P. falciparum Modulates Erythroblast Cell Gene Expression in Signaling and Erythrocyte Production Pathways

Global, genomic responses of erythrocytes to infectious agents have been difficult to measure because these cells are e-nucleated. We have previously demonstrated that in vitro matured, nucleated erythroblast cells at the orthochromatic stage can be efficiently infected by the human malaria parasite Plasmodium falciparum. We now show that infection of orthochromatic cells induces change in 609 host genes. 592 of these transcripts are up-regulated and associated with metabolic and chaperone pathways unique to P. falciparum infection, as well as a wide range of signaling pathways that are also induced in related apicomplexan infections of mouse hepatocytes or human fibroblast cells. Our data additionally show that polychromatophilic cells, which precede the orthochromatic stage and are not infected when co-cultured with P. falciparum, up-regulate a small set of genes, at least two of which are associated with pathways of hematopoiesis and/or erythroid cell development. These data support the idea that P. falciparum affects erythropoiesis at multiple stages during erythroblast differentiation. Further P. falciparum may modulate gene expression in bystander erythroblasts and thus influence pathways of erythrocyte development. This study provides a benchmark of the host erythroblast cell response to infection by P. falciparum

The Malaria Secretome: From Algorithms to Essential Function in Blood Stage Infection

The malaria agent Plasmodium falciparum is predicted to export a “secretome” of several hundred proteins to remodel the host erythrocyte. Prediction of protein export is based on the presence of an ER-type signal sequence and a downstream Host-Targeting (HT) motif (which is similar to, but distinct from, the closely related Plasmodium Export Element [PEXEL]). Previous attempts to determine the entire secretome, using either the HT-motif or the PEXEL, have yielded large sets of proteins, which have not been comprehensively tested. We present here an expanded secretome that is optimized for both P. falciparum signal sequences and the HT-motif. From the most conservative of these three secretome predictions, we identify 11 proteins that are preserved across human- and rodent-infecting Plasmodium species. The conservation of these proteins likely indicates that they perform important functions in the interaction with and remodeling of the host erythrocyte important for all Plasmodium parasites. Using the piggyBac transposition system, we validate their export and find a positive prediction rate of ∼70%. Even for proteins identified by all secretomes, the positive prediction rate is not likely to exceed ∼75%. Attempted deletions of the genes encoding the conserved exported proteins were not successful, but additional functional analyses revealed the first conserved secretome function. This gave new insight into mechanisms for the assembly of the parasite-induced tubovesicular network needed for import of nutrients into the infected erythrocyte. Thus, genomic screens combined with functional assays provide unexpected and fundamental insights into host remodeling by this major human pathogen

Antimalarial drug discovery and characterization with DNA microarrays.

Antimalarial drug discovery and characterization with DNA microarrays

<i>P. falciparum</i> Modulates Erythroblast Cell Gene Expression in Signaling and Erythrocyte Production Pathways

Global, genomic responses of erythrocytes to infectious agents have been difficult to measure because these cells are e-nucleated. We have previously demonstrated that in vitro matured, nucleated erythroblast cells at the orthochromatic stage can be efficiently infected by the human malaria parasite Plasmodium falciparum. We now show that infection of orthochromatic cells induces change in 609 host genes. 592 of these transcripts are up-regulated and associated with metabolic and chaperone pathways unique to P. falciparum infection, as well as a wide range of signaling pathways that are also induced in related apicomplexan infections of mouse hepatocytes or human fibroblast cells. Our data additionally show that polychromatophilic cells, which precede the orthochromatic stage and are not infected when co-cultured with P. falciparum, up-regulate a small set of genes, at least two of which are associated with pathways of hematopoiesis and/or erythroid cell development. These data support the idea that P. falciparum affects erythropoiesis at multiple stages during erythroblast differentiation. Further P. falciparum may modulate gene expression in bystander erythroblasts and thus influence pathways of erythrocyte development. This study provides a benchmark of the host erythroblast cell response to infection by P. falciparum.</p

An erythrocyte vesicle protein exported by the malaria parasite promotes tubovesicular lipid import from the host cell surface.

Plasmodium falciparum is the protozoan parasite that causes the most virulent of human malarias. The blood stage parasites export several hundred proteins into their host erythrocyte that underlie modifications linked to major pathologies of the disease and parasite survival in the blood. Unfortunately, most are 'hypothetical' proteins of unknown function, and those that are essential for parasitization of the erythrocyte cannot be 'knocked out'. Here, we combined bioinformatics and genome-wide expression analyses with a new series of transgenic and cellular assays to show for the first time in malaria parasites that microarray read out from a chemical perturbation can have predictive value. We thereby identified and characterized an exported P. falciparum protein resident in a new vesicular compartment induced by the parasite in the erythrocyte. This protein, named Erythrocyte Vesicle Protein 1 (EVP1), shows novel dynamics of distribution in the parasite and intraerythrocytic membranes. Evidence is presented that its expression results in a change in TVN-mediated lipid import at the host membrane and that it is required for intracellular parasite growth, but not invasion. This exported protein appears to be needed for the maintenance of an essential tubovesicular nutrient import pathway induced by the pathogen in the host cell. Our approach may be generalized to the analysis of hundreds of 'hypothetical' P. falciparum proteins to understand their role in parasite entry and/or growth in erythrocytes as well as phenotypic contributions to either antigen export or tubovesicular import. By functionally validating these unknowns, one may identify new targets in host-microbial interactions for prophylaxis against this major human pathogen

