Direct type I interferon signaling in hepatocytes controls malaria.

Malaria is a devastating disease impacting over half of the world's population. Plasmodium parasites that cause malaria undergo obligatory development and replication in hepatocytes before infecting red blood cells and initiating clinical disease. While type I interferons (IFNs) are known to facilitate innate immune control to Plasmodium in the liver, how they do so has remained unresolved, precluding the manipulation of such responses to combat malaria. Utilizing transcriptomics, infection studies, and a transgenic Plasmodium strain that exports and traffics Cre recombinase, we show that direct type I IFN signaling in Plasmodium-infected hepatocytes is necessary to control malaria. We also show that the majority of infected hepatocytes naturally eliminate Plasmodium infection, revealing the potential existence of anti-malarial cell-autonomous immune responses in such hepatocytes. These discoveries challenge the existing paradigms in Plasmodium immunobiology and are expected to inspire anti-malarial drugs and vaccine strategies.

Transcriptomics and proteomics reveal two waves of translational repression during the maturation of malaria parasite sporozoites.

Plasmodium sporozoites are transmitted from infected mosquitoes to mammals, and must navigate the host skin and vasculature to infect the liver. This journey requires distinct proteomes. Here, we report the dynamic transcriptomes and proteomes of both oocyst sporozoites and salivary gland sporozoites in both rodent-infectious Plasmodium yoelii parasites and human-infectious Plasmodium falciparum parasites. The data robustly define mRNAs and proteins that are upregulated in oocyst sporozoites (UOS) or upregulated in infectious sporozoites (UIS) within the salivary glands, including many that are essential for sporozoite functions in the vector and host. Moreover, we find that malaria parasites use two overlapping, extensive, and independent programs of translational repression across sporozoite maturation to temporally regulate protein expression. Together with gene-specific validation experiments, these data indicate that two waves of translational repression are implemented and relieved at different times during sporozoite maturation, migration and infection, thus promoting their successful development and vector-to-host transition.

Ribozyme-mediated, multiplex CRISPR gene editing and CRISPR interference (CRISPRi) in rodent-infectious Plasmodium yoelii.

Malaria remains a major global health issue, affecting millions and killing hundreds of thousands of people annually. Efforts to break the transmission cycle of the causal Plasmodium parasite, and to cure those that are afflicted, rely upon functional characterization of genes essential to the parasite's growth and development. These studies are often based upon manipulations of the parasite genome to disrupt or modify a gene of interest to understand its importance and function. However, these approaches can be limited by the availability of selectable markers and the time required to generate transgenic parasites. Moreover, there also is a risk of disrupting native gene regulatory elements with the introduction of exogenous sequences. To address these limitations, we have developed CRISPR-RGR, a Streptococcus pyogenes (Sp)Cas9-based gene editing system for Plasmodium that utilizes a ribozyme-guide-ribozyme (RGR) single guide RNA (sgRNA) expression strategy with RNA polymerase II promoters. Using rodent-infectious Plasmodium yoelii, we demonstrate that both gene disruptions and coding sequence insertions are efficiently generated, producing marker-free parasites with homology arms as short as 80-100 bp. Additionally, we find that the common practice of using one sgRNA can produce both unintended plasmid integration and desired locus replacement editing events, whereas the use of two sgRNAs results in only locus replacement editing. Lastly, we show that CRISPR-RGR can be used for CRISPR interference (CRISPRi) by binding catalytically dead SpCas9 (dSpCas9) to the region upstream of a gene of interest, resulting in a position-dependent, but strand-independent reduction in gene expression. This robust and flexible system facilitates efficient genetic characterizations of rodent-infectious Plasmodium species.

Plasmodium male gametocyte development and transmission are critically regulated by the two putative deadenylases of the CAF1/CCR4/NOT complex.

With relatively few known specific transcription factors to control the abundance of specific mRNAs, Plasmodium parasites may rely more on the regulation of transcript stability and turnover to provide sufficient gene regulation. Plasmodium transmission stages impose translational repression on specific transcripts in part to accomplish this. However, few proteins are known to participate in this process, and those that are characterized primarily affect female gametocytes. We have identified and characterized Plasmodium yoelii (Py) CCR4-1, a putative deadenylase, which plays a role in the development and activation of male gametocytes, regulates the abundance of specific mRNAs in gametocytes, and ultimately increases the efficiency of host-to-vector transmission. We find that when pyccr4-1 is deleted or its protein made catalytically inactive, there is a loss in the initial coordination of male gametocyte maturation and a reduction of parasite infectivity of the mosquito. Expression of only the N-terminal CAF1 domain of the essential CAF1 deadenylase leads to a similar phenotype. Comparative RNA-seq revealed that PyCCR4-1 affects transcripts important for transmission-related functions that are associated with male or female gametocytes, some of which directly associate with the immunoprecipitated complex. Finally, circular RT-PCR of one of the bound, dysregulated transcripts showed that deletion of the pyccr4-1 gene does not result in gross changes to its UTR or poly(A) tail length. We conclude that the two putative deadenylases of the CAF1/CCR4/NOT complex play critical and intertwined roles in gametocyte maturation and transmission.

