Biology subjects

Freville, A.

Publications and source records attributed to Freville, A..

6 recordsLinked to original sources

bioRxiv2024

Malaria parasites undergo a rapid and extensive metamorphosis after invasion of the host erythrocyte

Within the human host, the symptoms of malaria are caused by the replication of malaria parasites within erythrocytes. Growth inside the erythrocyte exposes the parasites to the normal surveillance of erythrocytes by the host organism, in particular the clearance of erythrocytes in the spleen. Here we show that the malaria parasite Plasmodium falciparum undergoes a rapid, multi-step metamorphosis that transforms the invasive merozoite into an amoeboid-shaped cell within minutes after invading erythrocytes. This transformation involves an increase in the parasite surface area and is mediated by factors already present in the merozoite, including the parasite phospholipid transfer protein PV6. Parasites lacking PV6 do not assume an amoeboid form and instead are spherical and have a smaller surface area than amoeboid forms. Furthermore, erythrocytes infected with parasites lacking PV6 undergo a higher loss of surface area upon infection by P. falciparum, which affects the traversal of infected erythrocytes through the spleen. This is the first evidence that after invasion, the parasite undergoes a rapid, complex metamorphosis within the host erythrocyte that promotes survival in the host.

microbiology↗

bioRxiv2023

Expression of the MSPDBL2 antigen in a discrete subset of Plasmodium falciparum schizonts is regulated by GDV1 but not linked to sexual commitment

The Plasmodium falciparum merozoite surface protein MSPDBL2 is a polymorphic antigen targeted by acquired immune responses, and normally expressed in only a minority of mature schizonts. The potential relationship of MSPDBL2 to sexual commitment is examined, as variable mspdbl2 transcript levels and proportions of MSPDBL2-positive mature schizonts in clinical isolates have previously correlated with levels of many sexual stage parasite gene transcripts, although not with the master regulator ap2-g. It is demonstrated that conditional overexpression of GDV1, which promotes sexual commitment, also substantially increases the proportion of MSPDBL2-positive schizonts in culture. Conversely, truncation of the gdv1 gene is shown to prevent any expression of MSPDBL2. However, across diverse P. falciparum cultured lines the variable proportions of MSPDBL2 positivity in schizonts does not correlate significantly with variable gametocyte conversion rates, indicating it is not involved in sexual commitment. Confirming this, examining a line with endogenous HA-tagged AP2-G showed that the individual schizonts expressing MSPDBL2 are mostly different to those expressing AP2-G. Using a selection-linked integration system, modified P. falciparum lines were engineered to express an intact or disrupted version of MSPDBL2, showing the protein is not required for sexual commitment or early gametocyte development. Asexual parasite multiplication rates were also not affected by expression of either intact or disrupted MSPDBL2 in a majority of schizonts. Occurring alongside sexual commitment, the role of the discrete MSPDBL2-positive schizont subpopulation requires further investigation in natural infections where it is under immune selection.

microbiology↗

bioRxiv2023

Identification of a non-exported Plasmepsin V substrate that functions in the parasitophorous vacuole of malaria parasites

Malaria parasites alter multiple properties of the host erythrocyte by exporting proteins into the host cell. Many exported proteins contain a five-amino acid motif called the Plasmodium Export Element (PEXEL) that is cleaved by the parasite protease Plasmepsin V (PM V). The presence of a PEXEL is considered a signature of protein export and has been used to identify a large number of exported proteins. The export of proteins becomes essential midway through the intraerythrocytic cycle - preventing protein export blocks parasite development 18-24 h after invasion. However, a genetic investigation revealed that the absence of the PEXEL protein PFA0210c causes parasite development to arrest immediately after invasion. We now show that this protein (renamed PV6) is cleaved by PM V but not exported into the host erythrocyte and instead functions in the parasitophorous vacuole. Furthermore, we show that the lysine residue that becomes the N terminus of PV6 after processing by PM V prevents export. This is the first example of a native Plasmodium falciparum PM V substrate that remains in the parasitophorous vacuole. We also provide evidence that the parasite produces at least one additional essential, non-exported PM V substrate. These results reveal that the presence of a PEXEL, and hence processing of a protein by PM V, does not always target a protein for export and that PM V has a broader function in parasite growth beyond processing exported proteins. Furthermore, we utilized this finding to investigate possible requirements for protein export further.

