Recruitment of erythrocyte membrane components by apicomplexan parasites Babesia divergens and Plasmodium falciparum

There are a few parasitic protozoa that infect erythrocyte and the erythrocyte does not play any essential or even obligatory role in the parasite survival and subsequent development for most of these, but for two apicomplexan parasites of the genera Plasmodium and Babesia. For these parasites, the host erythrocyte plays a vital role in survival and in associated pathogenicity (directly or indirectly). Moreover these two parasites are also known to alter the host cell differentially; for what seems customising it to specific requirements. Of these alterations one prominent is the formation of the unique vacuolar compartment ‘parasitophorus vacuole’ surrounded by ‘parasitophorus vacuole membrane. However our idea about the contribution of the host cell towards the PVM is limited. In absence of any marker for PVM, the formation and the fate too had not been studied in details but in a few apicomplexa. I took the advantage of a Babesia divergens strain, adapted to human erythrocytes (B. divergens normally infect cattle or immuno-suppressed humans) and used Plasmodium falciparum clone 3D7 (routinely cultured in human erythrocyte) and did a detailed comparative analysis between the PVM formed the invasion of these two related obligate intracellular apicomplexa in identical host cell (erythrocyte). Ultrastructure analysis of infected erythrocytes revealed that unlike Plasmodium falciparum, which remained inside the PV all along the intra-erythrocytic development, the Babesia divergens lost its PVM sometime soon after invasion: an observation, possibly indicating that the maintenance of the PV per se as a protective environment is not a prerequisite for this parasite growth. Thereafter with a strict selection of erythrocyte membrane proteins (membrane anchor containing, membrane spanning, cytoskeletal, cytoskeleton associated and erythrocyte surface receptor), reportedly internalized or discounted during the invasion by P. falciparum and present or absent on the newly formed PVM based on their association with erythrocyte cytoskeleton, I performed epifluorescence microscopy and biochemical analysis. I aimed to demonstrate the fate of these proteins parallaly in P. falciparum and B. divergens infected erythrocytes. I took help of immuno-electron microscopy to confirm my results. With fluorescence microscopy, I could show that both of the parasites took up labelled lipid components were from the labelled erythrocyte surface and recruited them onto their respective PVMs. However there was a difference in the recruitment of proteins between these two. A high copy number, erythrocyte membrane protein (Band 3) and a cytoskeletal protein (Spectrin) was found present in the PVM of Babesia divergens but not in PVM of Plasmodium falciparum. Parallel to this in B. divergens infected erythrocytes; PVM-localization could not be confirmed for few proteins, for which incorporation into the PVM of P. falciparum had been suggested in several reports. Altogether the results obtained from this study suggest that the recruitment or exclusion of specific membrane components is determined in a parasite specific manner and is not regulated by the intrinsic properties of the erythrocyte membrane. However incorporation or exclusion of different proteins may also reflect difference in the preferred entry sites for these parasites, leading ultimately to the difference in components of the PVM and show the possibility of using such parasites as molecular tools for understanding the inducible physiological processes, generally silent in such quiescent cells (erythrocyte)

Recruitment of erythrocyte membrane components by apicomplexan parasites Babesia divergens and Plasmodium falciparum

