The Myosin Family of Mechanoenzymes: From Mechanisms to Therapeutic Approaches.

Myosins are among the most fascinating enzymes in biology. As extremely allosteric chemomechanical molecular machines, myosins are involved in myriad pivotal cellular functions and are frequently sites of mutations leading to disease phenotypes. Human ß-cardiac myosin has proved to be an excellent target for small-molecule therapeutics for heart muscle diseases, and, as we describe here, other myosin family members are likely to be potentially unique targets for treating other diseases as well. The first part of this review focuses on how myosins convert the chemical energy of ATP hydrolysis into mechanical movement, followed by a description of existing therapeutic approaches to target human ß-cardiac myosin. The next section focuses on the possibility of targeting nonmuscle members of the human myosin family for several diseases. We end the review by describing the roles of myosin in parasites and the therapeutic potential of targeting them to block parasitic invasion of their hosts.

A Single Transcription Factor Drives Toxoplasma gondii Differentiation.

Microbes that cause persistent infections (e.g., herpes viruses) do so by switching from fast-growing lytic states to slow-growing latent states. Waldman et al. have identified a single transcription factor that governs the switch between the lytic and latent forms of Toxoplasma gondii, a parasite that causes a persistent brain infection.

Identification of a Master Regulator of Differentiation in Toxoplasma.

Toxoplasma gondii chronically infects a quarter of the world's population, and its recrudescence can cause life-threatening disease in immunocompromised individuals and recurrent ocular lesions in the immunocompetent. Acute-stage tachyzoites differentiate into chronic-stage bradyzoites, which form intracellular cysts resistant to immune clearance and existing therapies. The molecular basis of this differentiation is unknown, despite being efficiently triggered by stresses in culture. Through Cas9-mediated screening and single-cell profiling, we identify a Myb-like transcription factor (BFD1) necessary for differentiation in cell culture and in mice. BFD1 accumulates during stress and its synthetic expression is sufficient to drive differentiation. Consistent with its function as a transcription factor, BFD1 binds the promoters of many stage-specific genes and represents a counterpoint to the ApiAP2 factors that dominate our current view of parasite gene regulation. BFD1 provides a genetic switch to study and control Toxoplasma differentiation and will inform prevention and treatment of chronic infections.

PD-L1-PD-1 interactions limit effector regulatory T cell populations at homeostasis and during infection.

Phenotypic and transcriptional profiling of regulatory T (Treg) cells at homeostasis reveals that T cell receptor activation promotes Treg cells with an effector phenotype (eTreg) characterized by the production of interleukin-10 and expression of the inhibitory receptor PD-1. At homeostasis, blockade of the PD-1 pathway results in enhanced eTreg cell activity, whereas during infection with Toxoplasma gondii, early interferon-γ upregulates myeloid cell expression of PD-L1 associated with reduced Treg cell populations. In infected mice, blockade of PD-L1, complete deletion of PD-1 or lineage-specific deletion of PD-1 in Treg cells prevents loss of eTreg cells. These interventions resulted in a reduced ratio of pathogen-specific effector T cells: eTreg cells and increased levels of interleukin-10 that mitigated the development of immunopathology, but which could compromise parasite control. Thus, eTreg cell expression of PD-1 acts as a sensor to rapidly tune the pool of eTreg cells at homeostasis and during inflammatory processes.

A CTCF-binding site in the Mdm1-Il22-Ifng locus shapes cytokine expression profiles and plays a critical role in early Th1 cell fate specification.

Cytokine expression during T cell differentiation is a highly regulated process that involves long-range promoter-enhancer and CTCF-CTCF contacts at cytokine loci. Here, we investigated the impact of dynamic chromatin loop formation within the topologically associating domain (TAD) in regulating the expression of interferon gamma (IFN-γ) and interleukin-22 (IL-22); these cytokine loci are closely located in the genome and are associated with complex enhancer landscapes, which are selectively active in type 1 and type 3 lymphocytes. In situ Hi-C analyses revealed inducible TADs that insulated Ifng and Il22 enhancers during Th1 cell differentiation. Targeted deletion of a 17 bp boundary motif of these TADs imbalanced Th1- and Th17-associated immunity, both in vitro and in vivo, upon Toxoplasma gondii infection. In contrast, this boundary element was dispensable for cytokine regulation in natural killer cells. Our findings suggest that precise cytokine regulation relies on lineage- and developmental stage-specific interactions of 3D chromatin architectures and enhancer landscapes.

