Transcriptional changes in absence of SEMP1 (microarray data).

<p>Transcriptional changes in absence of SEMP1 (microarray data).</p

Requirements for SEMP1 export into the RBC.

<p>A: Lysates of SEMP1-KO parasites expressing full-length and truncated or mutated forms of SEMP1 C-terminally fused to GFP were generated by saponin lysis and analysed by immunoblotting using mouse α-GFP antibodies. Lane 1: SEMP1_1–123-GFP (full-length), lane 2: SEMP1_17–123-GFP, lane3: SEMP1_72–123GFP, lane4: SEMP1_1–97-GFP, lane 5: MSP1_1–16SEMP1_17–123-GFP, lane 6: MAHRP2_1–16SEMP1_17–123-GFP. B: Immunofluorescence assays of MeOH-fixed RBCs infected with SEMP1-KO parasites expressing full-length and truncated or mutated forms of SEMP1 C-terminally fused to GFP. Expressed SEMP1 was labelled with rabbit α-GFP antibodies. The transmembrane domain is depicted in red (TM), a MSP1 signal peptide in brown (MSP1) and the MAHRP2 N-terminus in blue (M2).</p

Identification of potential SEMP1 interaction partners by Co-IP and MS analysis.

<p>Identification of potential SEMP1 interaction partners by Co-IP and MS analysis.</p

Identification of potential SEMP1 interaction partners by Co-IP.

<p>Co-IP was performed with 3D7 parasites expressing SEMP1 with a C-terminal 3xHA tag. Cultures were cross-linked with 1% formaldehyde and parasites were released by saponin treatment, lysed in 1%SDS followed by sonication. Lysate (Input) was incubated with α-HA affinity matrix. After centrifugation the matrix was washed three times with washing buffer (Wash) and proteins were eluted from by HA peptide elution. As a negative control, an excess of soluble HA peptides was added to the input to block the HA binding sites of the matrix (c-). Samples were analysed by Western blot with α-HA antibodies (A) and by silver staining (B). Proteins co-eluted with SEMP1-3xHA were identified by MS analysis of both TCA precipitated total elution (precipitation) and Coomassie-stained gel slices (gel extraction).</p

Nanomimics of Host Cell Membranes Block Invasion and Expose Invasive Malaria Parasites

The fight against most infectious diseases, including malaria, is often hampered by the emergence of drug resistance and lack or limited efficacies of vaccines. Therefore, new drugs, vaccines, or other strategies to control these diseases are needed. Here, we present an innovative nanotechnological strategy in which the nanostructure itself represents the active substance with no necessity to release compounds to attain therapeutic effect and which might act in a drug- and vaccine-like dual function. Invasion of <i>Plasmodium falciparum</i> parasites into red blood cells was selected as a biological model for the initial validation of this approach. Stable nanomimicspolymersomes presenting receptors required for parasite attachment to host cellswere designed to efficiently interrupt the life cycle of the parasite by inhibiting invasion. A simple way to build nanomimics without postformation modifications was established. First, a block copolymer of the receptor with a hydrophobic polymer was synthesized and then mixed with a polymersome-forming block copolymer. The resulting nanomimics bound parasite-derived ligands involved in the initial attachment to host cells and they efficiently blocked reinvasion of malaria parasites after their egress from host cells <i>in vitro</i>. They exhibited efficacies of more than 2 orders of magnitude higher than the soluble form of the receptor, which can be explained by multivalent interactions of several receptors on one nanomimic with multiple ligands on the infective parasite. In the future, our strategy might offer interesting treatment options for severe malaria or a way to modulate the immune response

Localization of potential SEMP1 interacting proteins.

<p>Immunofluorescence assays of MeOH-fixed RBCs infected with 3D7 expressing SEMP1/PF3D7_0702500/PF3D7_0601900 with a C-terminal 3xHA tag and 3D7 expressing PIESP2 with a C-terminal GFP-tag, co-labelled with mouse α-SEMP1 and rat α-HA (PF3D7_0702500-3xHA & PF3D7_0601900-3xHA) /α-GFP (PIESP2-GFP). For co-labelling of SEMP1-3xHA with REX1 and Pf332, rat α-HA and rabbit α-REX1 / mouse α-Pf332 antibodies were used.</p

Transcriptional changes in absence of SEMP1 identified by microarray analysis.

<p>Summary of all significantly (p<0.05) up- and down-regulated parasite genes with a respective average fold change (RAFC) >1.5 or <0.6. The RAFC is thereby the average FC over the significantly up- / down-regulated time points (TPs) only. A graphic depiction of their FCs throughout the four time points (TPs) is shown in form of a heat map. Up-regulation (FC>1) is indicated in red, down-regulation (FC<1) in green. The time points were 6–14 hpi (TP1), 16–24 hpi (TP2), 26–34 hpi (TP3) and 36–44 hpi (TP4). Exported proteins are highlighted in yellow.</p

SEMP1 expression and localization.

<p>A: schematic representation of the <i>semp1</i> gene (top) and SEMP1 protein structure (bottom). TM, transmembrane protein. B: Live cell imaging of 3D7 parasites expressing SEMP1 with C-terminal tagged GFP. C: Immunofluorescence assays of MeOH-fixed RBCs infected with 3D7 expressing SEMP1 with a C-terminal 3xHA tag, co-labelled with rat α-HA and either rabbit α-MAHRP1 or rabbit α-REX1 antibodies. D: Scatter plot of co-localization of SEMP1 and REX1 in SEMP1-3xHA parasites. E: Electron microscopy (EM) of RBCs infected with 3D7 expressing SEMP1 with a C-terminal GFP tag labelled with rabbit α-GFP antibodies and decorated with 5 nm gold conjugated Protein A.</p

Conditional Knockdown of SEMP1.

<p>A: <i>EcoR</i>I-digested gDNA isolated from SEMP1-DD parasites (DD) was analysed by Southern blot and probed with radioactively labelled <i>hdhfr</i> B: A culture of 3D7 parasites expressing SEMP1-DD was split and cultured for 2 weeks in presence (Shld +) or absence (Shld -) of the small molecule Shield-1. Whole parasite-lysates were generated after saponin lysis and analysed by Western blot using α-SEMP1. As a loading control the blot was additionally probed with antibodies against the housekeeping protein GAPDH.</p

Characterization of the Small Exported <i>Plasmodium falciparum</i> Membrane Protein SEMP1

<div><p>Survival and virulence of the human malaria parasite <i>Plasmodium falciparum</i> during the blood stage of infection critically depend on extensive host cell refurbishments mediated through export of numerous parasite proteins into the host cell. The parasite-derived membranous structures called Maurer's clefts (MC) play an important role in protein trafficking from the parasite to the red blood cell membrane. However, their specific function has yet to be determined. We identified and characterized a new MC membrane protein, termed small exported membrane protein 1 (SEMP1). Upon invasion it is exported into the RBC cytosol where it inserts into the MCs before it is partly translocated to the RBC membrane. Using conventional and conditional loss-of-function approaches we showed that SEMP1 is not essential for parasite survival, gametocytogenesis, or PfEMP1 export under culture conditions. Co-IP experiments identified several potential interaction partners, including REX1 and other membrane-associated proteins that were confirmed to co-localize with SEMP1 at MCs. Transcriptome analysis further showed that expression of a number of exported parasite proteins was up-regulated in SEMP1-depleted parasites. By using Co-IP and transcriptome analysis for functional characterization of an exported parasite protein we provide a new starting point for further detailed dissection and characterisation of MC-associated protein complexes.</p></div