Dynamic Activation and Repression of the Plasmodium falciparum rif Gene Family and Their Relation to Chromatin Modification

The regulation of variant gene expression in Plasmodium falciparum is still only partially understood. Regulation of var genes, the most studied gene family involved in antigenic variation, is orchestrated by a dynamic pattern of inherited chromatin states. Although recent evidence pointed to epigenetic regulation of transcribed and repressed rif loci, little is known about specific on/off associated histone modifications of individual rif genes. To investigate the chromatin marks for transcribed and repressed rif loci, we cultivated parasites and evaluated the transcriptional status of chosen rif targets by qRT-PCR and performed ChIP assays using H3K9ac and H3K9me3 antibodies. We then monitored changes in the epigenetic patterns in parasites after several reinvasions and also evaluated the “poised” mark in trophozoites and schizonts of the same erythrocytic cycle by ChIP using H3K4me2 specific antibodies. Our results show that H3K9 is acetylated in transcribed rif loci and trimethylated or even unmodified in repressed rif loci. These transcriptional and epigenetic states are inherited after several reinvasions. The poised modification H3K4me2 showed a tendency to be more present in loci in trophozoites that upon progression to schizonts strongly transcribe the respective locus. However, this effect was not consistently observed for all monitored loci. While our data show important similarities to var transcription-associated chromatin modifications, the observed swiftly occurring modifications at rif loci and the absence of H3K9 modification point to a different dynamic of recruitment of chromatin modifying enzymes

A Major Role for the Plasmodium falciparum ApiAP2 Protein PfSIP2 in Chromosome End Biology

The heterochromatic environment and physical clustering of chromosome ends at the nuclear periphery provide a functional and structural framework for antigenic variation and evolution of subtelomeric virulence gene families in the malaria parasite Plasmodium falciparum. While recent studies assigned important roles for reversible histone modifications, silent information regulator 2 and heterochromatin protein 1 (PfHP1) in epigenetic control of variegated expression, factors involved in the recruitment and organization of subtelomeric heterochromatin remain unknown. Here, we describe the purification and characterization of PfSIP2, a member of the ApiAP2 family of putative transcription factors, as the unknown nuclear factor interacting specifically with cis-acting SPE2 motif arrays in subtelomeric domains. Interestingly, SPE2 is not bound by the full-length protein but rather by a 60kDa N-terminal domain, PfSIP2-N, which is released during schizogony. Our experimental re-definition of the SPE2/PfSIP2-N interaction highlights the strict requirement of both adjacent AP2 domains and a conserved bipartite SPE2 consensus motif for high-affinity binding. Genome-wide in silico mapping identified 777 putative binding sites, 94% of which cluster in heterochromatic domains upstream of subtelomeric var genes and in telomere-associated repeat elements. Immunofluorescence and chromatin immunoprecipitation (ChIP) assays revealed co-localization of PfSIP2-N with PfHP1 at chromosome ends. Genome-wide ChIP demonstrated the exclusive binding of PfSIP2-N to subtelomeric SPE2 landmarks in vivo but not to single chromosome-internal sites. Consistent with this specialized distribution pattern, PfSIP2-N over-expression has no effect on global gene transcription. Hence, contrary to the previously proposed role for this factor in gene activation, our results provide strong evidence for the first time for the involvement of an ApiAP2 factor in heterochromatin formation and genome integrity. These findings are highly relevant for our understanding of chromosome end biology and variegated expression in P. falciparum and other eukaryotes, and for the future analysis of the role of ApiAP2-DNA interactions in parasite biology

Longevity and Composition of Cellular Immune Responses Following Experimental Plasmodium falciparum Malaria Infection in Humans

Cellular responses to Plasmodium falciparum parasites, in particular interferon-gamma (IFNγ) production, play an important role in anti-malarial immunity. However, clinical immunity to malaria develops slowly amongst naturally exposed populations, the dynamics of cellular responses in relation to exposure are difficult to study and data about the persistence of such responses are controversial. Here we assess the longevity and composition of cellular immune responses following experimental malaria infection in human volunteers. We conducted a longitudinal study of cellular immunological responses to sporozoites (PfSpz) and asexual blood-stage (PfRBC) malaria parasites in naïve human volunteers undergoing single (n = 5) or multiple (n = 10) experimental P. falciparum infections under highly controlled conditions. IFNγ and interleukin-2 (IL-2) responses following in vitro re-stimulation were measured by flow-cytometry prior to, during and more than one year post infection. We show that cellular responses to both PfSpz and PfRBC are induced and remain almost undiminished up to 14 months after even a single malaria episode. Remarkably, not only ‘adaptive’ but also ‘innate’ lymphocyte subsets contribute to the increased IFNγ response, including αβT cells, γδT cells and NK cells. Furthermore, results from depletion and autologous recombination experiments of lymphocyte subsets suggest that immunological memory for PfRBC is carried within both the αβT cells and γδT compartments. Indeed, the majority of cytokine producing T lymphocytes express an CD45RO+ CD62L- effector memory (EM) phenotype both early and late post infection. Finally, we demonstrate that malaria infection induces and maintains polyfunctional (IFNγ+IL-2+) EM responses against both PfRBC and PfSpz, previously found to be associated with protection. These data demonstrate that cellular responses can be readily induced and are long-lived following infection with P. falciparum, with a persisting contribution by not only adaptive but also (semi-)innate lymphocyte subsets. The implications hereof are positive for malaria vaccine development, but focus attention on those factors potentially inhibiting such responses in the field