There are a few parasitic protozoa that infect erythrocyte and the erythrocyte does not play any essential or even obligatory role in the parasite survival and subsequent development for most of these, but for two apicomplexan parasites of the genera Plasmodium and Babesia. For these parasites, the host erythrocyte plays a vital role in survival and in associated pathogenicity (directly or indirectly). Moreover these two parasites are also known to alter the host cell differentially; for what seems customising it to specific requirements. Of these alterations one prominent is the formation of the unique vacuolar compartment ‘parasitophorus vacuole’ surrounded by ‘parasitophorus vacuole membrane. However our idea about the contribution of the host cell towards the PVM is limited. In absence of any marker for PVM, the formation and the fate too had not been studied in details but in a few apicomplexa. I took the advantage of a Babesia divergens strain, adapted to human erythrocytes (B. divergens normally infect cattle or immuno-suppressed humans) and used Plasmodium falciparum clone 3D7 (routinely cultured in human erythrocyte) and did a detailed comparative analysis between the PVM formed the invasion of these two related obligate intracellular apicomplexa in identical host cell (erythrocyte). Ultrastructure analysis of infected erythrocytes revealed that unlike Plasmodium falciparum, which remained inside the PV all along the intra-erythrocytic development, the Babesia divergens lost its PVM sometime soon after invasion: an observation, possibly indicating that the maintenance of the PV per se as a protective environment is not a prerequisite for this parasite growth. Thereafter with a strict selection of erythrocyte membrane proteins (membrane anchor containing, membrane spanning, cytoskeletal, cytoskeleton associated and erythrocyte surface receptor), reportedly internalized or discounted during the invasion by P. falciparum and present or absent on the newly formed PVM based on their association with erythrocyte cytoskeleton, I performed epifluorescence microscopy and biochemical analysis. I aimed to demonstrate the fate of these proteins parallaly in P. falciparum and B. divergens infected erythrocytes. I took help of immuno-electron microscopy to confirm my results. With fluorescence microscopy, I could show that both of the parasites took up labelled lipid components were from the labelled erythrocyte surface and recruited them onto their respective PVMs. However there was a difference in the recruitment of proteins between these two. A high copy number, erythrocyte membrane protein (Band 3) and a cytoskeletal protein (Spectrin) was found present in the PVM of Babesia divergens but not in PVM of Plasmodium falciparum. Parallel to this in B. divergens infected erythrocytes; PVM-localization could not be confirmed for few proteins, for which incorporation into the PVM of P. falciparum had been suggested in several reports. Altogether the results obtained from this study suggest that the recruitment or exclusion of specific membrane components is determined in a parasite specific manner and is not regulated by the intrinsic properties of the erythrocyte membrane. However incorporation or exclusion of different proteins may also reflect difference in the preferred entry sites for these parasites, leading ultimately to the difference in components of the PVM and show the possibility of using such parasites as molecular tools for understanding the inducible physiological processes, generally silent in such quiescent cells (erythrocyte)

Alternative Protein Secretion in the Malaria Parasite <i>Plasmodium falciparum</i>

<div><p><i>Plasmodium falciparum</i> invades human red blood cells, residing in a parasitophorous vacuole (PV), with a parasitophorous vacuole membrane (PVM) separating the PV from the host cell cytoplasm. Here we have investigated the role of <i>N</i>-myristoylation and two other N-terminal motifs, a cysteine potential S-palmitoylation site and a stretch of basic residues, as the driving force for protein targeting to the parasite plasma membrane (PPM) and subsequent translocation across this membrane. <i>Plasmodium falciparum</i> adenylate kinase 2 (<i>Pf</i> AK2) contains these three motifs, and was previously proposed to be targeted beyond the parasite to the PVM, despite the absence of a signal peptide for entry into the classical secretory pathway. Biochemical and microscopy analyses of <i>Pf</i>AK2 variants tagged with green fluorescent protein (GFP) showed that these three motifs are involved in targeting the protein to the PPM and translocation across the PPM to the PV. It was shown that the N-terminal 37 amino acids of <i>Pf</i>AK2 alone are sufficient to target and translocate GFP across the PPM. As a control we examined the N-myristoylated <i>P</i>. <i>falciparum</i> ADP-ribosylation factor 1 (<i>Pf</i>ARF1). <i>Pf</i>ARF1 was found to co-localise with a Golgi marker. To determine whether or not the putative palmitoylation and the cluster of lysine residues from the N-terminus of <i>Pf</i>AK2 would modulate the subcellular localization of <i>Pf</i>ARF1, a chimeric fusion protein containing the N-terminus of <i>Pf</i>ARF1 and the two additional <i>Pf</i>AK2 motifs was analysed. This chimeric protein was targeted to the PPM, but not translocated across the membrane into the PV, indicating that other features of the N-terminus of <i>Pf</i>AK2 also play a role in the secretion process.</p></div

Subcellular locations of <i>Pf</i>AK2^C4A/GFP in which the cysteine at position 4 of AK2 has been replaced with alanine, and <i>Pf</i>AK2^G2AC4A/GFP in which the glycine (position 2) and cysteine (position 4) residues were each replaced with alanine.