Characterization of the Neutralizing Antibody Response in a Case of Genetically Linked HIV Superinfection.

This report describes the identification of a genetically confirmed linked heterosexual human immunodeficiency virus (HIV) superinfection (HIV-SI) in a woman with chronic HIV infection who acquired a second strain of the virus from her husband. Serum neutralizing antibody (NAb) responses against their homologous and heterologous viruses, including the superinfecting strain, in the woman and her husband were examined before and after onset of HIV-SI. The woman displayed a moderately potent and broad anti-HIV NAb response prior to superinfection but did not possess NAb activity against the superinfecting strain. This case highlights the unique potential of linked HIV-SI studies to examine natural protection from HIV infection.

Gemin4 is an essential gene in mice, and its overexpression in human cells causes relocalization of the SMN complex to the nucleoplasm.

Gemin4 is a member of the Survival Motor Neuron (SMN) protein complex, which is responsible for the assembly and maturation of Sm-class small nuclear ribonucleoproteins (snRNPs). In metazoa, Sm snRNPs are assembled in the cytoplasm and subsequently imported into the nucleus. We previously showed that the SMN complex is required for snRNP import in vitro, although it remains unclear which specific components direct this process. Here, we report that Gemin4 overexpression drives SMN and the other Gemin proteins from the cytoplasm into the nucleus. Moreover, it disrupts the subnuclear localization of the Cajal body marker protein, coilin, in a dose-dependent manner. We identified three putative nuclear localization signal (NLS) motifs within Gemin4, one of which is necessary and sufficient to direct nuclear import. Overexpression of Gemin4 constructs lacking this NLS sequestered Gemin3 and, to a lesser extent Gemin2, in the cytoplasm but had little effect on the nuclear accumulation of SMN. We also investigated the effects of Gemin4 depletion in the laboratory mouse, Mus musculusGemin4 null mice die early in embryonic development, demonstrating that Gemin4 is an essential mammalian protein. When crossed onto a severe SMA mutant background, heterozygous loss of Gemin4 failed to modify the early postnatal mortality phenotype of SMA type I (Smn-/-;SMN2+/+ ) mice. We conclude that Gemin4 plays an essential role in mammalian snRNP biogenesis, and may facilitate import of the SMN complex (or subunits thereof) into the nucleus.

ALBA4 modulates its stage-specific interactions and specific mRNA fates during Plasmodium yoelii growth and transmission.

Transmission of the malaria parasite occurs in an unpredictable moment, when a mosquito takes a blood meal. Plasmodium has therefore evolved strategies to prepare for transmission, including translationally repressing and protecting mRNAs needed to establish the infection. However, mechanisms underlying these critical controls are not well understood, including whether Plasmodium changes its translationally repressive complexes and mRNA targets in different stages. Efforts to understand this have been stymied by severe technical limitations due to substantial mosquito contamination of samples. Here using P. yoelii, for the first time we provide a proteomic comparison of a protein complex across asexual blood, sexual and sporozoite stages, along with a transcriptomic comparison of the mRNAs that are affected in these stages. We find that the Apicomplexan-specific ALBA4 RNA-binding protein acts to regulate development of the parasite's transmission stages, and that ALBA4 associates with both stage-specific and stage-independent partners to produce opposing mRNA fates. These efforts expand our understanding and ability to interrogate both sexual and sporozoite transmission stages and the molecular preparations they evolved to perpetuate their infectious cycle.

Evaluation of postpartum HIV superinfection and mother-to-child transmission.

