<em>In Vivo</em> Assessment of Rodent <em>Plasmodium</em> Parasitemia and Merozoite Invasion by Flow Cytometry

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an ...

Macquarie University

Complex traits are under the genetic control of multiple genes, often with weak effects and strong epistatic interactions. We developed two new collections of mouse strains to improve genetic dissection of complex traits. They are derived from several backcrosses of the Mus spretus SEG/Pas or STF/Pas strains on the C57BL/6J background. Each of the 55 interspecific recombinant congenic strains (IRCSs) carries up to eight SEG/Pas chromosomal segments with an average size of 11.7 Mb, totalizing 1.37% of the genome. The complete series covers 39.7% of the SEG/Pas genome. As a complementary resource, six partial or complete interspecific consomic strains were developed and increased genome coverage to 45.6%. To evaluate the usefulness of these strains for QTL mapping, 16 IRCSs were compared with C57BL/6J for seven hematological parameters. Strain 66H, which carries three SEG/Pas chromosomal segments, had lower red blood cell volume and higher platelet count than C57BL/6J. Each chromosomal segment was isolated in a congenic strain to evaluate individual effects. Congenic strains were combined to assess epistasis. Our data show that both traits were controlled by several genes with complex epistatic interactions. IRCSs are therefore useful to unravel QTL with small effects and gene-by-gene interactions.

Interspecific recombinant congenic strains between C57BL/6 and mice of the Mus spretus species: a powerful tool to dissect genetic control of complex traits.

Recurrent Spontaneous Abortion (RSA) is a frequent pathology affecting 1 to 5% of couples. In approximately 50 % of cases, the aetiology is unknown suggesting a subtle interaction between genetic and environmental factors. Previous attempts to describe genetic factors using the candidate gene approach have been relatively unsuccessful due to the physiological, cellular and genetic complexity of mammalian reproduction. Indeed, fertility can be considered as a quantitative feature resulting from the interaction of genetic, epigenetic and environmental factors. Herein, we identified Quantitative Trait Loci (QTL) associated with diverse embryonic lethality phenotypes and the subsequent embryonic resorption in 39 inter-specific recombinant congenic mice strains, using in vivo ultrasound bio-microscopy. The short chromosomal intervals related to the phenotypes will facilitate the study of a restricted number of candidate genes which are potentially dysregulated in patients affected by RSA.

Identification of Quantitative Trait Loci responsible for embryonic lethality in mice assessed by ultrasonography.

To assess the genetic basis of the skull shape variation and morphological integration in mice, we have used a tool based on the cross between the distantly related mouse species Mus spretus (SEG/Pas strain) and the laboratory strain C57BL/6 called interspecific recombinant congenic strains (IRCSs). The genome of each IRCS consists on average of 1.3% of SEG/Pas derived sequences, located on multiple chromosomes as small-sized, DNA segments. Quantitative trait loci (QTL) on the skull shape, separated into dorsal and ventral sides, were analyzed in 17 IRCSs by a Procrustes superimposition method using 3D landmarks. The shapes of 16 strains differed significantly from C57BL/6. Discrepancy in the QTLs effects was found between the dorsal side and the anterior region of the ventral side due to a differential effect of the SEG/Pas alleles on the skull shape. A comprehensive analysis of all allelic combinations of the BCG-66H strain showed strong epistatic interactions between SEG/Pas segment acting on both skull sides. Epistatic pleiotropy and covariation between sides were dependent in SEG/Pas alleles direction and contributed to the strong morphological integration between sides. Introduction of Mus spretus alleles in a C57BL/6 background induced strong morphological changes mostly in SEG/Pas alleles direction and provided evidence for high level of morphological integration.

Genetic analysis of skull shape variation and morphological integration in the mouse using interspecific recombinant congenic strains between C57BL/6 and mice of the mus spretus species.

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Malaria is a disease that infects over 500 million people, causing at least 1 million deaths every year, with the majority occurring in developing countries. The current antimalarial arsenal is becoming dulled due to the rapid rate of resistance of the parasite. However, in populations living in malaria-endemic regions there are many examples of genetic-based resistance to the severe effects of the parasite Plasmodium. Defining the genetic factors behind host resistance has been an area of great scientific interest over the last few decades; this review summarizes the current knowledge of the genetic loci involved. Perhaps the lessons learned from the natural variation in both the human populations and experimental mouse models of infection may pave the way for novel resistance-proof antimalarials.

Host resistance to malaria: using mouse models to explore the host response.