<p>(A) The AK2^C4A/GFP and AK2^G2AC4A/GFP proteins, respectively, were expressed using the CRT promoter (construct indicated above the images). The intracellular location of the AK2^C4A/GFP protein detected by epifluorescence microscopy in early and late trophozoite stages showed some localised patch-like structures within the parasite, a different pattern from the signal seen for <i>Pf</i>AK2/GFP or <i>Pf</i>AK2^G2A/GFP. The intracellular location of the AK2^G2AC4A/GFP was uniform throughout the parasite cytoplasm, and reminiscent of the distribution of <i>Pf</i>AK2^G2A/GFP. Details as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125191#pone.0125191.g001" target="_blank">Fig 1</a>. (B) SLO and saponin lysis, combined with a protease protection assay for AK2^C4A/GFP. Western blot analysis was performed with equal cell equivalents using anti-GFP, anti-SERP and anti-aldolase antibodies; labelling as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125191#pone.0125191.g001" target="_blank">Fig 1B</a>. No degradation of this fusion protein was detected following Proteinase K treatment of either the SLO or the saponin pellet fractions. (C) The <i>Pf</i>AK2^C4A/GFP and <i>Pf</i>AK2^G2AC4A/GFP transgenic parasites were subjected to hypotonic lysis, divided into soluble (SF) and membrane (MF) fractions following centrifugation, and then analysed by Western blot using anti-GFP, anti-Exp1 and anti-aldolase antibodies. (D) The pellet fractions from the SLO and saponin lysates of the AK2^G2AC4A/GFP expressing parasites, were divided equally into four samples and either untreated, treated by addition of Proteinase K, treated by addition of Proteinase K and Triton X-100, or treated by addition of Triton X-100 and protease inhibitors (PIC/PMSF). Triton X-100 was used to dissolve the PPM so that Proteinase K was able to digest the GFP chimera once it was accessible. These data show the efficacy of protein degradation by Proteinase K (disappearance of the protein band in the corresponding fraction). Western blot analysis was performed using anti-GFP, anti-SERP and anti-aldolase antibodies. No Proteinase K degradation of the AK2^G2AC4A/GFP fusion protein was detected after treatment of either the SLO or the saponin pellet fractions.</p

Subcellular location of <i>Pf</i>AK2/GFP and the non-myristoylated G2A variant, as determined by live fluorescence microscopy and cell fractionation using SLO and saponin lysis together with a protease protection assay.

<p>(A) The AK2/GFP and the AK2^G2A/GFP fusion proteins were expressed using the CRT promoter from an episomal plasmid (constructs indicated above the images). AK2/GFP is located at the periphery of the intracellular parasite as judged by epifluorescence microscopy, and is associated with one or two protuberances towards the host cell cytoplasm present on each parasite (indicated by white arrow). In contrast, the AK2^G2A/GFP chimera is located within the parasite cytosol. The infected cell was visualised by differential interference contrast (DIC), intrinsic fluorescence of the GFP identified the location of the AK2/GFP fusion protein, and parasite nuclei were detected by Hoechst staining. Overlay: green (GFP), blue (DNA). Scale bar—3 μm. (B) The <i>Pf</i>AK2/GFP transgenic parasite and (C) the G2A variant parasite were both subjected to SLO and saponin lysis, separated into soluble supernatant (SN) and pellet (P) fractions, and then part of the pellet fraction was treated with Proteinase K (PrK). Western blot analysis with equal cell equivalents was performed using anti-GFP, anti-SERP (a soluble PV protein), and anti-aldolase (a parasite cytoplasm protein) antibodies. Size markers are in kDa. The absence of the protein band corresponding to the <i>Pf</i>AK2/GFP fusion protein in the saponin pellet fraction treated with Proteinase K indicates secretion of the protein beyond the PPM.</p

