Daniel P. Beiting

Infection with Cryptosporidium is a leading cause of diarrheal disease and early childhood mortality. This apicomplexan parasite undergoes asexual and sexual replication within the same host, and recent studies have shown an intrinsic developmental program of obligate transition to male and female gametes and sex. While factors were identified that control male fate and development, how female gene expression is orchestrated remains largely unknown. Here, we use the Cryptosporidium Single Cell Atlas to discover an RNA-binding protein (F-RBP) as one of the earliest markers of female identity in C. parvum . Reporter parasites engineered based on this gene allowed us to calibrate transcriptional pseudotime against the real time of female development, revealing a significant window of transcriptional fate ambiguity. While F-RBP is an early transcript, the protein persists throughout female development and into the zygote. Conditional ablation of the F-RBP gene showed it to be dispensable for sex determination and early female development in vitro . However, the gene is essential in vivo, and its loss results in rapid cure. Cell biological experiments link this loss to the production of oocysts that release sporozoites incapable of host-cell invasion. F-RBP binds transcripts highly expressed in the female gamete, enriched for a sequence motif recognized by a Y-box protein family member, and forms mRNA-protein complexes in late females akin to processing or P bodies. We propose F-RBP’s essential role to be in the regulation of long-term homeostasis of maternally inherited RNA required for sporozoite infectivity. IMPORTANCE: The parasite Cryptosporidium is responsible for millions of cases of severe diarrheal disease in young children, and the infection is both a consequence of and a risk factor for chronic malnutrition. There are no vaccines available, and a single approved treatment is largely ineffective. Infection relies on ingestion of the oocyst, an environmentally stable spore, which contains an invasive form of the parasite, the sporozoite. Oocysts and sporozoites are the products of parasite sex and the fusion of female and male Cryptosporidium parasites. Here, we report the critical role of an RNA-binding protein in female mRNA homeostasis. Maternal transcripts orchestrate the complex process of sexual replication and oocyst formation. Loss of the protein results in spores that fail to initiate the next round of the life cycle, and conditional ablation using a small-molecule trigger quickly cures infected animals, demonstrating the essential role of parasite sex in continued infection.

Infection with Cryptosporidium is a leading cause of diarrheal disease and early childhood mortality. This apicomplexan parasite undergoes asexual and sexual replication within the same host and recent studies have shown an intrinsic developmental program of obligate transition to male and female gametes and sex. While factors were identified that control male fate and development, how female gene expression is orchestrated remains largely unknown. Here we use the Cryptosporidium Single Cell Atlas to discover an RNA binding protein (F-RBP) as one of the earliest markers of female identity. Reporter parasites engineered based on this gene allowed us to calibrate transcriptional pseudotime against the real time of female development revealing a significant window of transcriptional fate ambiguity. While F-RBP is an early transcript, the protein persists throughout female development and into the zygote. Conditional ablation of the F-RBP gene showed it to be dispensable for sex determination and early female development in vitro . However, the gene is essential in vivo and its loss results in rapid cure. Cell biological experiments link this loss to the production of sterile oocysts which release sporozoites incapable of host cell invasion. F-RBP binds transcripts highly expressed in the female gamete enriched for a YBOX primary sequence motif and forms mRNA protein complexes in late females akin to processing or P bodies. We propose F-RBP’s essential role to be in the regulation of long-term homeostasis of maternally inherited RNA required for sporozoite infectivity. Importance: The parasite Cryptosporidium is responsible for millions of cases of severe diarrheal disease in young children and is both a consequence of, and a risk factor for, chronic malnutrition. There are no vaccines available and a single approved treatment is largely ineffective. Infection relies on ingestion of the oocyst, an environmentally stable spore, which contains an invasive form of the parasite, the sporozoite. Oocyst and sporozoites are the product of parasite sex and the fusion of female and male Cryptosporidium parasites. Here we report the critical role of an RNA- binding protein in female mRNA homeostasis. Maternal transcripts orchestrate the complex process of sexual replication and oocyst formation. Loss of the protein results in sterile spores that fail to initiate the next round of the lifecycle, and conditional ablation using a small molecule trigger quickly cures infected animals demonstrating the essential role of parasite sex in continued infection.

Supplementary Tables 1, 2, and 3

Supplementary Figures 1-7 with legends

Fragility of regulatory T (Treg) cells manifested by the loss of neuropilin-1 (NRP1) and expression of IFNγ undermines the immune suppressive functions of Treg cells and contributes to the success of immune therapies against cancers. Intratumoral Treg cells somehow avoid fragility; however, the mechanisms by which Treg cells are protected from fragility in the tumor microenvironment are not well understood. Here, we demonstrate that the IFNAR1 chain of the type I IFN (IFN1) receptor was downregulated on intratumoral Treg cells. Downregulation of IFNAR1 mediated by p38α kinase protected Treg cells from fragility and maintained NRP1 levels, which were decreased in response to IFN1. Genetic or pharmacologic inactivation of p38α and stabilization of IFNAR1 in Treg cells induced fragility and inhibited their immune suppressive and protumorigenic activities. The inhibitor of sumoylation TAK981 (Subasumstat) upregulated IFNAR1, eliciting Treg fragility and inhibiting tumor growth in an IFNAR1-dependent manner. These findings describe a mechanism by which intratumoral Treg cells retain immunosuppressive activities and suggest therapeutic approaches for inducing Treg fragility and increasing the efficacy of immunotherapies.