Le paludisme est une maladie qui infecte plus de 500 millions de personnes, causant au moins 1 million de d&eacute;c&egrave;s chaque ann&eacute;e, avec la majorit&eacute; qui se produisent dans les pays en d&eacute;veloppement. L&#39;arsenal actuel antipaludique est devient &eacute;mouss&eacute;e en raison du rythme rapide de la r&eacute;sistance du parasite. Cependant, chez les populations vivant dans les r&eacute;gions o&ugrave; le paludisme est end&eacute;mique, il y a beaucoup d&#39;exemples de r&eacute;sistance g&eacute;n&eacute;tique aux effets graves du parasite Plasmodium. D&eacute;finir les facteurs g&eacute;n&eacute;tiques de r&eacute;sistance de l&#39;h&ocirc;te a &eacute;t&eacute; une zone de grand int&eacute;r&ecirc;t scientifique durant les derni&egrave;res d&eacute;cennies ; Cette revue r&eacute;sume les connaissances actuelles des loci g&eacute;n&eacute;tiques impliqu&eacute;s. Peut-&ecirc;tre les le&ccedil;ons tir&eacute;es de la variation naturelle dans les populations humaines et des mod&egrave;les exp&eacute;rimentaux de souris de l&#39;infection pourraient ouvrir la voie pour le roman &agrave; l&#39;&eacute;preuve de la r&eacute;sistance aux antipaludiques.

R&eacute;sistance au paludisme de l&#39;h&ocirc;te: &agrave; l&#39;aide de mod&egrave;les murins pour &eacute;tudier la r&eacute;ponse de l&#39;h&ocirc;te.

Malaria ist eine Erkrankung, die mehr als 500 Millionen Menschen, die mindestens 1 Million Todesopfer fordert jedes Jahr mit den meisten vorkommenden in Entwicklungsl&auml;ndern infiziert. Aktuelle Malariamedikamente Arsenal ist aufgrund der raschen Widerstand des Parasiten abgestumpft werden. Allerdings in Populationen, die in Malaria-endemischen Regionen Leben es gibt viele Beispiele f&uuml;r genetische basierende Widerstand gegen die schweren Auswirkungen der Parasit Plasmodium. Definieren der genetischen Faktoren hinter Host Widerstand wurde ein Gebiet von gro&szlig;em wissenschaftlichen Interesse in den letzten Jahrzehnten; diesen Bericht fasst das aktuelle Wissen der genetischen Loci beteiligt. Vielleicht die Lehren aus der nat&uuml;rlichen Schwankungen der beiden Bev&ouml;lkerungsgruppen und experimentelle Mausmodelle der Infektion k&ouml;nnen ebnen den Weg f&uuml;r neuartige Widerstand-Beweis-Malariamittel.

Resistenz gegen Malaria zu hosten: mit Maus-Modellen der Wirtsantwort erkunden.

La malaria &egrave; una malattia che infetta oltre 500 milioni di persone, causando almeno 1 milione di morti ogni anno, con la maggioranza che si verificano nei paesi in via di sviluppo. L&#39;attuale arsenale antimalarico &egrave; sempre offuscato a causa del rapido tasso di resistenza del parassita. Tuttavia, in popolazioni che vivono nelle regioni endemiche malaria ci sono molti esempi di resistenza genetica basata per i gravi effetti del parassita Plasmodium. Definire i fattori genetici dietro resistenza host &egrave; stata un&#39;area di grande interesse scientifico negli ultimi decenni; questa recensione riassume le conoscenze attuali dei loci genetici coinvolti. Forse le lezioni apprese dalla variazione naturale in entrambe le popolazioni umane e modelli sperimentali del mouse dell&#39;infezione possono spianare la strada a nuovi antimalarici prova di resistenza.

Ospitare resistenza alla malaria: utilizzo di modelli di mouse per esplorare la risposta dell&#39;ospite.

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La malaria es una enfermedad que infecta a m&aacute;s de 500 millones de personas, causando al menos 1 mill&oacute;n de muertes cada a&ntilde;o, con la mayor&iacute;a que ocurre en los pa&iacute;ses en desarrollo. El actual arsenal antimal&aacute;rico es convertirse en desafilar debido a la r&aacute;pida tasa de resistencia del par&aacute;sito. Sin embargo, en las poblaciones que viven en regiones donde la malaria es end&eacute;mica, hay muchos ejemplos de resistencia gen&eacute;tica basada en los graves efectos del par&aacute;sito Plasmodium. Definir los factores gen&eacute;ticos detr&aacute;s de resistencia anfitri&oacute;n ha sido un &aacute;rea de gran inter&eacute;s cient&iacute;fico en las &uacute;ltimas d&eacute;cadas; Esta revisi&oacute;n resume el conocimiento actual de los loci gen&eacute;ticos implicados. Quiz&aacute;s las lecciones aprendidas de la variaci&oacute;n natural en las poblaciones humanas de ambos y modelos de infecci&oacute;n experimental de rat&oacute;n pueden allanar el camino para nuevos medicamentos antimal&aacute;ricos de prueba de resistencia.

