Kasturi Haldar

Vacuolar uptake of host components, and a role for cholesterol and sphingomyelin in malarial infection

Erythrocytes, which are incapable of endocytosis or phagocytosis, can be infected by the malaria parasite Plasmodium falciparum. We find that a transmembrane protein (Duffy), glycosylphosphatidylinositol (GPI)‐anchored and cytoplasmic proteins, associated with detergent‐resistant membranes (DRMs) that are characteristic of microdomains in host cell membranes, are internalized by vacuolar parasites, while the major integral membrane and cytoskeletal proteins are not. The internalized host proteins and a plasmodial transmembrane resident parasitophorous vacuolar membrane (PVM) protein are detected in DRMs associated with vacuolar parasites. This is the first report of a host transmembrane protein being recruited into an apicomplexan vacuole and of the presence of vacuolar DRMs; it establishes that integral association does not preclude protein internalization into the P.falciparum vacuole. Rather, as shown for Duffy, intracellular accumulation occurs at the same rate as that seen for a DRM‐associated GPI‐anchored protein. Furthermore, novel mechanisms regulated by the DRM lipids, sphingomyelin and cholesterol, mediate (i) the uptake of host DRM proteins and (ii) maintenance of the intracellular vacuole in the non‐endocytic red cell, which may have implications for intracellular parasitism and pathogenesis.

Identification and localization of ERD2 in the malaria parasite Plasmodium falciparum: separation from sites of sphingomyelin synthesis and implications for organization of the Golgi.

The ERD2 gene product in mammalian cells and yeast is a receptor required for protein retention in the endoplasmic reticulum (ER); immunolocalization studies indicate that the protein is concentrated in the cis Golgi. We have identified a homologue of ERD2 in the malaria parasite, Plasmodium falciparum (PfERD2). The deduced protein sequence is 42% identical to mammalian and yeast homologues and bears striking homology in its proposed tertiary structure. PfERD2 is tightly confined to a single focus of staining in the perinuclear region as seen by indirect immunofluorescence. This is redistributed by brefeldin A (BFA) to a diffuse pattern similar to that of parasite BiP, a marker for the ER; removal of the drug results in recovery of the single focus, consistent with the localization of PfERD2 to the parasite Golgi and its participation in a retrograde transport pathway to the ER. Sphingomyelin synthesis is a second resident activity of the cis Golgi whose organization is sensitive to BFA in mammalian cells. Within the parasite it again localizes to a perinuclear region but does not reorganize upon BFA treatment. The results strongly suggest that these two activities are in distinct compartments of the Golgi in the malaria parasite.

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.

Identification of a stomatin orthologue in vacuoles induced in human erythrocytes by malaria parasites. A role for microbial raft proteins in apicomplexan vacuole biogenesis.

When the human malaria parasite Plasmodium falciparum infects erythrocytes, proteins associated with host-derived detergent-resistant membrane (DRM) rafts are selectively recruited into the newly formed vacuole, but parasite proteins that contribute to raft-based vacuole development are unknown. In mammalian cells, DRM-associated integral membrane proteins such as caveolin-1 and flotillin-1 that form oligomers have been linked to the formation of DRM-based invaginations called caveolae. Here we show that the P. falciparum genome does not encode caveolins or flotillins but does contain an orthologue of human band 7 stomatin, a protein known to oligomerize, associate with non-caveolar DRMs and is distantly related to flotillins. Stomatins are members of a large protein family conserved in evolution and P. falciparum (Pf) stomatin appears to be a prokaryotic-like molecule. Evidence is presented that it associates with DRMs and may oligomerize, suggesting that these features are conserved in the stomatin family. Further, Pfstomatin is an integral membrane protein concentrated at the apical end of extracellular parasites, where it co-localizes with invasion-associated rhoptry organelles. A resident rhoptry protein, RhopH2 also resides in DRMs. This provides the first evidence that rhoptries of an apicomplexan parasite contain DRM rafts. Further, when the parasite invades erythrocytes, rhoptry Pfstomatin and RhopH2 are inserted into the newly formed vacuole. Thus, like caveolin-1 and flotillin-1, a stomatin may also associate with non-clathrin coated, DRM-enriched vacuoles. We propose a new model of invasion and vacuole formation involving DRM-based interactions of both host and parasite molecules.

Salmonella enterica Serovar Typhimurium Requires Nonsterol Precursors of the Cholesterol Biosynthetic Pathway for Intracellular Proliferation

ABSTRACT We have previously shown that Salmonella enterica serovar Typhimurium infection perturbs the host cholesterol biosynthetic pathway. Here we show that inhibiting the first step of this pathway (3-hydroxy-3-methylglutaryl coenzyme A reductase) reduces the growth of intracellular S. enterica serovar Typhimurium and has no effect on extracellular bacterial growth. Selectively inhibiting synthesis of downstream sterol components has no effect on infection, suggesting that the effect of statins on host nonsterol intermediates is detrimental to bacterial growth. Furthermore, statins also reduce bacterial proliferation in the S. enterica serovar Typhimurium mouse model. This suggests that blocking the production of nonsterol precursors in the host cell can be used to reduce infection.

Cooperative domains define a unique host cell-targeting signal in Plasmodium falciparum-infected erythrocytes.