Functional Profiling of a Plasmodium Genome Reveals an Abundance of Essential Genes.

The genomes of malaria parasites contain many genes of unknown function. To assist drug development through the identification of essential genes and pathways, we have measured competitive growth rates in mice of 2,578 barcoded Plasmodium berghei knockout mutants, representing >50% of the genome, and created a phenotype database. At a single stage of its complex life cycle, P. berghei requires two-thirds of genes for optimal growth, the highest proportion reported from any organism and a probable consequence of functional optimization necessitated by genomic reductions during the evolution of parasitism. In contrast, extreme functional redundancy has evolved among expanded gene families operating at the parasite-host interface. The level of genetic redundancy in a single-celled organism may thus reflect the degree of environmental variation it experiences. In the case of Plasmodium parasites, this helps rationalize both the relative successes of drugs and the greater difficulty of making an effective vaccine.

Alarmin S100A11 initiates a chemokine response to the human pathogen Toxoplasma gondii.

Toxoplasma gondii is a common protozoan parasite that infects up to one third of the world's population. Notably, very little is known about innate immune sensing mechanisms for this obligate intracellular parasite by human cells. Here, by applying an unbiased biochemical screening approach, we show that human monocytes recognized the presence of T. gondii infection by detecting the alarmin S100A11 protein, which is released from parasite-infected cells via caspase-1-dependent mechanisms. S100A11 induced a potent chemokine response to T. gondii by engaging its receptor RAGE, and regulated monocyte recruitment in vivo by inducing expression of the chemokine CCL2. Our experiments reveal a sensing system for T. gondii by human cells that is based on the detection of infection-mediated release of S100A11 and RAGE-dependent induction of CCL2, a crucial chemokine required for host resistance to the parasite.

A Genome-wide CRISPR Screen in Toxoplasma Identifies Essential Apicomplexan Genes.

Apicomplexan parasites are leading causes of human and livestock diseases such as malaria and toxoplasmosis, yet most of their genes remain uncharacterized. Here, we present the first genome-wide genetic screen of an apicomplexan. We adapted CRISPR/Cas9 to assess the contribution of each gene from the parasite Toxoplasma gondii during infection of human fibroblasts. Our analysis defines ∼200 previously uncharacterized, fitness-conferring genes unique to the phylum, from which 16 were investigated, revealing essential functions during infection of human cells. Secondary screens identify as an invasion factor the claudin-like apicomplexan microneme protein (CLAMP), which resembles mammalian tight-junction proteins and localizes to secretory organelles, making it critical to the initiation of infection. CLAMP is present throughout sequenced apicomplexan genomes and is essential during the asexual stages of the malaria parasite Plasmodium falciparum. These results provide broad-based functional information on T. gondii genes and will facilitate future approaches to expand the horizon of antiparasitic interventions.

In vitro production of cat-restricted Toxoplasma pre-sexual stages.

Sexual reproduction of Toxoplasma gondii, confined to the felid gut, remains largely uncharted owing to ethical concerns regarding the use of cats as model organisms. Chromatin modifiers dictate the developmental fate of the parasite during its multistage life cycle, but their targeting to stage-specific cistromes is poorly described1,2. Here we found that the transcription factors AP2XII-1 and AP2XI-2 operate during the tachyzoite stage, a hallmark of acute toxoplasmosis, to silence genes necessary for merozoites, a developmental stage critical for subsequent sexual commitment and transmission to the next host, including humans. Their conditional and simultaneous depletion leads to a marked change in the transcriptional program, promoting a full transition from tachyzoites to merozoites. These in vitro-cultured pre-gametes have unique protein markers and undergo typical asexual endopolygenic division cycles. In tachyzoites, AP2XII-1 and AP2XI-2 bind DNA as heterodimers at merozoite promoters and recruit MORC and HDAC3 (ref. 1), thereby limiting chromatin accessibility and transcription. Consequently, the commitment to merogony stems from a profound epigenetic rewiring orchestrated by AP2XII-1 and AP2XI-2. Successful production of merozoites in vitro paves the way for future studies on Toxoplasma sexual development without the need for cat infections and holds promise for the development of therapies to prevent parasite transmission.

