Targeted nanoparticles modify neutrophil function in vivo.

Neutrophils play critical roles in a broad spectrum of clinical conditions. Accordingly, manipulation of neutrophil function may provide a powerful immunotherapeutic approach. However, due to neutrophils characteristic short half-life and their large population number, this possibility was considered impractical. Here we describe the identification of peptides which specifically bind either murine or human neutrophils. Although the murine and human neutrophil-specific peptides are not cross-reactive, we identified CD177 as the neutrophil-expressed binding partner in both species. Decorating nanoparticles with a neutrophil-specific peptide confers neutrophil specificity and these neutrophil-specific nanoparticles accumulate in sites of inflammation. Significantly, we demonstrate that encapsulating neutrophil modifying small molecules within these nanoparticles yields specific modulation of neutrophil function (ROS production, degranulation, polarization), intracellular signaling and longevity both in vitro and in vivo. Collectively, our findings demonstrate that neutrophil specific targeting may serve as a novel mode of immunotherapy in disease.

A nuclear redox sensor modulates gene activation and var switching in Plasmodium falciparum.

The virulence of Plasmodium falciparum, which causes the deadliest form of human malaria, is attributed to its ability to evade the human immune response. These parasites "choose" to express a single variant from a repertoire of surface antigens called PfEMP1, which are placed on the surface of the infected red cell. Immune evasion is achieved by switches in expression between var genes, each encoding a different PfEMP1 variant. While the mechanisms that regulate mutually exclusive expression of var genes are still elusive, antisense long-noncoding RNAs (lncRNAs) transcribed from the intron of the active var gene were implicated in the "choice" of the single active var gene. Here, we show that this lncRNA colocalizes with the site of var mRNA transcription and is anchored to the var locus via DNA:RNA interactions. We define the var lncRNA interactome and identify a redox sensor, P. falciparum thioredoxin peroxidase I (PfTPx-1), as one of the proteins associated with the var antisense lncRNA. We show that PfTPx-1 localizes to a nuclear subcompartment associated with active transcription on the nuclear periphery, in ring-stage parasite, when var transcription occurs. In addition, PfTPx-1 colocalizes with S-adenosylmethionine synthetase (PfSAMS) in the nucleus, and its overexpression leads to activation of var2csa, similar to overexpression of PfSAMS. Furthermore, we show that PfTPx-1 knockdown alters the var switch rate as well as activation of additional gene subsets. Taken together, our data indicate that nuclear PfTPx-1 plays a role in gene activation possibly by providing a redox-controlled nuclear microenvironment ideal for active transcription.

FIT-PNAs as RNA-Sensing Probes for Drug-Resistant Plasmodium falciparum.

Detecting RNA at single-nucleotide resolution is a formidable task. Plasmodium falciparum is the deadliest form of malaria in humans and has shown to gain resistance to essentially all antimalarial drugs including artemisinin and chloroquine. Some of these drug resistances are associated with single-nucleotide polymorphisms (SNPs). Forced-intercalation peptide nucleic acids (FIT-PNAs) are DNA mimics that are designed as RNA-sensing molecules that fluoresce upon hybridization to their complementary (RNA) targets. We have previously designed and synthesized FIT-PNAs that target the C580Y SNP in the K13 gene of P. falciparum. In addition, we have now prepared FIT-PNAs that target the K76T SNP in the CRT gene of P. falciparum. Both SNPs are common ones associated with artemisinin and chloroquine drug resistance, respectively. Our FIT-PNAs are conjugated to a simple cell-penetrating peptide (CPP) that consists of eight d-lysines (dK8), which renders these FIT-PNAs cell-permeable to infected red blood cells (iRBCs). Herein, we demonstrate that FIT-PNAs clearly discriminate between wild-type (WT) strains (NF54-WT: artemisinin-sensitive or chloroquine-sensitive) and mutant strains (NF54-C580Y: artemisinin-resistant or Dd2: chloroquine-resistant) of P. falciparum parasites. Simple incubation of FIT-PNAs with live blood-stage parasites results in a substantial difference in fluorescence as corroborated by FACS analysis and confocal microscopy. We foresee FIT-PNAs as molecular probes that will provide a fast, simple, and cheap means for the assessment of drug resistance in malaria─a tool that would be highly desirable for the optimal choice of antimalarial treatment in endemic countries.