Sede de resistencia a la malaria: utilizar modelos de rat&oacute;n para explorar la respuesta del hu&eacute;sped.

Genetic determinism of cranial morphology in the mouse is still largely unknown, despite the localization of putative QTLs and the identification of genes associated with Mendelian skull malformations. To approach the dissection of this multigenic control, we have used a set of interspecific recombinant congenic strains (IRCS) produced between C57BL/6 and mice of the distant species Mus spretus (SEG/Pas). Each strain has inherited 1.3% of its genome from SEG/Pas under the form of few, small-sized, chromosomal segments.

Nasal bone shape is under complex epistatic genetic control in mouse interspecific recombinant congenic strains.

The blood stage of the plasmodium parasite life cycle is responsible for the clinical symptoms of malaria. Epidemiological studies have identified coincidental malarial endemicity and multiple red blood cell (RBC) disorders. Many RBC disorders result from mutations in genes encoding cytoskeletal proteins and these are associated with increased protection against malarial infections. However the mechanisms underpinning these genetic, host responses remain obscure. We have performed an N-ethyl-N-nitrosourea (ENU) mutagenesis screen and have identified a novel dominant (haploinsufficient) mutation in the Ank-1 gene (Ank1(MRI23420)) of mice displaying hereditary spherocytosis (HS). Female mice, heterozygous for the Ank-1 mutation showed increased survival to infection by Plasmodium chabaudi adami DS with a concomitant 30% decrease in parasitemia compared to wild-type, isogenic mice (wt). A comparative in vivo red cell invasion and parasite growth assay showed a RBC-autonomous effect characterised by decreased proportion of infected heterozygous RBCs. Within approximately 6-8 hours post-invasion, TUNEL staining of intraerythrocytic parasites, showed a significant increase in dead parasites in heterozygotes. This was especially notable at the ring and trophozoite stages in the blood of infected heterozygous mutant mice compared to wt (p<0.05). We conclude that increased malaria resistance due to ankyrin-1 deficiency is caused by the intraerythrocytic death of P. chabaudi parasites.

A novel ENU-mutation in ankyrin-1 disrupts malaria parasite maturation in red blood cells of mice.

Recurrent spontaneous abortion (RSA) is defined as the loss of three or more consecutive pregnancies during the first trimester of embryonic intrauterine development. This kind of human infertility is frequent among the general population since it affects 1 to 5% of women. In half of the cases the etiology remains unelucidated. In the present study, we used interspecific recombinant congenic mouse strains (IRCS) in the aim to identify genes responsible for embryonic lethality. Applying a cartographic approach using a genotype/phenotype association, we identified a minimal QTL region, of about 6 Mb on chromosome 1, responsible for a high rate of embryonic death (∼30%). Genetic analysis suggests that the observed phenotype is linked to uterine dysfunction. Transcriptomic analysis of the uterine tissue revealed a preferential deregulation of genes of this region compared to the rest of the genome. Some genes from the QTL region are associated with VEGF signaling, mTOR signaling and ubiquitine/proteasome-protein degradation pathways. This work may contribute to elucidate the molecular basis of a multifactorial and complex human disorder as RSA.

Refined mapping of a quantitative trait locus on chromosome 1 responsible for mouse embryonic death.

Morphological integration and modularity within semi-autonomous modules are essential mechanisms for the evolution of morphological traits. However, the genetic makeup responsible for the control of variational modularity is still relatively unknown. In our study, we tested the hypothesis that the genetic variation for mandible shape clustered into two morphogenetic components: the alveolar group and the ascending ramus. We used the mouse as a model system to investigate genetics determinants of mandible shape. To do this, we used a combination of geometric morphometric tools and a set of 18 interspecific recombinant congenic strains (IRCS) derived from the distantly related species, Mus spretus SEG/Pas and Mus musculus C57BL/6. Quantitative trait loci (QTL) analysis comparing mandible morphometry between the C57BL/6 and the IRCSs identified 42 putative SEG/Pas segments responsible for the genetic variation. The magnitude of the QTL effects was dependent on the proportion of SEG/Pas genome inherited. Using a multivariate correlation coefficient adapted for modularity assessment and a two-block partial least squares analysis to explore the morphological integration, we found that these QTL clustered into two well-integrated morphogenetic groups, corresponding to the ascending ramus and the alveolar region. Together, these results provide evidence that the mouse mandible is subjected to genetic coordination in a modular manner.

Exploration of the genetic organization of morphological modularity on the mouse mandible using a set of interspecific recombinant congenic strains between C57BL/6 and mice of the Mus spretus species.