OBJECTIVE: This study examined HIV superinfection in HIV-infected women postpartum, and its association with mother-to-child transmission (MTCT). DESIGN: Plasma samples were obtained from HIV-infected women who transmitted HIV to their infants after 6 weeks of age (transmitters, n = 91) and HIV-infected women who did not transmit HIV to their infants (nontransmitters, n = 91). These women were originally enrolled in a randomized trial for prevention of MTCT of HIV in Malawi (Post-Exposure Prophylaxis of Infants trial in Malawi). METHODS: Two HIV genomic regions (p24 and gp41) were analyzed by next-generation sequencing for HIV superinfection. HIV superinfection was established if the follow-up sample contained a new, phylogenetically distinct viral population. HIV superinfection and transmission risk were examined by multiple logistic regression, adjusted for Post-Exposure Prophylaxis of Infants study arm, baseline viral load, baseline CD4 cell count, time to resumption of sex, and breastfeeding duration. RESULTS: Transmitters had lower baseline CD4 cell counts (P = 0.001) and higher viral loads (P < 0.0001) compared with nontransmitters. There were five cases of superinfection among transmitters (rate of superinfection = 4.7/100 person-years) compared with five cases among the nontransmitters (rate of superinfection = 4.4/100 person-years; P = 0.78). HIV superinfection was not associated with increased risk of postnatal MTCT of HIV after controlling for maternal age, baseline viral load, and CD4 cell count (adjusted odds ratio = 2.32, P = 0.30). Longer breastfeeding duration was independently associated with a lower risk of HIV superinfection after controlling for study arm and baseline viral load (P = 0.05). CONCLUSION: There was a significant level of HIV superinfection in women postpartum, but this was not associated with an increased risk of MTCT via breastfeeding.

Reversible epigenetic histone modifications and Bdnf expression in neurons with aging and from a mouse model of Alzheimer's disease.

With aging and Alzheimer's disease (AD), there is an increased sensitivity to stress along with declines in the memory-associated neurotrophin brain-derived neurotrophic factor in AD. We have replicated this aging phenotype in cultured neurons from aged mice despite being grown in the same environmental conditions as young neurons. This led us to hypothesize that age-related differences in epigenetic acetylation and methylation of histones are associated with age-related gene regulation. We cultured hippocampal/cortical neurons from the 3xTg-AD mouse model and from non-transgenic mice to quantify single cell acetylation and methylation levels across the life span. In non-transgenic neurons, H3 acetylation was unchanged with age, while H4 acetylation decreased with age of the donor. Compared to non-transgenic neurons, 3xTg-AD neurons had higher levels of H3 and H4 acetylation beginning at 4 months of age. In contrast to non-transgenic neurons, 3xTg-AD neurons increased acetylation with age; 3xTg-AD neurons also responded differently to inhibition of histone deacetylases at an early age. Importantly, treatment of non-transgenic neurons with the AD peptide Aß also elevated levels of acetylation. We also examined the repressive function of histone H3 lysine 9 (H3K9) methylation. H3K9 methylation increased with age in non-transgenic neurons, which was amplified further in 3xTg-AD neurons. The dominant effect of higher H3K9 methylation was supported by lower Bdnf gene expression in non-transgenic and 3xTg-AD mice. These data show that the epigenetic states of non-transgenic and 3xTg-AD brain neurons are profoundly different and reversible, beginning at 4 months of age when the first memory deficits are reported.

An IGF1/insulin receptor substrate-1 pathway stimulates a mitotic kinase (cdk1) in the uterine epithelium during the proliferative response to estradiol.

Estrogens are potent mitogens for some target organs, such as the uterus, and cancers that develop in this organ might be linked to the proliferative action of these hormones. However, the mechanism by which estrogens influence the cell cycle machinery is not known. We found that a null mutation for the insulin receptor substrate (IRS)-1, a docking protein that is important for IGF1 signaling, compromised hormone-induced mitosis in the uterine epithelium; BrdU incorporation was not affected. This selective effect on mitosis was associated with a reduction in uterine cyclin B-associated kinase activity; cyclin A-associated kinase activity was not changed. The null mutation also reduced the extent of hormone-induced phosphorylation of endogenous uterine histone H1, as determined with phospho-specific antiserum. Uterine epithelial cyclin dependent kinase (cdk)1 was induced in response to hormone, but the level of the kinase protein, as determined by immunoblotting, was noticeably less in the irs1 null mutant than that in the wild-type (WT) mouse, especially around the time of peak mitosis (24 h). Since IRS-1 binds/activates phosphatidylinositol 3-kinase (PI3K), the absence of this docking protein could impair signaling of a known pathway downstream of AKT that stimulates translation of cell cycle components. Indeed, we found that phosphorylation of uterine AKT (Ser473) in irs1 null mutants was less than that in WTs following treatment. Based on earlier studies, it is also possible that an IGF1/IRS-1/PI3K/AKT pathway regulates posttranslational changes in cdk1. This model may provide insights as to how a growth factor pathway can mediate hormone action on cell proliferation.

