8273
E64	Sigma-Aldrich	E3132
Compound-1 (4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H pyrrol-3-yl]-pyridine)	A gift from Dr. Jeffrey Dvorin (Boston Children’s Hospital)
R59022 (Diacylglycerol Kinase Inhibitor I)	Sigma-Aldrich	D5919
Phenylmethylsulfonyl fluoride (PMSF)	Sigma-Aldrich	78830
N_α-Tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK)	Sigma-Aldrich	T7254
N-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK)	Sigma-Aldrich	P5318
Pepstatin A	Sigma-Aldrich	P5318
Chymostatin	Sigma-Aldrich	C7268
E64d	Selleckchem	S7393
ALLN (Calpain inhibitor 1)	Sigma-Aldrich (Calbiochem)	208719
H89	Sigma-Aldrich	B1427
BIPPO 5-benzyl-3-isopropyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one	A gift from Dr. Jeffrey Dvorin (Boston Children’s Hospital).
A23187	Sigma-Aldrich	C7522
Propranolol hydrochloride	Sigma-Aldrich	P0884
ML10	A gift from Dr. Simon Osborne (LifeArc)
Saponin	Calbiochem	558255
Alexa Fluor^TM 488 Phalloidin	Invitrogen	A12379
Shld1	Synthesized as per(Banaszynski et al., 2006)
TRIzol®	Invitrogen	15596026
D-(+)-Glucosamine hydrochloride (GlcN)	Sigma-Aldrich	G1514
Blasticidin-S	Invivogen	ant-bl-10p
Critical commercial assays
DNeasy Blood and Tissue Kit	Qiagen	69504
RNeasy Mini spin-columns	Qiagen	74106
P3 Primary Cell 4D-Nucleofector™ Kit	Lonza	V4XP-3024

Deposited data
Bulk synchronous RNAseq	NCBI SRA	PRJNA804502
Single-cell RNAseq	NCBI SRA	PRJNA803312
Code	GitHub	https://github.com/umbibio/Babesia_time_course


Experimental models: Cell lines
Babesia divergens strain Rouen 1987	Kindly provided by Kirk Deitsch and Laura Kirkman (Weill Cornell Medical College)

Oligonucleotides
See supplementary table 3 for primers and synthesis products

Recombinant DNA
pBdEF1-GFP-BSD	This study
pBdEF1-Cas9-BSD-PKG-T651Q	This study
pBdEF1-Cas9-BSD-CDPK4-HA-DD-glmS	This study
pBdEF1-Cas9-BSD-CDPK5-HA-DD-glmS	This study
pBdEF1-Cas9-BSD-CDPK7-HA-DD-glmS	This study
pBdEF1-Cas9-BSD-PKAc1-HA-DD-glmS	This study
pBdEF1-Cas9-BSD-PKAc2-HA-DD-glmS	This study
pBdEF1-Cas9-BSD-ASP2-HA-DD-glmS	This study
pBdEF1-Cas9-BSD-ASP3-HA-DD-glmS	This study
pBdEF1-Cas9-BSD-DPAP1-HA-DD-glmS	This study
pBdEF1-Cas9-BSD-PLP1-HA-DD-glmS	This study
pBdEF1-Cas9-BSD-PLP3-HA-DD-glmS	This study

Software and algorithms
Prism version 9	GraphPad
ImageJ/Fiji version 2.0	https://imagej.net/software/fiji/
Zen 2	Zeiss
VEuPathDB release 46 (PiroplasmaDB/PlasmoDB/ToxoDB)	https://veupathdb.org/veupathdb/app

Other
8/18-well glass bottom imaging chamber	Ibidi	80827/81817
1.2 µM Pall Acrodisc® Sterile Syringe Filters with Supor® Membrane	Pall	4656

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There is NO Competing Interest.

