Host Porphobilinogen Deaminase Deficiency Confers Malaria Resistance in Plasmodium chabaudi but Not in Plasmodium berghei or Plasmodium falciparum During Intraerythrocytic Growth

An important component in host resistance to malaria infection are inherited mutations that give rise to abnormalities and deficiencies in erythrocyte proteins and enzymes. Understanding how such mutations confer protection against the disease may be useful for developing new treatment strategies. A mouse ENU-induced mutagenesis screen for novel malaria resistance-conferring mutations identified a novel non-sense mutation in the gene encoding porphobilinogen deaminase (PBGD) in mice, denoted here as PbgdMRI58155. Heterozygote PbgdMRI58155 mice exhibited ~50% reduction in cellular PBGD activity in both mature erythrocytes and reticulocytes, although enzyme activity was ~10 times higher in reticulocytes than erythrocytes. When challenged with blood-stage P. chabaudi, which preferentially infects erythrocytes, heterozygote mice showed a modest but significant resistance to infection, including reduced parasite growth. A series of assays conducted to investigate the mechanism of resistance indicated that mutant erythrocyte invasion by P. chabaudi was normal, but that following intraerythrocytic establishment a significantly greater proportions of parasites died and therefore, affected their ability to propagate. The Plasmodium resistance phenotype was not recapitulated in Pbgd-deficient mice infected with P. berghei, which prefers reticulocytes, or when P. falciparum was cultured in erythrocytes from patients with acute intermittent porphyria (AIP), which had modest (20-50%) reduced levels of PBGD. Furthermore, the growth of Pbgd-null P. falciparum and Pbgd-null P. berghei parasites, which grew at the same rate as their wild-type counterparts in normal cells, were not affected by the PBGD-deficient background of the AIP erythrocytes or Pbgd-deficient mice. Our results confirm the dispensability of parasite PBGD for P. berghei infection and intraerythrocytic growth of P. falciparum, but for the first time identify a requirement for host erythrocyte PBGD by P. chabaudi during in vivo blood stage infection.The authors were supported by an International Macquarie University Research Excellence Scholarship (CS), the NHMRC (490037, 605524, APP1047090, and APP1066502), the Australian Research Council (DP120100061) and the National Collaborative Research Infrastructure (NCRIS) via the Australian Phenomics Network (APN)

Parasite-host interactions during the blood and liver stage of rodent malaria parasites

Thesis by publication.Cotutelle thesis with Universität Bern (the University of Bern (Switzerland)).Includes bibliographical references.1. Introduction -- 2. Publication and manuscripts -- 3. Discussion -- Appendices.Malaria is a parasitic disease which puts almost half of the world's population at risk of infection. It is caused by Plasmodium, a parasite transmitted by Anopheles mosquitoes. During its life cycle in the vertebrate host, the malaria parasite invades and multiplies in hepatocytes and in red blood cells. During invasion of sporozoites and merozoites, the host cell membrane invaginates around the parasite to form the parasitophorous vacuole membrane (PVM). The membrane is extensively remodeled by the parasite: host cell membrane proteins are removed and parasite proteins are incorporated. The PVM shields the parasite from a direct attack by cytosolic host cell immune responses and provides the host-parasite interface. Important roles of the PVM are nutrient acquisition, excretion of waste products and export of host-targeted proteins which add to parasite fitness and virulence. Finally, egress is critical for parasite release from its host cell and the first step is PVM disintegration. PVM biology and remodeling is of great interest, but despite all of these important tasks fulfilled by proteins in the PVM, the composition is not yet clear. Here, a proximity labeling technique (BioID) was used to identify PVM or PVM-interacting proteins in P. berghei blood stage parasites. Using this technique, many of the already known PVM proteins were found. Apart from known resident PVM proteins, I detected several proteins that were that were hitherto unknown as PVM proteins. Uncharacterized protein candidates were endogenously GFP-tagged and analyzed using live- and fixed-cell microscopy during the liver and the blood stage. Using this approach, I was able to identify several novel PVM proteins, and proteins that come in close proximity to the PVM. During the intraerythrocytic symptomatic blood stage, Plasmodium digests up to 70% of host hemoglobin. Heme is an essential component of a number of proteins including hemoglobin and is essential for life. It is synthesized by a pathway involving at least eight enzymatic steps, and deficiency of any of these result in porphyria. Despite the abundance of hemoglobin and heme during the blood stage, the parasite expresses all enzymes of its canonical heme pathway, which can facilitate the de novo heme synthesis. Previous research has indicated that the parasite heme pathway is non-essential during the blood stage, and that the deficiency of host heme enzymes can influence parasite growth. That Plasmodium can be greatly influenced by the host background is well established. Here we hypothesized that host porphobilinogen deaminase (PBGD) and coproporphyrinogen oxidase (CPOX), two additional heme pathway enzymes, play a role during the blood stage. To facilitate this goal, in-depth studies by using rodent malaria species were utilized to infect mice deficient in PBGD and CPOX. Blood from porphyric patients was infected with P. falciparum and parasite growth was significantly inhibited. Host PBGD was localized to the parasite periphery which could indicate that the parasite imports this enzyme during the blood stage. PBGD deficient mice are more resistant to malaria infection which suggests that Plasmodium relies on host PBGD to sustain its growth in erythrocytes and cause blood-stage infection. CPOX deficient mice displayed an increased survival, probably due to iron deficiency anemia, but the exact underlying mechanism remains to be elucidated. A novel role for PBGD and CPOX was described in this thesis adding to the understanding of host-pathogen interactions during the blood stage. Together this thesis has identified a number of novel PVM proteins during the blood and liver stage which will help to gain a deeper understanding of PVM biology. Deficiency of host enzymes can influence Plasmodium and here two novel host proteins, PBGD and CPOX are added to them. This thesis increased the understanding of host-parasite interactions during the blood and liver stage of Plasmodium.1 online resource (150 pages : illustrations, map

