Distinct effects of HIV protease inhibitors and ERAD inhibitors on zygote to ookinete transition of the malaria parasite

In an effort to eradicate malaria, new interventions are proposed to include compound/vaccine development against pre-erythrocytic, erythrocytic and mosquito stages of Plasmodium. Drug repurposing might be an alternative approach to new antimalarials reducing the cost and the time required for drug development. Previous in vitro studies have examined the effects of protease inhibitors on different stages of the Plasmodium parasite, although the clinical relevance of this remains unclear. In this study we tested the putative effect of three HIV protease inhibitors, two general aspartyl protease inhibitors and three AAA-p97 ATPase inhibitors on the zygote to ookinete transition of the Plasmodium parasite. Apart from the two general aspartyl inhibitors, all other compounds had a profound effect on the development of the parasites. HIVPIs inhibited zygote to ookinete conversion by 75%–90%, while the three AAA-p97 ATPase inhibitors blocked conversion by 50%–90% at similar concentrations, while electron microscopy highlighted nuclear and structural abnormalities. Our results highlight a potential of HIV protease inhibitors and p97 inhibitors as transmission blocking agents for the eradication of malaria

The splicing factor XAB2 interacts with ERCC1-XPF and XPG for R-loop processing

RNA splicing, transcription and the DNA damage response are intriguingly linked in mammals but the underlying mechanisms remain poorly understood. Using an in vivo biotinylation tagging approach in mice, we show that the splicing factor XAB2 interacts with the core spliceosome and that it binds to spliceosomal U4 and U6 snRNAs and pre-mRNAs in developing livers. XAB2 depletion leads to aberrant intron retention, R-loop formation and DNA damage in cells. Studies in illudin S-treated cells and Csb(m/m) developing livers reveal that transcription-blocking DNA lesions trigger the release of XAB2 from all RNA targets tested. Immunoprecipitation studies reveal that XAB2 interacts with ERCC1-XPF and XPG endonucleases outside nucleotide excision repair and that the trimeric protein complex binds RNA:DNA hybrids under conditions that favor the formation of R-loops. Thus, XAB2 functionally links the spliceosomal response to DNA damage with R-loop processing with important ramifications for transcription-coupled DNA repair disorders

DNA Damage: From Chronic Inflammation to Age-Related Deterioration

To lessen the wear and tear of existence, cells have evolved mechanisms that continuously sense DNA lesions, repair DNA damage and restore the compromised genome back to its native form. Besides genome maintenance pathways, multi-cellular organisms may also employ adaptive and innate immune mechanisms to guard themselves against bacteria or viruses. Recent evidence points to reciprocal interactions between DNA repair, DNA damage responses and aspects of immunity; both self-maintenance and defense responses share a battery of common players and signaling pathways aimed at safeguarding our bodily functions over time. In the short-term, this functional interplay would allow injured cells to restore damaged DNA templates or communicate their compromised state to the micro-environment. In the long-term, however, it may result in the (premature) onset of age-related degeneration, including cancer. Here, we discuss the beneficial and unrewarding outcomes of DNA damage-driven inflammation in the context of tissue-specific pathology and disease progression

DNA Damage Response and Metabolic Reprogramming in Health and Disease

Nuclear DNA damage contributes to cellular malfunction and the premature onset of age-related diseases, including cancer. Until recently, the canonical DNA damage response (DDR) was thought to represent a collection of nuclear processes that detect, signal and repair damaged DNA. However, recent evidence suggests that beyond nuclear events, the DDR rewires an intricate network of metabolic circuits, fine-tunes protein synthesis, trafficking, and secretion as well as balances growth with defense strategies in response to genotoxic insults. In this review, we discuss how the active DDR signaling mobilizes extranuclear and systemic responses to promote cellular homeostasis and organismal survival in health and disease

Transcription stress at telomeres leads to cytosolic DNA release and paracrine senescence.

Transcription stress has been linked to DNA damage -driven aging, yet the underlying mechanism remains unclear. Here, we demonstrate that Tcea1-/- cells, which harbor a TFIIS defect in transcription elongation, exhibit RNAPII stalling at oxidative DNA damage sites, impaired transcription, accumulation of R-loops, telomere uncapping, chromatin bridges, and genome instability, ultimately resulting in cellular senescence. We found that R-loops at telomeres causally contribute to the release of telomeric DNA fragments in the cytoplasm of Tcea1-/- cells and primary cells derived from naturally aged animals triggering a viral-like immune response. TFIIS-defective cells release extracellular vesicles laden with telomeric DNA fragments that target neighboring cells, which consequently undergo cellular senescence. Thus, transcription stress elicits paracrine signals leading to cellular senescence, promoting aging

R-loops trigger the release of cytoplasmic ssDNAs leading to chronic inflammation upon DNA damage

How DNA damage leads to chronic inflammation and tissue degeneration with aging remains to be fully resolved. Here, we show that DNA damage leads to cellular senescence, fibrosis, loss-of-tissue architecture, and chronic pancreatitis in mice with an inborn defect in the excision repair cross complementation group 1 (Ercc1) gene. We find that DNA damage-driven R-loops causally contribute to the active release and buildup of single-stranded DNAs (ssDNAs) in the cytoplasm of cells triggering a viral-like immune response in progeroid and naturally aged pancreata. To reduce the proinflammatory load, we developed an extracellular vesicle (EV)-based strategy to deliver recombinant S1 or ribonuclease H nucleases in inflamed Ercc1(-/-) pancreatic cells. Treatment of Ercc1(-/-) animals with the EV-delivered nuclease cargo eliminates DNA damage-induced R-loops and cytoplasmic ssDNAs alleviating chronic inflammation. Thus, DNA damage-driven ssDNAs causally contribute to tissue degeneration, Ercc1(-/-) paving the way for novel rationalized intervention strategies against age-related chronic inflammation

<i>s2p(-)</i> parasites show delayed transmission and growth, resulting in reduced virulence.