Phosphorylation of the Canonical Histone H2A Marks Foci of Damaged DNA in Malaria Parasites.

Plasmodium falciparum parasites proliferate within circulating red blood cells and are responsible for the deadliest form of human malaria. These parasites are exposed to numerous intrinsic and external sources that could cause DNA damage; therefore, they have evolved efficient mechanisms to protect their genome integrity and allow them to proliferate under such conditions. In higher eukaryotes, double-strand breaks rapidly lead to phosphorylation of the core histone variant H2A.X, which marks the site of damaged DNA. We show that in P. falciparum that lacks the H2A.X variant, the canonical P. falciparum H2A (PfH2A) is phosphorylated on serine 121 upon exposure to sources of DNA damage. We further demonstrate that phosphorylated PfH2A is recruited to foci of damaged chromatin shortly after exposure to sources of damage, while the nonphosphorylated PfH2A remains spread throughout the nucleoplasm. In addition, we found that PfH2A phosphorylation is dynamic and that over time, as the parasite activates the repair machinery, this phosphorylation is removed. Finally, we demonstrate that these phosphorylation dynamics could be used to establish a novel and direct DNA repair assay in P. falciparumIMPORTANCEPlasmodium falciparum is the deadliest human parasite that causes malaria when it reaches the bloodstream and begins proliferating inside red blood cells, where the parasites are particularly prone to DNA damage. The molecular mechanisms that allow these pathogens to maintain their genome integrity under such conditions are also the driving force for acquiring genome plasticity that enables them to create antigenic variation and become resistant to essentially all available drugs. However, mechanisms of DNA damage response and repair have not been extensively studied for these parasites. The paper addresses our recent discovery that P. falciparum that lacks the histone variant H2A.X phosphorylates its canonical core histone PfH2A in response to exposure to DNA damage. The process of DNA repair in Plasmodium was mostly studied indirectly. Our findings enabled us to establish a direct DNA repair assay for P. falciparum similar to assays that are widely used in model organisms.

A Unique Virulence Gene Occupies a Principal Position in Immune Evasion by the Malaria Parasite Plasmodium falciparum.

Mutually exclusive gene expression, whereby only one member of a multi-gene family is selected for activation, is used by the malaria parasite Plasmodium falciparum to escape the human immune system and perpetuate long-term, chronic infections. A family of genes called var encodes the chief antigenic and virulence determinant of P. falciparum malaria. var genes are transcribed in a mutually exclusive manner, with switching between active genes resulting in antigenic variation. While recent work has shed considerable light on the epigenetic basis for var gene activation and silencing, how switching is controlled remains a mystery. In particular, switching seems not to be random, but instead appears to be coordinated to result in timely activation of individual genes leading to sequential waves of antigenically distinct parasite populations. The molecular basis for this apparent coordination is unknown. Here we show that var2csa, an unusual and highly conserved var gene, occupies a unique position within the var gene switching hierarchy. Induction of switching through the destabilization of var specific chromatin using both genetic and chemical methods repeatedly led to the rapid and exclusive activation of var2csa. Additional experiments demonstrated that these represent "true" switching events and not simply de-silencing of the var2csa promoter, and that activation is limited to the unique locus on chromosome 12. Combined with translational repression of var2csa transcripts, frequent "default" switching to this locus and detection of var2csa untranslated transcripts in non-pregnant individuals, these data suggest that var2csa could play a central role in coordinating switching, fulfilling a prediction made by mathematical models derived from population switching patterns. These studies provide the first insights into the mechanisms by which var gene switching is coordinated as well as an example of how a pharmacological agent can disrupt antigenic variation in Plasmodium falciparum.

The molecular basis of antifolate resistance in Plasmodium falciparum: looking beyond point mutations.

Drugs that target the folate-synthesis pathway have a long history of effectiveness against a variety of pathogens. As antimalarials, the antifolates were safe and well tolerated, but resistance emerged quickly and has persisted even with decreased drug pressure. The primary determinants of resistance in Plasmodium falciparum are well-described point mutations in the enzymes dihydropteroate synthase and dihydrofolate reductase targeted by the combination sulfadoxine-pyrimethamine. Recent work has highlighted the contributions of additional parasite adaptation to antifolate resistance. In fact, the evolution of antifolate-resistant parasites is multifaceted and complex. Gene amplification of the first enzyme in the parasite folate synthesis pathway, GTP-cyclohydrolase, is strongly associated with resistant parasites and potentially contributes to persistence of resistant parasites. Further understanding of how parasites adjust flux through the folate pathway is important to the further development of alternative agents targeting this crucial synthesis pathway.