The parasite Cryptosporidium is a leading cause of life-threatening diarrheal disease, and effective treatment is not available. The discovery of potent anti- Cryptosporidium activity of clofazimine, offered the opportunity to repurpose a drug already used to treat leprosy and tuberculosis. However, clofazimine failed in a human trial, which was attributed to poor bioavailability. Here, we observed differential susceptibility among parasite isolates which we exploit to map the mode of action to type II NADH dehydrogenase (NDH2) in an unbiased genetic cross. Targeted genetic ablation of NDH2 resulted in profound clofazimine resistance, and biochemical studies demonstrated NDH2 mediated electron transfer to clofazimine. Through genomic analyses we uncovered heterogeneity at the NDH2 locus for C. parvum and C. hominis and widespread carriage of a conserved attenuated allele across multiple continents. This heterogeneity allows parasites genomically linked through frequent sexual recombination to adjust to changing NDH2 requirements and predisposes Cryptosporidium to evade clofazimine treatment.

A select group of pathogens infects neurons in the brain. Prior dogma held that neurons were “defenseless” against infecting microbes, but many studies suggest that neurons can mount anti-microbial defenses. However, a knowledge gap in understanding how neurons respond in vitro and in vivo to different classes of microorganisms remains. To address this gap, we compared a transcriptional data set derived from primary neuron cultures (PNCs) infected with the neurotropic intracellular parasite Toxoplasma gondii with a data set derived from neurons injected with T. gondii protein in vivo . These curated responses were then compared to the transcriptional responses of PNCs infected with the single-stranded RNA viruses, West Nile virus or Zika virus. These analyses highlighted a conserved response to infection associated with chemokines ( Cxcl10, Ccl2 ) and cytokines (interferon signaling). However, T. gondii had diminished IFN-α signaling in vitro compared to the viral data sets and was uniquely associated with a decrease in neuron-specific genes ( Snap25 , Slc17a7 , Prkcg ). These data underscore that neurons participate in infection-induced neuroinflammation and illustrate that neurons possess both pathogen-specific and pathogen-conserved responses. IMPORTANCE: Though neurons are commonly the target of pathogens that infect the central nervous system (CNS), few data sets assess the neuronal response to infection. This paucity of data is likely because neurons are perceived to have diminished immune capabilities. However, to understand the role of neurons in neuroinflammation and their immune capabilities, their responses must be investigated. Here, we analyzed publicly accessible, neuron-specific data sets to compare neuron responses to a eukaryotic pathogen vs two Orthoflaviviruses. A better understanding of neuron responses to different infections will allow us to develop methods for inhibiting pathways that lead to neuron dysfunction, enhancing those that limit pathogen survival, and mitigating infection-induced damage to the CNS.

A select group of pathogens infect neurons in the brain. Prior dogma held that neurons were “defenseless” against infecting microbes, but many studies suggest that neurons can mount anti-microbial defenses. However, a knowledge gap in understanding how neurons respond in vitro and in vivo to different classes of micro-organisms remains. To address this gap, we compared a transcriptional dataset derived from primary neuron cultures (PNCs) infected with the neurotropic intracellular parasite Toxoplasma gondii with a dataset derived from neurons injected with T. gondii protein in vivo . These curated responses were then compared to the transcriptional responses of PNCs infected with the single stranded RNA viruses West Nile Virus (WNV) or Zika Virus (ZKV). These analyses highlighted a conserved response to infection associated with chemokines ( Cxcl10, Ccl2 ) and cytokines (interferon signaling). However, T. gondii had diminished IFN-α signaling in vitro compared to the viral datasets and was uniquely associated with a decrease in neuron-specific genes ( Snap25 , Slc17a7 , Prkcg ). These data underscore that neurons participate in infection-induced neuroinflammation and illustrate that neurons possess both pathogen-specific and pathogen-conserved responses. Importance: Though neurons are commonly the target of pathogens that infect the CNS, few datasets assess the neuronal response to infection. This paucity of data is likely because neurons are perceived to have diminished immune capabilities. However, to understand the role of neurons in neuroinflammation and their immune capabilities, their responses must be investigated. Here we analyzed publicly accessible, neuron-specific datasets to compare neuron responses to a eukaryotic pathogen versus two Orthoflaviviruses. A better understanding of neuron responses to different infections will allow us to develop methods for inhibiting pathways that lead to neuron dysfunction, enhancing those that limit pathogen survival, and mitigating infection-induced damage to the CNS.