SPOTting stress at the mitochondrial outer membrane.

Li et al. (2022) discover that Toxoplasma infection triggers remodeling of the mitochondrial outer membrane through generation of a mitochondrial subdomain termed "structure positive for outer mitochondrial membrane" (SPOT).

Essential role for GABARAP autophagy proteins in interferon-inducible GTPase-mediated host defense.

Mammalian autophagy-related 8 (Atg8) homologs consist of LC3 proteins and GABARAPs, all of which are known to be involved in canonical autophagy. In contrast, the roles of Atg8 homologs in noncanonical autophagic processes are not fully understood. Here we show a unique role of GABARAPs, in particular gamma-aminobutyric acid (GABA)-A-receptor-associated protein-like 2 (Gabarapl2; also known as Gate-16), in interferon-γ (IFN-γ)-mediated antimicrobial responses. Cells that lacked GABARAPs but not LC3 proteins and mice that lacked Gate-16 alone were defective in the IFN-γ-induced clearance of vacuolar pathogens such as Toxoplasma. Gate-16 but not LC3b specifically associated with the small GTPase ADP-ribosylation factor 1 (Arf1) to mediate uniform distribution of interferon-inducible GTPases. The lack of GABARAPs reduced Arf1 activation, which led to formation of interferon-inducible GTPase-containing aggregates and hampered recruitment of interferon-inducible GTPases to vacuolar pathogens. Thus, GABARAPs are uniquely required for antimicrobial host defense through cytosolic distribution of interferon-inducible GTPases.

Rapid Enhancer Remodeling and Transcription Factor Repurposing Enable High Magnitude Gene Induction upon Acute Activation of NK Cells.

Innate immune responses rely on rapid and precise gene regulation mediated by accessibility of regulatory regions to transcription factors (TFs). In natural killer (NK) cells and other innate lymphoid cells, competent enhancers are primed during lineage acquisition, and formation of de novo enhancers characterizes the acquisition of innate memory in activated NK cells and macrophages. Here, we investigated how primed and de novo enhancers coordinate to facilitate high-magnitude gene induction during acute activation. Epigenomic and transcriptomic analyses of regions near highly induced genes (HIGs) in NK cells both in vitro and in a model of Toxoplasma gondii infection revealed de novo chromatin accessibility and enhancer remodeling controlled by signal-regulated TFs STATs. Acute NK cell activation redeployed the lineage-determining TF T-bet to de novo enhancers, independent of DNA-sequence-specific motif recognition. Thus, acute stimulation reshapes enhancer function through the combinatorial usage and repurposing of both lineage-determining and signal-regulated TFs to ensure an effective response.

Differentiation of type 1 ILCs from a common progenitor to all helper-like innate lymphoid cell lineages.

Innate lymphoid cells (ILCs) are a recently recognized group of lymphocytes that have important functions in protecting epithelial barriers against infections and in maintaining organ homeostasis. ILCs have been categorized into three distinct groups, transcriptional circuitry and effector functions of which strikingly resemble the various T helper cell subsets. Here, we identify a common, Id2-expressing progenitor to all interleukin 7 receptor-expressing, "helper-like" ILC lineages, the CHILP. Interestingly, the CHILP differentiated into ILC2 and ILC3 lineages, but not into conventional natural killer (cNK) cells that have been considered an ILC1 subset. Instead, the CHILP gave rise to a peculiar NKp46(+) IL-7Rα(+) ILC lineage that required T-bet for specification and was distinct of cNK cells or other ILC lineages. Such ILC1s coproduced high levels of IFN-γ and TNF and protected against infections with the intracellular parasite Toxoplasma gondii. Our data significantly advance our understanding of ILC differentiation and presents evidence for a new ILC lineage that protects barrier surfaces against intracellular infections.