Origin of robustness in generating drug-resistant malaria parasites.

Biological robustness allows mutations to accumulate while maintaining functional phenotypes. Despite its crucial role in evolutionary processes, the mechanistic details of how robustness originates remain elusive. Using an evolutionary trajectory analysis approach, we demonstrate how robustness evolved in malaria parasites under selective pressure from an antimalarial drug inhibiting the folate synthesis pathway. A series of four nonsynonymous amino acid substitutions at the targeted enzyme, dihydrofolate reductase (DHFR), render the parasites highly resistant to the antifolate drug pyrimethamine. Nevertheless, the stepwise gain of these four dhfr mutations results in tradeoffs between pyrimethamine resistance and parasite fitness. Here, we report the epistatic interaction between dhfr mutations and amplification of the gene encoding the first upstream enzyme in the folate pathway, GTP cyclohydrolase I (GCH1). gch1 amplification confers low level pyrimethamine resistance and would thus be selected for by pyrimethamine treatment. Interestingly, the gch1 amplification can then be co-opted by the parasites because it reduces the cost of acquiring drug-resistant dhfr mutations downstream in the same metabolic pathway. The compensation of compromised fitness by extra GCH1 is an example of how robustness can evolve in a system and thus expand the accessibility of evolutionary trajectories leading toward highly resistant alleles. The evolution of robustness during the gain of drug-resistant mutations has broad implications for both the development of new drugs and molecular surveillance for resistance to existing drugs.

Direct evidence for the adaptive role of copy number variation on antifolate susceptibility in Plasmodium falciparum.

Resistance to antimalarials targeting the folate pathway is widespread. GTP-cyclohydrolase (gch1), the first enzyme in this pathway, exhibits extensive copy number variation (CN) in parasite isolates from areas with a history of longstanding antifolate use. Increased CN of gch1 is associated with a greater number of point mutations in enzymes targeted by the antifolates, pyrimethamine and sulphadoxine. While these observations suggest that increases in gch1 CN are an adaptation to drug pressure, changes in CN have not been experimentally demonstrated to directly alter drug susceptibility. To determine if changes in gch1 expression alone modify pyrimethamine sensitivity, we manipulated gch1 CN in several parasite lines to test the effect on drug sensitivity. We report that increases in gch1 CN alter pyrimethamine resistance in most parasites lines. However we find evidence of a detrimental effect of very high levels of gch1 overexpression in parasite lines with high endogenous levels of gch1 expression, revealing the importance of maintaining balance in the folate pathway and implicating changes in gch1 expression in preserving proper metabolic flux. This work expands our understanding of parasite adaptation to drug pressure and provides a possible mechanism for how specific mutations become fixed within parasite populations.

p27 deficiency is associated with migration defects in PDGF-expressing gliomas in vivo.

p27(Kip1) is a cyclin dependent kinase inhibitor that functions as a tumor suppressor in a variety of different cancers. While p27 has a well established role in regulating the cell cycle, it has also been shown to regulate cellular migration by influencing the activation state of the small GTPase RhoA. We recently demonstrated that loss of p27 enhances tumor progression and leads to a dramatic decrease in survival in PDGF-induced oligodendrogliomas. Here we show that p27 deficient PDGF-expressing glial cells contained elevated levels of Rho-GTP and were less migratory than wild type cells. Migration defects in p27 deficient cells were rescued by either Rho kinase inhibition or expression of p27 or CK(-), a mutant of p27 that cannot bind cyclins/cdks. The RCAS/tv-a retroviral system was used to specifically induce PDGF-expressing gliomas in mice. Many of the p27 deficient mice died earlier than wild type mice and displayed hydrocephalus which was associated with periventricular tumors that failed to invade the normal brain parenchyma. Invasion failure was reversed by co-expression of PDGF with either the GAP domain of p190(RhoGAP), a negative regulator of Rho, or p27, or CK(-). These results suggest that p27 mediated regulation of the Rho pathway is cell cycle independent and demonstrate for the first time a migration defect in cancer cells that is associated with p27 deficiency in vivo in a mouse tumor model.