Malaria Parasite clag3 Genes Determine Channel-Mediated Nutrient Uptake by Infected Red Blood Cells

SummaryDevelopment of malaria parasites within vertebrate erythrocytes requires nutrient uptake at the host cell membrane. The plasmodial surface anion channel (PSAC) mediates this transport and is an antimalarial target, but its molecular basis is unknown. We report a parasite gene family responsible for PSAC activity. We used high-throughput screening for nutrient uptake inhibitors to identify a compound highly specific for channels from the Dd2 line of the human pathogen P. falciparum. Inheritance of this compound's affinity in a Dd2 × HB3 genetic cross maps to a single parasite locus on chromosome 3. DNA transfection and in vitro selections indicate that PSAC-inhibitor interactions are encoded by two clag3 genes previously assumed to function in cytoadherence. These genes are conserved in plasmodia, exhibit expression switching, and encode an integral protein on the host membrane, as predicted by functional studies. This protein increases host cell permeability to diverse solutes.PaperFlic

The inner membrane complex through development of Toxoplasma gondii and Plasmodium

Plasmodium spp. and Toxoplasma gondii are important human and veterinary pathogens. These parasites possess an unusual double membrane structure located directly below the plasma membrane named the inner membrane complex (IMC). First identified in early electron micrograph studies, huge advances in genetic manipulation of the Apicomplexa have allowed the visualization of a dynamic, highly structured cellular compartment with important roles in maintaining the structure and motility of these parasites. This review summarizes recent advances in the field and highlights the changes the IMC undergoes during the complex life cycles of the Apicomplexa

A Novel Family of Toxoplasma IMC Proteins Displays a Hierarchical Organization and Functions in Coordinating Parasite Division

Apicomplexans employ a peripheral membrane system called the inner membrane complex (IMC) for critical processes such as host cell invasion and daughter cell formation. We have identified a family of proteins that define novel sub-compartments of the Toxoplasma gondii IMC. These IMC Sub-compartment Proteins, ISP1, 2 and 3, are conserved throughout the Apicomplexa, but do not appear to be present outside the phylum. ISP1 localizes to the apical cap portion of the IMC, while ISP2 localizes to a central IMC region and ISP3 localizes to a central plus basal region of the complex. Targeting of all three ISPs is dependent upon N-terminal residues predicted for coordinated myristoylation and palmitoylation. Surprisingly, we show that disruption of ISP1 results in a dramatic relocalization of ISP2 and ISP3 to the apical cap. Although the N-terminal region of ISP1 is necessary and sufficient for apical cap targeting, exclusion of other family members requires the remaining C-terminal region of the protein. This gate-keeping function of ISP1 reveals an unprecedented mechanism of interactive and hierarchical targeting of proteins to establish these unique sub-compartments in the Toxoplasma IMC. Finally, we show that loss of ISP2 results in severe defects in daughter cell formation during endodyogeny, indicating a role for the ISP proteins in coordinating this unique process of Toxoplasma replication

Regulation of in vitro and in vivo Hepatic Stellate Cell Activation by the Ayrl Hydrocarbon Receptor

Liver fibrosis is a pathological condition characterized by the excessive deposition of extracellular matrix material by activated hepatic stellate cells (HSCs). We recently reported that activation of the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increases HSC activation in vitro and in mouse models of experimental liver fibrosis. The goal of this project was to determine the mechanism by which AhR activation impacts HSC activation and the subsequent development of liver fibrosis. It is possible that HSCs are direct cellular targets for TCDD. Alternatively, TCDD could increase HSC activation indirectly by exacerbating hepatocyte damage and inflammation. To investigate this, we generated mice in which the AhR was selectively removed from either hepatocytes or HSCs to determine the ramifications on liver injury, inflammation, and HSC activation in an experimental model of liver fibrosis elicited by chronic administration of TCDD. Results from these studies indicate that TCDD does not directly activate HSCs in the mouse liver to produce fibrosis. Instead, it appears that TCDD-induced changes in hepatocytes, such as the development of steatosis, are what ultimately stimulate HSC activation and produce fibrosis. A second focus of this project was to investigate an endogenous role for AhR signaling in the regulation of HSC activation in the absence of liver injury and inflammation. To this end, I used CRISPR/Cas9 technology to knock down the AhR in the human HSC cell line, LX-2. I discovered that a functional AhR is required for optimal proliferation of activated HSCs. However, other endpoints of HSC activation, such as the production of collagen type I, were not impacted by the removal of AhR signaling. These findings are important because the AhR has been shown to be a druggable target, and there is growing interest in therapeutically modulating AhR activity to prevent or reverse HSC activation. Collectively, results from this project indicate that therapeutically targeting AhR signaling in hepatocytes, instead of AhR signaling in HSCs, might be a preferred approach for limiting HSC activation and preventing or diminishing liver fibrosis

Proteolysis at a specific extracellular residue implicates integral membrane CLAG3 in malaria parasite nutrient channels.