Platelets restrict the growth of intraerythrocytic malaria parasites by binding to parasitized cells and killing the parasite within. Here, we show that the platelet molecule platelet factor 4 (PF4 or CXCL4) and the erythrocyte Duffy-antigen receptor (Fy) are necessary for platelet-mediated killing of Plasmodium falciparum parasites. PF4 is released by platelets on contact with parasitized red cells, and the protein directly kills intraerythrocytic parasites. This function for PF4 is critically dependent on Fy, which binds PF4. Genetic disruption of Fy expression inhibits binding of PF4 to parasitized cells and concomitantly prevents parasite killing by both human platelets and recombinant human PF4. The protective function afforded by platelets during a malarial infection may therefore be compromised in Duffy-negative individuals, who do not express Fy.

Platelet factor 4 and Duffy antigen required for platelet killing of Plasmodium falciparum.

Platelets, as well as regulating blood hemostasis, are an important component of the body's defense against invading microbial pathogens. We previously reported that platelets protect during malaria infection by binding Plasmodium-infected erythrocytes (IE) and killing the parasite within. More recent studies have now revealed the platelet plasmocidal factor, platelet factor 4 (PF4) and the red cell-expressed Duffy-antigen molecule as the central players in the parasite killing activity of platelets.

New insights into the protective power of platelets in malaria infection.

Malaria treatments are becoming less effective due to the rapid spread of drug resistant parasites. Increased understanding of the host/parasite interaction is crucial in order to develop treatments that will be less prone to resistance. Parasite invasion of the red blood cell (RBC) is a critical aspect of the parasite life cycle and is, therefore, a promising target for the development of malaria treatments. Assays for analysing parasite invasion in vitro have been developed, but no equivalent assays exist for in vivo studies. This article describes a novel flow cytometric in vivo parasite invasion assay.

A flow cytometric assay to quantify invasion of red blood cells by rodent Plasmodium parasites in vivo.

The human erythrocyte contains an abundance of the thiol-dependant peroxidase Peroxiredoxin-2 (Prx2), which protects the cell from the pro-oxidant environment it encounters during its 120 days of life in the blood stream. In malarial infections, the Plasmodium parasite invades red cells and imports Prx2 during intraerythrocytic development, presumably to supplement in its own degradation of peroxides generated during cell metabolism, especially hemoglobin (Hb) digestion. Here we demonstrate that an irreversible Prx2 inhibitor, Conoidin A (2,3-bis(bromomethyl)-1,4-dioxide-quinoxaline; BBMQ), has potent cytocidal activity against cultured P. falciparum. Parasite growth was also inhibited in red cells that were treated with BBMQ and then washed prior to parasite infection. These cells remained susceptible to merozoite invasion, but failed to support normal intraerythrocytic development. In addition the potency of chloroquine (CQ), an antimalarial drug that prevents the detoxification of Hb-derived heme, was significantly enhanced in the presence of BBMQ. CQ IC50 values decreased an order of magnitude when parasites were either co-incubated with BBMQ, or introduced into BBMQ-pretreated cells; these effects were equivalent for both drug-resistant and drug-sensitive parasite lines. Together these results indicate that treatment of red cells with BBMQ renders them incapable of supporting parasite growth and increases parasite sensitivity to CQ. We also propose that molecules such as BBMQ that target host cell proteins may constitute a novel host-directed therapeutic approach for treating malaria.

Treatment of erythrocytes with the 2-cys peroxiredoxin inhibitor, Conoidin A, prevents the growth of Plasmodium falciparum and enhances parasite sensitivity to chloroquine.

Many red cell polymorphisms are a result of selective pressure by the malarial parasite. Here, we add another red cell disease to the panoply of erythrocytic changes that give rise to resistance to malaria. Erythrocytes from individuals with erythropoietic protoporphyria (EPP) have low levels of the final enzyme in the heme biosynthetic pathway, ferrochelatase. Cells from these patients are resistant to the growth of Plasmodium falciparum malarial parasites. This phenomenon is due to the absence of ferrochelatase and not an accumulation of substrate, as demonstrated by the normal growth of P falciparum parasites in the EPP phenocopy, X-linked dominant protoporphyria, which has elevated substrate, and normal ferrochelatase levels. This observation was replicated in a mouse strain with a hypomorphic mutation in the murine ferrochelatase gene. The parasite enzyme is not essential for parasite growth as Plasmodium berghei parasites carrying a complete deletion of the ferrochelatase gene grow normally in erythrocytes, which confirms previous studies. That ferrochelatase is essential to parasite growth was confirmed by showing that inhibition of ferrochelatase using the specific competitive inhibitor, N-methylprotoporphyrin, produced a potent growth inhibition effect against cultures of P falciparum. This raises the possibility of targeting human ferrochelatase in a host-directed antimalarial strategy.

Gaetan Burgio

Australian School of Advanced Medicine

Macquarie University

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

Gaetan Burgio

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Australian School of Advanced Medicine

Macquarie University

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

Australian School of Advanced Medicine