Direct and indirect effects of dorsolateral prefrontal cortex and emotion regulation strategy use on mental health during Covid-19

Background. Covid-19 and associated restrictions have been linked to negative mental health outcomes across the globe. Cognitive emotion regulation strategies constitute means to mitigating negative affect resulting from stressful life events, possibly offering an opportunity to change negative consequences associated with pandemics. Neuronally, emotion regulation is supported by prefrontal and limbic brain regions, but a direct investigation of brain structural characteristics in association with contextual emotion regulation during prolonged stressful events is yet missing. Methods. Variations in cognitive emotion regulation strategy use, anxiety and depression scores were assessed in 43 adults (31♀/12♂, age=35.14±9.20y) during the first months following Covid-19 onset and again at the end of 2020 (seven repeated measures assessments). Pre-pandemic behavioral and neuroimaging measures were available for all participants, allowing the investigation of mediating effects of pre-pandemic emotion regulatory brain structures and use of cognitive emotion regulation strategies during the pandemic on mental well-being at the beginning and end of the first pandemic year. Results. Heightened, but varying levels of anxiety and depression were observed across 2020. While adaptive emotion regulation strategies were most frequently employed, maladaptive strategies explained the highest variation in negative mental health outcomes (i.e., anxiety and depression scores). Adaptive strategies had a positive, maladaptive strategies a negative effect, however, this direction varied when considering long-term mental health effects. Emotion regulation strategy use mediated the association between pre-pandemic emotion regulatory brain structure (i.e., cortical thickness) in right dorsolateral prefrontal cortex and mental health, with prefrontal-amygdala coupling as a possible driving factor. Additionally, early mental health measures impacted later mental well-being. Conclusion. Maladaptive emotion regulation strategies have a negative effect on mental health during prolonged stress as induced by pandemics. Interventions targeting the reduction of maladaptive emotion regulation strategies may provide a way to counteract negative effects, thus offering a window of opportunity for action

Mental well-being during the first months of Covid-19 in adults and children: behavioral evidence and neural precursors