Genomic Expression Analyses Reveal Lysosomal, Innate Immunity Proteins, as Disease Correlates in Murine Models of a Lysosomal Storage Disorder

<div><p>Niemann-Pick Type C (NPC) disease is a rare, genetic, lysosomal disorder with progressive neurodegeneration. Poor understanding of the pathophysiology and a lack of blood-based diagnostic markers are major hurdles in the treatment and management of NPC and several additional, neurological lysosomal disorders. To identify disease severity correlates, we undertook whole genome expression profiling of sentinel organs, brain, liver, and spleen of <em>Balb/c Npc1^−/−</em> mice relative to <em>Npc1^+/−</em> at an asymptomatic stage, as well as early- and late-symptomatic stages. Unexpectedly, we found prominent up regulation of innate immunity genes with age-dependent change in their expression, in all three organs. We shortlisted a set of 12 secretory genes whose expression steadily increased with age in both brain and liver, as potential plasma correlates of neurological and/or liver disease. Ten were innate immune genes with eight ascribed to lysosomes. Several are known to be elevated in diseased organs of murine models of other lysosomal diseases including Gaucher’s disease, Sandhoff disease and MPSIIIB. We validated the top candidate lysozyme, in the plasma of <em>Npc1^−/−</em> as well as <em>Balb/c Npc1^nmf164</em> mice (bearing a point mutation closer to human disease mutants) and show its reduction in response to an emerging therapeutic. We further established elevation of innate immunity in <em>Npc1^−/−</em> mice through multiple functional assays including inhibition of bacterial infection as well as cellular analysis and immunohistochemistry. These data revealed neutrophil elevation in the <em>Npc1</em>^−/− spleen and liver (where large foci were detected proximal to damaged tissue). Together our results yield a set of lysosomal, secretory innate immunity genes that have potential to be developed as pan or specific plasma markers for neurological diseases associated with lysosomal storage and where diagnosis is a major problem. Further, the accumulation of neutrophils in diseased organs (hitherto not associated with NPC) suggests their role in pathophysiology and disease exacerbation.</p> </div

Stage-specific susceptibility of human erythroblasts to Plasmodium falciparum malaria infection

Malaria parasites are known to invade and develop in erythrocytes and reticulocytes, but little is known about their infection of nucleated erythroid precursors. We used an in vitro cell system that progressed through basophilic, polychromatic, orthochromatic, and reticulocyte stages to mature erythrocytes. We show that orthochromatic cells are the earliest stages that may be invaded by Plasmodium falciparum, the causative agent of fatal human malaria. Susceptibility to invasion is distinct from intracellular survival and occurs at a time of extensive erythroid remodeling. Together these data suggest that the potential for complexity of host interactions involved in infection may be vastly greater than hitherto realized

An erythrocyte vesicle protein exported by the malaria parasite promotes tubovesicular lipid import from the host cell surface.

Plasmodium falciparum is the protozoan parasite that causes the most virulent of human malarias. The blood stage parasites export several hundred proteins into their host erythrocyte that underlie modifications linked to major pathologies of the disease and parasite survival in the blood. Unfortunately, most are 'hypothetical' proteins of unknown function, and those that are essential for parasitization of the erythrocyte cannot be 'knocked out'. Here, we combined bioinformatics and genome-wide expression analyses with a new series of transgenic and cellular assays to show for the first time in malaria parasites that microarray read out from a chemical perturbation can have predictive value. We thereby identified and characterized an exported P. falciparum protein resident in a new vesicular compartment induced by the parasite in the erythrocyte. This protein, named Erythrocyte Vesicle Protein 1 (EVP1), shows novel dynamics of distribution in the parasite and intraerythrocytic membranes. Evidence is presented that its expression results in a change in TVN-mediated lipid import at the host membrane and that it is required for intracellular parasite growth, but not invasion. This exported protein appears to be needed for the maintenance of an essential tubovesicular nutrient import pathway induced by the pathogen in the host cell. Our approach may be generalized to the analysis of hundreds of 'hypothetical' P. falciparum proteins to understand their role in parasite entry and/or growth in erythrocytes as well as phenotypic contributions to either antigen export or tubovesicular import. By functionally validating these unknowns, one may identify new targets in host-microbial interactions for prophylaxis against this major human pathogen.</p

List of 12 potential biomarker genes.

<p>Genes marked in bold code for secretory lysosomal proteins.</p

Top 20 up regulated genes in spleen of <i>Npc1^−/−</i> mice across three age groups (20–71 days).

<p>Genes marked in bold are related to innate immunity and the genes marked in bold and also underlined are innate immunity genes catalogued by InnateDB.</p