Reduced viability, fertility and fecundity in mice lacking the cajal body marker protein, coilin.

BACKGROUND: Coilin is the signature protein of the Cajal body, a conserved nuclear organelle involved in multiple aspects of small ribonucleoprotein (RNP) biogenesis. Coilin is required for Cajal body homeostasis in both plants and animals. Mice lacking coilin are viable when the mutation is crossed to an outbred strain but only partially viable when crossed to inbred lines. METHODOLOGY/PRINCIPAL FINDINGS: In order to clarify this issue, we backcrossed the coilin deletion onto the C57BL6/J background for ten generations and then investigated the consequences of coilin removal on overall viability and reproductive success. We conclude that semi-lethal phenotype observed in mixed-background crosses is due to loss of the Coilin gene (or a very tightly-linked locus). Interestingly, coilin knockout embryos die relatively late in gestation, between E13.5 and birth. We show that the maternal contribution of coilin is not important for organismal viability. Importantly, coilin knockout mice display significant fertility and fecundity defects. Mutant males that escape the embryonic lethality display reduced testis size, however, both males and females contribute to the observed reduction in reproductive fitness. CONCLUSIONS/SIGNIFICANCE: The evolutionary conservation of coilin from plants to animals suggests that the protein plays an important role, perhaps coordinating the activities of various RNA-processing machineries. Our observations are consistent with the idea that coilin functions to ensure robust organismal development, especially during periods of rapid growth.

SMN complex localizes to the sarcomeric Z-disc and is a proteolytic target of calpain.

Spinal muscular atrophy (SMA) is a recessive neuromuscular disease caused by mutations in the human survival motor neuron 1 (SMN1) gene. The human SMN protein is part of a large macromolecular complex involved in the biogenesis of small ribonucleoproteins. Previously, we showed that SMN is a sarcomeric protein in flies and mice. In this report, we show that the entire mouse Smn complex localizes to the sarcomeric Z-disc. Smn colocalizes with alpha-actinin, a Z-disc marker protein, in both skeletal and cardiac myofibrils. Furthermore, this localization is both calcium- and calpain-dependent. Calpains are known to release proteins from various regions of the sarcomere as a part of the normal functioning of the muscle; however, this removal can be either direct or indirect. Using mammalian cell lysates, purified native SMN complexes, as well as recombinant SMN protein, we show that SMN is a direct target of calpain cleavage. Finally, myofibers from a mouse model of severe SMA, but not controls, display morphological defects that are consistent with a Z-disc deficiency. These results support the view that the SMN complex performs a muscle-specific function at the Z-discs.

A Drosophila melanogaster model of spinal muscular atrophy reveals a function for SMN in striated muscle.

Mutations in human survival motor neurons 1 (SMN1) cause spinal muscular atrophy (SMA) and are associated with defects in assembly of small nuclear ribonucleoproteins (snRNPs) in vitro. However, the etiological link between snRNPs and SMA is unclear. We have developed a Drosophila melanogaster system to model SMA in vivo. Larval-lethal Smn-null mutations show no detectable snRNP reduction, making it unlikely that these animals die from global snRNP deprivation. Hypomorphic mutations in Smn reduce dSMN protein levels in the adult thorax, causing flightlessness and acute muscular atrophy. Mutant flight muscle motoneurons display pronounced axon routing and arborization defects. Moreover, Smn mutant myofibers fail to form thin filaments and phenocopy null mutations in Act88F, which is the flight muscle-specific actin isoform. In wild-type muscles, dSMN colocalizes with sarcomeric actin and forms a complex with alpha-actinin, the thin filament crosslinker. The sarcomeric localization of Smn is conserved in mouse myofibrils. These observations suggest a muscle-specific function for SMN and underline the importance of this tissue in modulating SMA severity.

Conditional transgenic system for mouse aurora a kinase: degradation by the ubiquitin proteasome pathway controls the level of the transgenic protein.