When the malaria parasite Plasmodium falciparum infects an erythrocyte, it resides in a parasitophorous vacuole and remarkably exports proteins into the periphery of its host cell. Two of these proteins, the histidine-rich proteins I and II (PfHRPI and PfHRPII), are exported to the erythrocyte cytoplasm. PfHRPI has been linked to cell-surface “knobby” protrusions that mediate cerebral malaria and are a frequent cause of death. PfHRPII has been implicated in (i) the production of hemozoin, the black pigment associated with disease, as well as (ii) interactions with the erythrocyte cytoskeleton. Here we show that a tripartite signal that is comprised of an endoplasmic reticulum-type signal sequence followed by a bipartite vacuolar translocation signal derived from HRPII and HRPI exports GFP from the parasitophorous vacuole to the host cytoplasm. The bipartite vacuolar translocation signal is comprised of unique, peptidic (≈40-aa) sequences. A domain within it contains the signal for export to “cleft” transport intermediates in the host erythrocyte and may thereby regulate the pathway of export to the host cytoplasm. A signal for posttranslational, vacuolar exit of proteins has hitherto not been described in eukaryotic secretion.

The Salmonella-containing vacuole is a major site of intracellular cholesterol accumulation and recruits the GPI-anchored protein CD55

Intracellular, pathogenic Salmonella typhimurium avoids phago‐lysosome fusion, and exists within a unique vacuolar niche that resembles a late endosome. This model has emerged from studying the trafficking of host proteins to the Salmonella‐containing vacuole (SCV). Very little is known about the role of major host lipids during infection. Here, we show using biochemical analyses as well as fluorescence microscopy, that intracellular infection perturbs the host sterol biosynthetic pathway and induces cholesterol accumulation in the SCV. Cholesterol accumulation is seen in both macrophages and epithelial cells: at the terminal stages of infection, as much as 30% of the total cellular cholesterol resides in the SCV. We find that accumulation of cholesterol in the SCV is linked to intracellular bacterial replication and may be dependent on Salmonella pathogenicity island 2 (SPI‐2). Furthermore, the construction of a three‐dimensional space‐filling model yields novel insights into the structure of the SCV: bacteria embedded in cholesterol‐rich membranes. Finally, we show that the glycosylphosphatidylinositol (GPI)‐anchored protein CD55 is recruited to the SCV. These data suggest that, in contrast to prevailing models, the SCV accumulates components of cholesterol‐rich early endocytic pathways during intracellular bacterial replication.

Esterification of cholesterol by a type III secretion effector during intracellular Salmonella infection

Survival of Salmonella typhimurium within a vacuole in host cells depends on secreted virulence factors encoded by the Salmonella pathogenicity island 2 (SPI‐2). High levels of cholesterol are detected at the Salmonella‐containing vacuole (SCV). Here we show that the SPI‐2 effector SseJ esterifies cholesterol in vitro, in cells and during infection. Intracellular infections with wild‐type as compared with ΔsseJ bacteria led to higher levels of cholesterol ester production in HeLa cells and RAW macrophages and were shown to increase levels of lipid droplets (structures enriched in cholesterol esters). Ectopic expression of SseJ reduced cholesterol levels in cellular membranes and antagonized a major membrane activity of a second bacterial effector known to be important to the stability of the SCV. Previous studies in mouse models of infection have established a virulence defect in ΔsseJ bacteria and have suggested a role for SseJ in regulating SCV dynamics. Our data indicating the molecular activity of SseJ suggest that cholesterol and its esterification at the SCV are functionally important for intracellular bacterial survival.

Common infection strategies of pathogenic eukaryotes

Pathogenic eukaryotes belong to several distinct phylogenetic lineages and have evolved the ability to colonize a range of hosts, including animals and plants. Pathogenic lifestyles have evolved repeatedly in eukaryotes, indicating that unique molecular processes are involved in host infection. However, evidence is now emerging that divergent eukaryotic pathogens might share common mechanisms of pathogenicity. The results from recent studies demonstrate that Plasmodium falciparum and Phytophthora infestans use equivalent host-targeting signals to deliver virulence adhesins and avirulence gene products into human and plant cells, respectively. Remodelling of host cells by different eukaryotic pathogens might therefore share some common features.

Erythrocyte remodeling by malaria parasites

Purpose of reviewPlasmodium falciparum causes the most virulent form of human malarias. It is a protozoan parasite that infects human erythrocytes and the erythrocytic stages are responsible for all symptoms and pathologies of the disease. Critical to infection is the formation of a parasitophorous vacuolar membrane at the time of entry and within which the intracellular parasite proliferates. Since erythrocytes lack endocytic machinery, it is surprising that they can be infected by pathogens. This review summarizes recent studies of the erythrocyte–malaria interaction that have provided insights into properties of erythrocyte membranes as well as parasite mechanisms that remodel the erythrocyte. Recent findingsThemes revealed by recent literature suggest that both parasite and erythrocyte components regulate parasite entry and intracellular growth by extensively remodeling host membranes. These remodeling events include the invagination of the host cell membrane during parasite entry that results in the creation and maintenance of a vacuole that surrounds the intracellular organism, and the development of antigenic, structural and transport alterations during intracellular parasite development. SummaryThe implications are that malarial erythrocyte remodeling events occur at a significant cost to the human host since many of the associated virulence events have been linked to severe disease pathologies.