The Crk4-Cyc4 complex regulates G2/M transition in Toxoplasma gondii.

A versatile division of apicomplexan parasites and a dearth of conserved regulators have hindered the progress of apicomplexan cell cycle studies. While most apicomplexans divide in a multinuclear fashion, Toxoplasma gondii tachyzoites divide in the traditional binary mode. We previously identified five Toxoplasma CDK-related kinases (Crk). Here, we investigated TgCrk4 and its cyclin partner TgCyc4. We demonstrated that TgCrk4 regulates conventional G2 phase processes, such as repression of chromosome rereplication and centrosome reduplication, and acts upstream of the spindle assembly checkpoint. The spatial TgCyc4 dynamics supported the TgCrk4-TgCyc4 complex role in the coordination of chromosome and centrosome cycles. We also identified a dominant TgCrk4-TgCyc4 complex interactor, TgiRD1 protein, related to DNA replication licensing factor CDT1 but played no role in licensing DNA replication in the G1 phase. Our results showed that TgiRD1 also plays a role in controlling chromosome and centrosome reduplication. Global phosphoproteome analyses identified TgCrk4 substrates, including TgORC4, TgCdc20, TgGCP2, and TgPP2ACA. Importantly, the phylogenetic and structural studies suggest the Crk4-Cyc4 complex is limited to a minor group of the binary dividing apicomplexans.

Interferon-induced guanylate-binding proteins in inflammasome activation and host defense.

Traditional views of the inflammasome highlight the assembly of pre-existing core components shortly after infection or tissue damage. Emerging work, however, suggests that the inflammasome machinery is also subject to 'tunable' or inducible signals that might accelerate its autocatalytic properties and dictate where inflammasome assembly takes place in the cell. Many of these signals operate downstream of interferon receptors to elicit inflammasome regulators, including a new family of interferon-induced GTPases called 'guanylate-binding proteins' (GBPs). Here we investigate the critical roles of interferon-induced GBPs in directing inflammasome subtype-specific responses and their consequences for cell-autonomous immunity to a wide variety of microbial pathogens. We discuss emerging mechanisms of action and the potential effect of these GBPs on predisposition to sepsis and other infectious or inflammatory diseases.

Id2 reinforces TH1 differentiation and inhibits E2A to repress TFH differentiation.

The differentiation of helper T cells into effector subsets is critical to host protection. Transcription factors of the E-protein and Id families are important arbiters of T cell development, but their role in the differentiation of the TH1 and TFH subsets of helper T cells is not well understood. Here, TH1 cells showed more robust Id2 expression than that of TFH cells, and depletion of Id2 via RNA-mediated interference increased the frequency of TFH cells. Furthermore, TH1 differentiation was blocked by Id2 deficiency, which led to E-protein-dependent accumulation of effector cells with mixed characteristics during viral infection and severely impaired the generation of TH1 cells following infection with Toxoplasma gondii. The TFH cell-defining transcriptional repressor Bcl6 bound the Id2 locus, which provides a mechanism for the bimodal Id2 expression and reciprocal development of TH1 cells and TFH cells.

Unique Properties of Apicomplexan Mitochondria.