Pandemics such as the Covid-19 pandemic have shown to impact our physical and mental well-being, with particular challenges for children and families. We describe data from 43 adults (31♀, ages = 22-51; 21 mothers) and 26 children (10♀, ages = 7-17 years) including pre-pandemic brain function and seven assessment points during the first months of the pandemic. We investigated (1) changes in child and adult well-being, (2) mother-child associations of mental well-being, and (3) associations between pre-pandemic brain activation during mentalizing and later fears or burden. In adults the prevalence of clinically significant anxiety-levels was 34.88% and subthreshold depression 32.56%. Caregiver burden in parents was moderately elevated. Overall, scores of depression, anxiety, and caregiver burden decreased across the 11 weeks after Covid-19-onset. Children's behavioral and emotional problems during Covid-19 did not significantly differ from pre-pandemic levels and decreased during restrictions. Mothers' subjective burden of care was associated with children's emotional and behavioral problems, while depression levels in mothers were related to children's mood. Furthermore, meeting friends was a significant predictor of children's mood during early restrictions. Pre-pandemic neural correlates of mentalizing in prefrontal regions preceded later development of fear of illnesses and viruses in all participants, while temporoparietal activation preceded higher subjective burden in mothers

BioID Reveals Novel Proteins of the Parasitophorous Vacuole Membrane.

During their development within the vertebrate host, parasites infect hepatocytes and red blood cells. Within these cells, parasites are surrounded by a parasitophorous vacuole membrane (PVM). The PVM plays an essential role for the interaction of parasites with their host cells; however, only a limited number of proteins of this membrane have been identified so far. This is partially because systematic proteomic analysis of the protein content of the PVM has been difficult in the past, due to difficulties encountered in attempts to separate the PVM from other membranes such as the parasite plasma membrane. In this study, we adapted the BioID technique to -cultivated blood stage parasites and utilized the promiscuous biotin ligase BirA* fused to PVM-resident exported protein 1 to biotinylate proteins of the PVM. These we further processed by affinity purification, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and label-free quantitation, leading to a list of 61 known and candidate PVM proteins. Seven proteins were analyzed further during blood and liver stage development. This resulted in the identification of three novel PVM proteins, which were the serine/threonine protein phosphatase UIS2 (PlasmoDB accession no. PBANKA_1328000) and two conserved proteins with unknown functions (PBANKA_0519300 and PBANKA_0509000). In conclusion, our report expands the number of known PVM proteins and experimentally validates BioID as a powerful method to screen for novel constituents of specific cellular compartments in . Intracellular pathogens are often surrounded by a host-cell derived membrane. This membrane is modified by the pathogens to their own needs and is crucial for their intracellular lifestyle. In parasites, this membrane is referred to as the PVM and only a limited number of its proteins are known so far. Here, we applied in rodent parasites a method called BioID, which is based on biotinylation of proximal and interacting proteins by the promiscuous biotin ligase BirA*, and demonstrated its usefulness in identification of novel PVM proteins

BioID Reveals Novel Proteins of the Plasmodium Parasitophorous Vacuole Membrane

During their development within the vertebrate host, parasites infect hepatocytes and red blood cells. Within these cells, parasites are surrounded by a parasitophorous vacuole membrane (PVM). The PVM plays an essential role for the interaction of parasites with their host cells; however, only a limited number of proteins of this membrane have been identified so far. This is partially because systematic proteomic analysis of the protein content of the PVM has been difficult in the past, due to difficulties encountered in attempts to separate the PVM from other membranes such as the parasite plasma membrane. In this study, we adapted the BioID technique to -cultivated blood stage parasites and utilized the promiscuous biotin ligase BirA* fused to PVM-resident exported protein 1 to biotinylate proteins of the PVM. These we further processed by affinity purification, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and label-free quantitation, leading to a list of 61 known and candidate PVM proteins. Seven proteins were analyzed further during blood and liver stage development. This resulted in the identification of three novel PVM proteins, which were the serine/threonine protein phosphatase UIS2 (PlasmoDB accession no. PBANKA_1328000) and two conserved proteins with unknown functions (PBANKA_0519300 and PBANKA_0509000). In conclusion, our report expands the number of known PVM proteins and experimentally validates BioID as a powerful method to screen for novel constituents of specific cellular compartments in . Intracellular pathogens are often surrounded by a host-cell derived membrane. This membrane is modified by the pathogens to their own needs and is crucial for their intracellular lifestyle. In parasites, this membrane is referred to as the PVM and only a limited number of its proteins are known so far. Here, we applied in rodent parasites a method called BioID, which is based on biotinylation of proximal and interacting proteins by the promiscuous biotin ligase BirA*, and demonstrated its usefulness in identification of novel PVM proteins