<p>(A) Kaplan-Meier analysis of time to patency after inoculation of 10,000 WT or <i>s2p(-)</i> salivary gland sporozoites into C57BL/6 mice (WT n = 9, <i>s2p(-)</i> n = 13). **, <i>P</i> < 0.01 (Log rank [Mantel-Cox] test). (B) Blood stage growth curve of the same mice showing the difference in growth rate of WT <i>vs</i>. <i>s2p(-)</i> parasites, alongside blood stage development of mice infected through bite back from infected <i>A</i>. <i>stephensi</i> mosquitoes (Wt bb <i>vs</i>. <i>s2p(-)</i> bb). In all cases mean values (± SD) are shown. **<i>P</i> < 0.01, ***<i>P</i> < 0.001 (Multiple t-test comparison). Significance is shown as follows: Black stars: Sporozoite injection experiment, Grey stars: Bite back experiment. (C) Kaplan-Meier curve of mice developing experimental cerebral malaria (ECM) over time after injection of 10,000 WT or <i>s2p(-)</i> salivary gland sporozoites (WT n = 9, <i>s2p(-)</i> n = 13). ***, <i>P</i> < 0.001 (Log rank [Mantel-Cox] test). (D, E) Parasitaemia levels of C57BL/6 mice after infection with (D) 10,000 (<i>n</i> = 3 each) or (E) 1,000 (<i>n</i> = 5 each) WT or <i>s2p(-)</i> iRBCs, respectively, as determined by Giemsa stained blood smears. <i>s2p(-)</i> parasites exhibit slower growth rates compared to the WT line. Mean values (± SD) are shown *<i>P</i> < 0.05 **<i>P</i> < 0.01, ***<i>P</i> < 0.001 (Multiple t-test comparison). (F) Kaplan-Meier curve of time to ECM development after patency. A one day delay in ECM symptoms was observed in mice infected with 10,000 <i>s2p(-)</i> iRBCs (<i>P</i> = 0.11 (Log rank [Mantel-Cox] test). Mice infected with 1,000 WT iRBC developed ECM symptoms at day 8 (4/5) and 9 (1/5), while 2/5 <i>s2p(-)</i> infected mice developed ECM at day 11 and 3/5 remained free of ECM symptoms. **<i>P</i> < 0.01 (Log rank [Mantel-Cox] test).</p

<i>s2p(-)</i> parasites show no defects in sexual development nor sporogony.

<p>(A) Exflagellation assay showing formation of exflagellation centres. Mean values (±SD) from three independent experiments are shown. Differences were non-significant (Mann-Whitney test). (B) Ookinete conversion rates of WT and <i>s2p(-)</i> parasites after staining with an antibody against the surface antigen P28 and enumeration of ookinetes, zygotes and macrogametes. Shown are mean values (±SD) from three independent experiments. Differences were non-significant (2way-ANOVA). (C) Immunofluorescence analysis of <i>Anopheles gambiae</i> epithelia sheets infected with WT or <i>s2p(-)</i> parasites. Both strains induce an epithelial response as shown by the SRPN6 antibody (red). Ookinetes are stained with an antibody against surface protein P28 (green) and nuclei are stained with TO-PRO 3 (blue). Scale bar 10 μM. (D) Oocyst numbers of <i>s2p(-)</i> strain compared to the parental WT line after standard membrane feeding assay of <i>An</i>. <i>gambiae</i> mosquitoes from two independent experiments. Black bars show mean values (±SEM). Differences were non-significant (Mann-Whitney test).</p

Expression and localisation of <i>Pb</i>S2P.

<p>(A) Relative expression levels of <i>PbS2P</i> as determined by qRT-PCR from cDNAs of schizonts (schz), sporozoites (spz), 24h liver stages (LS24) and 48h liver stages (LS48). Transcript levels were normalised to <i>PbHSP70</i> and <i>GFP</i>. (B) Western blot analysis of <i>Pb</i>S2P-HA whole protein extract from purified schizonts of transgenic <i>PbS2P-HA</i> parasites using an α-HA antibody. <i>Pb</i>S2P-HA migrates at 35kDa. (C) Immunofluorescence analysis (IFA) of <i>Pb</i>S2P-HA merozoites, ookinete, oocyst, and salivary gland sporozoites using α-HA (3F10) for detection of <i>Pb</i>S2P (red) and Hoechst stain for the nucleus (blue). For delineation of parasites the following antibodies (green) were used: α-HSP70, schizonts/merozoites; α-MTIP, ookinete and sporozoite; α-PbCap380, oocyst. Prominent localisation of <i>Pb</i>S2P in proximity to the nucleus is present in all invasive stages. Star, apical end of ookinete. Scale bar 5 μM.</p

<i>Pb</i>S2P shows partial co-localisation with the cis-Golgi marker ERD2.

<p>(A) Double labelling IFA of <i>P</i>. <i>berghei</i> schizont cultures using α-HA (3F10) for detection of <i>Pb</i>S2P (red) and α-ERD2 as a Golgi marker (green) showing partial, or in some cases complete, co-localisation. Nuclei are stained with Hoechst (blue). Scale bar 5 μM.</p