The subcellular location of <i>Pf</i>ARF1/GFP and GFP fused to the modified version of the N-terminus of ARF1, <i>Pf</i>ARF^1-17/+4/-5AK2^18-37/GFP.

<p>(A) <i>Pf</i>ARF1/GFP was expressed using the CRT promoter (construct indicated above the images) and detected by epifluorescence microscopy. <i>Pf</i>ARF1/GFP was largely located in discrete dots; Figure details as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125191#pone.0125191.g001" target="_blank">Fig 1</a>. (B) The chimeric <i>Pf</i>ARF^1-17/+4/-5AK2^18-37/GFP was expressed using the CRT promoter (construct indicated above the images). Live cell imaging of the <i>Pf</i>ARF^1-17/+4/-5AK2^18-37/GFP parasite line at the late trophozoite stage showed that the pattern of GFP expression was similar to that of <i>Pf</i>AK2/GFP. Figure details are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125191#pone.0125191.g001" target="_blank">Fig 1</a>. The <i>Pf</i>ARF^1-17/+4/-5AK2^18-37/GFP transgenic parasite was subjected to (C) hypotonic, or (D) SLO and saponin lysis and cell fractionation as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125191#pone.0125191.g002" target="_blank">Fig 2</a>. No degradation of the fusion protein was detected after Proteinase K treatment of either the SLO or the saponin pellet fractions, indicating that the protein is protected from the protease and therefore within the PPM. The labelling is as in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125191#pone.0125191.g001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125191#pone.0125191.g002" target="_blank">2</a>.</p

The <i>Pf</i>AK2 N-terminus targets GFP to the outside of the parasite plasma membrane.

<p>(A) The AK2^1-37/GFP was expressed using the CRT promoter (construct indicated above the images). Live cell imaging of the <i>Pf</i>AK2^1-37/GFP in trophozoite and schizont stages, showed a location of the GFP signal similar to that of <i>Pf</i>AK2/GFP. In the schizont stage of these parasites a clear signal for the fusion protein was visible around each of the individual daughter merozoites. Details as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125191#pone.0125191.g001" target="_blank">Fig 1</a>. The AK2^1-37/GFP transgenic parasites were subjected to (B) hypotonic, or (C) SLO and saponin lysis and cell fractionation as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125191#pone.0125191.g002" target="_blank">Fig 2</a>. The absence of the protein band corresponding to the <i>Pf</i>AK2^1-37/GFP fusion protein in the saponin pellet fraction treated with Proteinase K indicates the secretion of the protein beyond the PPM. The labelling is as in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125191#pone.0125191.g001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125191#pone.0125191.g002" target="_blank">2</a>.</p

Deletion of the stretch of basic residues at the N-terminus of <i>Pf</i>AK2 alters the subcellular location of the protein.

<p>(A) The AK2^Δ21-30/GFP was expressed using the CRT promoter (construct indicated above the images). Live cell imaging of the <i>Pf</i>AK2^Δ21-30/GFP parasite line in a late trophozoite stage parasite; details as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125191#pone.0125191.g001" target="_blank">Fig 1</a>. The AK2^Δ21-30/GFP transgenic parasites were subjected to (B) hypotonic, or (C) SLO and saponin lysis and cell fractionation as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125191#pone.0125191.g002" target="_blank">Fig 2</a>. No degradation of the fusion protein was detected after Proteinase K treatment of either the SLO or the saponin pellet fractions. The labelling is as in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125191#pone.0125191.g001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125191#pone.0125191.g002" target="_blank">2</a>.</p