VideoS1..avi
Video S1. 8-Br-cGMP induced egress
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Video S2. 8-Br-cGMP induced egress of Cytochalasin D treated parasites
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Video S3. IC and EC with calcium plus saponin
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Video S4. IC and EC without calcium plus saponin
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Video S5. ASP2 knockdown plus 8-Br-cGMP
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Video S6. ASP3 knockdown plus 8-Br-cGMP
TableS1.xlsx
Supplementary Table 1. Orthologs of known apicomplexan egress and invasion genes and putative novel egress and invasion genes in B. divergens
TableS2Transfectionsummary.docx
Supplementary Table 2. Summary of selection drugs, resistance markers and transfection methods used for B. divergens.
TableS3Primers.docx
Supplementary Table 3. Primers used in this study
SuppFigures.pdf
Supplementary Figure 1. Defining the molecular mediators of egress using small molecule inhibitors. (A) Diagram of a lytic cycle of B. divergens. One lytic cycle of B. divergens can contain multiple replication cycles and includes egress and invasion. The number of replication cycles per lytic cycle is variable. One replication cycle represents a parasite dividing to form two daughters. The percentage shown in brackets represent the approximate percentage of parasites that egress after one, two or more replication cycles. (B) Diagram of the flow cytometry-based egress assay. Egress is measured as the reduction in infected cells. (C) Model of the egress signaling pathway in B. divergens with activators shown with a green arrow, and inhibitors with a red line. (D) Percentage of parasites that egress when treated with each compound, as measured by flow cytometry. Data is normalized so that 0% is the RPMI only control (E) IC₅₀of each compound against proliferation in a 72 hours assay. (F) Induced egress was measured over time with three concentrations of 8-Br-cGMP. (G) Invasion inhibition by each compound. Isolated merozoites were pre-incubated with the inhibitor for 10 min before adding RBCs. (H). Screen for inhibitors of 8-Br-cGMP mediated induced egress. Data is normalized to 100% as a no 8-Br-cGMP control and 0% being RPMI plus 500 µM 8-Br-cGMP. (I) P. falciparum parasites egressing in the presence of phalloidin, which stains the RBC cytoskeleton. Top. A parasite that has begun egress and has permeabilized the RBC. The merozoites remain in the permeabilized RBC. Bottom. Merozoites that have completed egress. The RBC cytoskeleton has fragmented to release the merozoites. (J) Pre-treatment with E64d does not alter 8-Br-cGMP induced egress. For D-H the data is the mean ± SD of three biological experiments performed in technical triplicate. Supplementary Figure 2. The B. divergens transcriptome reveals expression of putative IMC genes and conserved apicomplexan egress and invasion genes. (A) PCA plot of the single-cell transcriptomics. (B) Expression profiles of IMC proteins fall into three clusters. (C) An outline of the approaches used to identify novel egress and invasion genes. ^54,112-115 Supplementary Figure 3. Genetic validation of CRISPR/Cas9 modified parasite lines. (A) Schematic of GFP reporter plasmid. (B) Schematic of CRISPR/Cas9 plasmid with a repair template. (C) Live cell imaging of B. divergens parasites expressing GFP. (D) The number of WT or PKG-DD-glmS parasites, with knockdown (no Shld1) or without knockdown induced for 24 h, that egress when treated with varying concentrations of 8-Br-cGMP. Data is the mean ± SD of three biological experiments performed in technical triplicate (E) Sanger sequencing of pkg in the uncloned parasite population after transfection. Positions 1-7 are the alternative bases in the homology repair template where editing is expected. (F) RFLP analysis of the uncloned parasite population after transfection with either a plasmid containing a 500 bp repair template or with a HA-DD-glmS tag flanked by two 500 bp HR regions. (G) Alignment of the CDPKs and PKG surrounding the gatekeeper residue (boxed amino acid). (H) PCR to confirm integration of the knockdown tag in each line. The amplified region spans the 3’ tag, with primers outside of the HR (primers are shown in Figure 3A). The increased size of the PCR product in the transgenic line corresponds to the correctly integrated tag. The non-clonal population was used to test CDPK5, with the majority of the population being correctly integrated. (I) The number of parasites per infected RBC over time with PKG knockdown, assessed by flow cytometry. A single representative experiment is shown of three biological repeats in technical duplicate. Supplementary Figure 4. Structure of all compounds used in this study

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Babesia divergens egress from host cells is orchestrated by essential and druggable kinases and proteases

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Abstract

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Introduction

Results

Chemical inhibition of PKG, proteases and calcium release all impair egress

Identification of putative egress, motility and invasion genes through transcriptomic analyses

PKG and CDPK4 are essential for egress and their depletion results in continued intracellular replication

The proteases ASP2 and ASP3 are required for egress and invasion

PKG, ASP2 and ASP3 are druggable targets required for egress and/or invasion

Discussion

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Methods

References

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