Identification and Genome-Wide Prediction of DNA Binding Specificities for the ApiAP2 Family of Regulators from the Malaria Parasite

The molecular mechanisms underlying transcriptional regulation in apicomplexan parasites remain poorly understood. Recently, the Apicomplexan AP2 (ApiAP2) family of DNA binding proteins was identified as a major class of transcriptional regulators that are found across all Apicomplexa. To gain insight into the regulatory role of these proteins in the malaria parasite, we have comprehensively surveyed the DNA-binding specificities of all 27 members of the ApiAP2 protein family from Plasmodium falciparum revealing unique binding preferences for the majority of these DNA binding proteins. In addition to high affinity primary motif interactions, we also observe interactions with secondary motifs. The ability of a number of ApiAP2 proteins to bind multiple, distinct motifs significantly increases the potential complexity of the transcriptional regulatory networks governed by the ApiAP2 family. Using these newly identified sequence motifs, we infer the trans-factors associated with previously reported plasmodial cis-elements and provide evidence that ApiAP2 proteins modulate key regulatory decisions at all stages of parasite development. Our results offer a detailed view of ApiAP2 DNA binding specificity and take the first step toward inferring comprehensive gene regulatory networks for P. falciparum

Brief communication: Extended chronology of the Cordón Caulle volcanic eruption beyond 2011 reveals toxic impacts

Aside of immediate impacts, the 2011 Puyehue–Cordón Caulle volcano (PCC) eruption also caused persisting chemical impacts. By 2012, toxicity resulted in overt dental fluorosis in deer, with bone fluoride increasing > 38-fold to 5175 ppm. Sheep, horses and cattle also succumbed to fluorosis. Due to eolian redeposition of tephra, exposure of ruminants continued, bone fluoride reached 10 396 ppm, and by 2014 skeletal fluorosis was found. Nonskeletal fluorosis resulted in reduced wool growth and major losses among periparturient cattle. Peculiarities of digestive processes make ruminants susceptible to fluoride-containing tephra, which averaged 548 ppm from PCC. Moreover, recent volcanic eruptions causing fluorosis could be aggravated by local iodine deficiency, which increases the incidence and harshness of fluorosis, and deficiency of selenium, which, among other things, also results in secondary deficiency of iodine. Notwithstanding, several measures are available to livestock producers to minimize chemical impacts of fluoride

The next frontier for recovering endangered huemul (Hippocamelus bisulcus): how to avoid recurrent misdiagnoses of health status and risks

Context. The currently remaining 350-500 huemuls in Argentina are not recovering. We evaluated live huemuls, along with animals that died soon after confinement, or those that had died recently. Although information on the health status is highly valuable, repeated misdiagnoses of the health status indicate a need for other strategies. Aims. . Discrepancies between clinical and postmortem diagnoses are critical for improving subsequent management decisions. Methods. Initial clinical interpretations and risk assessments were reinterpreted on the basis of necropsies and other data. Results. Two debilitated huemul individuals examined by veterinarians died soon afterwards, supposedly one being intoxicated and one being without lesions. Necropsies showed osteopathology and fluorosis (fluorine concentrations of 2209 and 2979 mg/kg). Another male was tied up, with authorities and veterinarians arriving the next day. After being sedated, and judged healthy, the animal was translocated. Because there was no reversal, this animal died 22 h postcapture. Exhumation showed severe osteopathology. Elsewhere, huemuls were considered adequate in selenium because values below the detection limit were excluded. However, when all values were included, 75% of the animals were selenium-deficient; this population had numerous cases of osteopathology. Recently, specialists went to Torres del Paine Park suspecting caseous lymphadenitis, reporting of which has been obligatory since 1937. However, many cases documented in 1999-2007 have not elicited responses since that time by health professionals. Selenium deficiency negatively affects antibody responses against caseous lymphadenitis. One province had denied huemul capture (2012 and 2013) on recommendation of scientific advisors. Because of the right for transparency, it was found out in 2016 that authorities had requested advice from only one veterinarian who assessed that darting was too risky. Another 2016 project proposed to dart the first huemul in Argentina. Two weeks earlier, that same team was called to rescue a tied-up huemul; the team opted not to involve a laboratory with drugs and radios that was only 1 h away. This huemul died and was left in the woods. Finally, the first huemul enclosure in Argentina was proposed (1995), but the permission was denied. Again, in 2000, the first huemul centre with private funding secured for 30 years was proposed. However, the Regional Delegation for Patagonian National Parks prevented aerial surveys, and advised not to provide a permit for the centre. Conclusions. Future assessments should consider osteopathology. Risk assessments should be transparent and based on assessment by multiple qualified professionals. Implications. Clinical misdiagnoses may reduce life expectancy, in contrast to taking individuals to enclosures, which would also allow valuable reintroductions. Not permitting captures, censusses and enclosures has resulted in unwarranted delays in conservation progress

An Alternative Interpretation of Plasma Selenium Data from Endangered Patagonian Huemul Deer (Hippocamelus bisulcus)

A group of huemul deer from a Chilean population was sampled for trace mineral analysis. Here we show that most huemul plasma Se values were severely deficient (64%) or deficient (9%), with a minority of the values marginal (18%) or adequate (9%). The role of Se deficiency thus merits further consideration in explaining huemul declines and in conservation efforts, especially given that soils in huemul habitat are Se deficient