Aurora A is a mitotic kinase that localizes to centrosomes. Expression of this protein is normally limited to the mitotic stage (G(2)-M) of the cell cycle, whereas human cancer cells frequently exhibit overexpression of Aurora A protein regardless of the cell cycle stage. In the present study, Aurora A transgenic mouse lines were generated with a new conditional expression system (cytomegalovirus immediate early enhancer-chicken beta-actin hybrid promoter-Z-enhanced green fluorescent protein) in order to analyze the function of this protein. Although transcripts for Aurora A were elevated in multiple organs of the transgenic mice, the corresponding protein was not detected in extracts analyzed by immunoblotting. The treatment of transgenic-derived embryonic fibroblasts (MEF) with proteasome inhibitors markedly increased the protein level of transgenic Aurora A, indicating that the transgenic Aurora A protein is readily degraded in normal mouse tissues. Under the exponential growth conditions of MEF cells, transgenic Aurora A was detected within the mitotic stage of the cell cycle and localized to centrosomes. In contrast, the marker of the transgenic promoter (enhanced green fluorescent protein) was continuously expressed throughout the cell cycle, indicating the constitutive transcription of transgenic mRNA. These results indicate that transgenic Aurora A is protected from degradation within G(2)-M but is immediately degraded after translation in the G(1)-S stage of the cell cycle. The findings obtained with this transgenic model and derived cells support that the transition from protection to degradation by the ubiquitin proteasome system at the end of mitosis is an important step in controlling the level of Aurora A protein during the cell cycle.

Mechanistic differences in inhibition of ubiquinol cytochrome c reductase by the proximal Qo-site inhibitors famoxadone and methoxyacrylate stilbene.

Famoxadone (FAM) is a newly commercialized antibiotic for use against plant pathogenic fungi. It inhibits mitochondria ubiquinol:cytochrome c oxidoreductase (EC 1.10.2.2, bc(1) complex) function by binding to the proximal niche of the quinol oxidation site on the enzyme. FAM has effects on the enzyme characteristic of both type Ia (E-beta-methoxyacrylates) and type Ic (stigmatellin) inhibitors. Steady-state and tight-binding inhibition kinetics; as well as direct binding measurements with famoxadone (FAM) and methoxyacrylate stilbene (MOAS), indicated that FAM is a non-competitive inhibitor of the enzyme while methoxyacrylate stilbene (MOAS) is better described as a mixed-competitive inhibitor with respect to substrate. Mixed-competitive and non-competitive inhibition kinetics predicts a ternary enzyme-substrate-inhibitor (ESI) intermediate in the reaction sequence. Current views of the Qo domain architecture propose substrate binding niches in both distal and proximal regions of the domain. Since both inhibitors bind within the proximal niche, the formation of an ESI complex implicates substrate binding within the distal niche near the iron-sulfur protein (ISP) and cytochrome c(1) (C1). In the presence of saturating FAM, addition of substrate led to a slow, nearly stoichiometric reduction of C1 that was enzyme dependent, and independent of O(2)(-) production. Similar experiments with saturating MOAS led to a slow, sub-stoichiometric reduction of C1 by substrate. A comparison of the stoichiometries of reduction, and the apparent second order rate constants (K(cat)/K(m)) indicated that saturating MOAS elicits two distinct enzyme-inhibitor (EI) intermediates. One form does not bind substrate, but the other does. In contrast, saturating FAM leads to a predominant EI form capable of binding substrate. We suggest that these differences can be correlated to the respective effects of each inhibitor on the position of the ISP, and the integrity of a distal substrate binding site. The results also indicate that binding of these inhibitory substrate analogues to the proximal niche of the Qo domain significantly increases the DeltaG(double dagger) for reduction of C1.

Mammary gland branching morphogenesis is diminished in mice with a deficiency of insulin-like growth factor-I (IGF-I), but not in mice with a liver-specific deletion of IGF-I.

The development of the mouse mammary gland occurs postnatally. Hormonal activation of local growth factor pathways stimulates rapid elongation and branching of the rudimentary gland through the fatty stroma. Earlier studies showed that GH is required for mammary gland ductal morphogenesis and that IGF-I mediates this action of GH. In the present study we show that adult IGF-I(m/m) mutant mice exhibit a marked reduction in levels of mammary gland and liver igf1 transcripts compared with controls. Whole mounts of the adult IGF-I(m/m) mammary glands revealed ducts that extended to the limits of the fat pad; however, the number of bifurcation branch points in the ductal tree of the mutants was reduced by half compared with that of wild-type glands. In contrast, adult mutant mice with a liver-specific deletion of the igf1 gene obtained by Cre/loxP recombination strategy maintained the normal levels of mammary gland igf1 transcripts and did not exhibit a branching deficit in this organ. It was previously reported that this specific loss of liver IGF-I causes serum levels of IGF-I (endocrine) to decrease by approximately 75%, whereas the levels of tissue igf1 transcripts remain unchanged. On the basis of these findings, we propose that paracrine, not endocrine, IGF-I is important for mammary branching morphogenesis.