Apicomplexan parasites constitute more than 6,000 species infecting a wide range of hosts. These include important pathogens such as those causing malaria and toxoplasmosis. Their evolutionary emergence coincided with the dawn of animals. Mitochondrial genomes of apicomplexan parasites have undergone dramatic reduction in their coding capacity, with genes for only three proteins and ribosomal RNA genes present in scrambled fragments originating from both strands. Different branches of the apicomplexans have undergone rearrangements of these genes, with Toxoplasma having massive variations in gene arrangements spread over multiple copies. The vast evolutionary distance between the parasite and the host mitochondria has been exploited for the development of antiparasitic drugs, especially those used to treat malaria, wherein inhibition of the parasite mitochondrial respiratory chain is selectively targeted with little toxicity to the host mitochondria. We describe additional unique characteristics of the parasite mitochondria that are being investigated and provide greater insights into these deep-branching eukaryotic pathogens.

A conserved complex of microneme proteins mediates rhoptry discharge in Toxoplasma.

Apicomplexan parasites discharge specialized organelles called rhoptries upon host cell contact to mediate invasion. The events that drive rhoptry discharge are poorly understood, yet essential to sustain the apicomplexan parasitic life cycle. Rhoptry discharge appears to depend on proteins secreted from another set of organelles called micronemes, which vary in function from allowing host cell binding to facilitation of gliding motility. Here we examine the function of the microneme protein CLAMP, which we previously found to be necessary for Toxoplasma gondii host cell invasion, and demonstrate its essential role in rhoptry discharge. CLAMP forms a distinct complex with two other microneme proteins, the invasion-associated SPATR, and a previously uncharacterized protein we name CLAMP-linked invasion protein (CLIP). CLAMP deficiency does not impact parasite adhesion or microneme protein secretion; however, knockdown of any member of the CLAMP complex affects rhoptry discharge. Phylogenetic analysis suggests orthologs of the essential complex components, CLAMP and CLIP, are ubiquitous across apicomplexans. SPATR appears to act as an accessory factor in Toxoplasma, but despite incomplete conservation is also essential for invasion during Plasmodium falciparum blood stages. Together, our results reveal a new protein complex that mediates rhoptry discharge following host-cell contact.

Both maternal IFNγ exposure and acute prenatal infection with Toxoplasma gondii activate fetal hematopoietic stem cells.

Infection directly influences adult hematopoietic stem cell (HSC) function and differentiation, but the fetal hematopoietic response to infection during pregnancy is not well-studied. Here, we investigated the fetal hematopoietic response to maternal infection with Toxoplasma gondii (T. gondii), an intracellular parasite that elicits Type II IFNγ-mediated maternal immunity. While it is known that maternal infection without direct pathogen transmission can affect fetal immune development, the effects of maternal IFNγ on developing HSCs and the signals that mediate these interactions have not been investigated. Our investigation reveals that the fetal HSCs respond to T. gondii infection with virulence-dependent changes in proliferation, self-renewal potential, and lineage output. Furthermore, maternal IFNγ crosses the fetal-maternal interface, where it is perceived by fetal HSCs. By comparing the effects of maternal IFNγ injection with maternal T. gondii infection, we reveal that the effects of IFNγ treatment mimic some aspects of the fetal HSC response to infection. Moreover, our findings illuminate that the fetal HSC response to prenatal infection is distinct from the adult HSC response to IFNγ-induced inflammation. Altogether, our data disentangle the role of infection-induced inflammatory cytokines in driving the expansion of downstream hematopoietic progenitors.

Epigenetic Reprogramming in Host-Parasite Coevolution: The Toxoplasma Paradigm.

Like many intracellular pathogens, the protozoan parasite Toxoplasma gondii has evolved sophisticated mechanisms to promote its transmission and persistence in a variety of hosts by injecting effector proteins that manipulate many processes in the cells it invades. Specifically, the parasite diverts host epigenetic modulators and modifiers from their native functions to rewire host gene expression to counteract the innate immune response and to limit its strength. The arms race between the parasite and its hosts has led to accelerated adaptive evolution of effector proteins and the unconventional secretion routes they use. This review provides an up-to-date overview of how T. gondii effectors, through the evolution of intrinsically disordered domains, the formation of supramolecular complexes, and the use of molecular mimicry, target host transcription factors that act as coordinating nodes, as well as chromatin-modifying enzymes, to control the fate of infected cells and ultimately the outcome of infection.