The plasmodial surface anion channel mediates uptake of nutrients and other solutes into erythrocytes infected with malaria parasites. The clag3 genes of P. falciparum determine this channel's activity in human malaria, but how the encoded proteins contribute to transport is unknown. Here, we used proteases to examine the channel's composition and function. While proteases with distinct specificities all cleaved within an extracellular domain of CLAG3, they produced differing degrees of transport inhibition. Chymotrypsin-induced inhibition depended on parasite genotype, with channels induced by the HB3 parasite affected to a greater extent than those of the Dd2 clone. Inheritance of functional proteolysis in the HB3×Dd2 genetic cross, DNA transfection, and gene silencing experiments all pointed to the clag3 genes, providing independent evidence for a role of these genes. Protease protection assays with a Dd2-specific inhibitor and site-directed mutagenesis revealed that a variant L1115F residue on a CLAG3 extracellular loop contributes to inhibitor binding and accounts for differences in functional proteolysis. These findings indicate that surface-exposed CLAG3 is the relevant pool of this protein for channel function. They also suggest structural models for how exposed CLAG3 domains contribute to pore formation and parasite nutrient uptake

Effects of proteases on CLAG3 and PSAC-mediated transport.

<p>(A) Immunoblots showing CLAG3 hydrolysis in HB3 and Dd2 parasites by chymotrypsin (“Ch”), trypsin (“Tr”), or pronase E (“PrE”); mouse anti-CLAG3 generated using a recombinant C-terminal fragment <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093759#pone.0093759-Nguitragool1" target="_blank">[23]</a>. The band at ∼160 kDa reflects uncleaved CLAG3; a C-terminal proteolysis fragment at ∼35 kDa is visible upon protease treatment. Addition of 2 mM PMSF abolishes cleavage by chymotrypsin. (B) Osmotic lysis kinetics for HB3- and Dd2-infected cells in sorbitol. Control traces represent matched samples not exposed to proteases or PMSF (black traces). While pronase E retards PSAC-mediated osmotic lysis and trypsin is without effect in both parasites (green and blue traces, respectively), chymotrypsin inhibits transport in HB3- but not Dd2-infected cells (red solid traces). (C) Mean ± S.E.M. PSAC inhibition determined from osmotic lysis experiments, normalized to 0% for no protease controls. (D) Mean ± S.E.M. inhibition resulting from chymotrypsin treatment of erythrocytes infected with indicated parasites.</p

A variant extracellular residue accounts for PSAC resistance to chymotrypsin in Dd2 parasites.

<p>(A) Multiple sequence alignment of an extracellular loop on indicated CLAG3.1 and CLAG3.2 sequences, representing geographically divergent parasites (Dd2, from Indochina; 3D7, probably Africa; 7G8 Brazil; HB3, Honduras). A variable segment is apparent in gray shading (Consensus); residues susceptible to chymotrypsin cleavage are shown in blue. Two sites refractory to cleavage in Dd2 CLAG3.1 are highlighted in red. (B) Schematic showing the allelic exchange strategy to introduce a single mutation in the Dd2 <i>clag3.1</i> gene. Plasmid carrying the mutation is shown at the top; single homologous recombination into the Dd2 genome produces an intact full-length gene with a single site mutation and unchanged UTR sequences. (C) Southern blot showing integration of plasmid into the Dd2^L1115F genome. Indicated DNA samples were digested and probed with an hDHFR-specific probe. Dd2 is not recognized by this probe, but Dd2^L1115F yields a single band whose size differs from that of the plasmid. (D) Osmotic lysis kinetics for Dd2^L1115F without and with 1h chymotrypsin treatment (black and red traces, respectively). Inset shows preferential expression of the mutated <i>clag3.1</i> gene in this clone. (E) Mean ± S.E.M. chymotrypsin-induced inhibition for indicated parasites. (F) Dose responses for inhibition of sorbitol permeability (<i>P</i>) by ISPA-28. Black and red symbols represent mean ± S.E.M. inhibition for Dd2 and Dd2^L1115F parasites, respectively. Solid lines represent best fits to the sum of two Langmuir isotherms.</p

<i>clag3</i> genes accounts for the differing sensitivities to chymotrypsin.

<p>(A) Mean ± S.E.M. block of sorbitol uptake by chymotrypsin treatment on indicated parental lines and progeny clones (black and gray bars, respectively). (B) Logarithm of odds (LOD) scores from a primary scan of QTL associated with PSAC inhibition. The peak at the 5′ end of chromosome 3 contains the two <i>clag3</i> genes. The <i>P</i> = 0.05 significance threshold (dashed horizontal line) was calculated from 1000 permutations. Inset shows results from a secondary scan for additional QTL after controlling for the <i>clag3</i> locus. No other loci reached the <i>P</i> = 0.05 threshold (dashed horizontal line). (C) Osmotic lysis kinetics for indicated parasites after selection for expression of a specific <i>clag3</i> gene. Black and red traces represent no protease control and chymotrypsin-treated cells, respectively. The ribbon schematic at the top of each panel shows the gene structure for the two <i>clag3</i> genes in each parasite with active transcription indicated by a bent arrow. The <i>clag3</i> gene resulting from allelic exchange in HB3<i>^3rec</i> has a gray shaded 3′ end to indicate the fragment derived from Dd2 (“<i>chimera</i>”). For each parasite, relative expression of the two paralogs is shown with an ethidium-stained gel at the bottom right of each panel. (D) Mean ± S.E.M. chymotrypsin-induced inhibition for each selected parasite.</p