microbiology↗

bioRxiv2022

Transcriptome analysis of diverse Plasmodium falciparum clinical isolates identifies genes correlating with highly variable expression of merozoite surface protein MSPDBL2

The merozoite surface protein MSPDBL2 of Plasmodium falciparum is under strong balancing selection and is a target of naturally acquired antibodies. Remarkably, MSPDBL2 is expressed in only a minority of mature schizonts of any cultured parasite line, and mspdbl2 gene transcription increases in response to overexpression of the gametocyte development inducer GDV1, so it is important to understand its natural expression. Here, MSPDBL2 in mature schizonts was analysed in the first ex vivo culture cycle of 96 clinical isolates from four populations with varying levels of infection endemicity in different West African countries, by immunofluorescence microscopy with antibodies against a conserved region of the protein. In most isolates, less than 1% of mature schizonts were positive for MSPDBL2 (median of 0.6% overall), but the frequency distribution was highly skewed as nine isolates had more than 3% schizonts positive and one had 73% positive. To investigate whether expression of other gene loci correlated with MSPDBL2 expression, whole transcriptome sequencing was performed on schizont-enriched material from 17 of the clinical isolates with a wide range of proportions of schizonts positive. Transcripts of particular parasite genes were highly significantly positively correlated with MSPDBL2 positivity in schizonts as well as with mspdbl2 gene transcript levels, with overrepresentation of genes previously implicated as likely to be involved in gametocytogenesis, but not including the gametocytogenesis master regulator ap2g. Although MSPDBL2 is apparently not directly involved in sexual commitment, it marks a co-occurring developmental subpopulation that may be functionally distinct within blood stage infections.

microbiology↗

bioRxiv2022

Variation in Plasmodium falciparum sexual commitment rates and responses to environmental modification

Asexual blood stage malaria parasites must produce sexual progeny in order to infect mosquitoes. It is therefore important to understand the scope of variation in sexual commitment rates of the most important human parasite Plasmodium falciparum, and to assess responses to conditions that may affect this commitment. First, using a genetically modified line with inducible elevated sexual commitment we compared two different methods of measuring the rates, either staining the Pfs16 marker in very early gametocytes or counting gametocytes after further development. The methods showed correlated estimates in evaluation across multiple independent replicate assays, with higher sensitivity and precision being achieved by Pfs16 marker detection in early gametocytes, so this method was used to survey a wide range of P. falciparum lines. A panel of six recently culture-established clinical isolates from Ghana showed mean sexual commitment rates per cycle ranging from 3.3% to 12.2%, with some significant differences among isolates as well as variation among biological replicates of each. Comparing 13 long-term laboratory-adapted lines of diverse origins, eight had sexual commitment rates similar to those of the recent clinical isolates, with means across multiple biological replicates for each line ranging from 4.7% to 14.5%, while four lines had significantly lower rates with means ranging from 0.3 to 1.6%, and one line with a non-functional ap2-g gene always showed zero sexual commitment. Among a subset of parasite lines, adding choline to suppress commitment had quantitatively variable effects, although these were significant in most assays of lines that had relatively high rates. This study indicates the importance of multiple assay replicates and comparisons of diverse isolates to understand natural and culture-induced variation in this key reproductive trait, relevant to investigating potential effects of parasite adaptation on transmission. Author SummaryOnly sexual malaria parasites are transmitted from humans to mosquitoes, so it is vital to understand variation in sexual commitment rates of blood stage malaria parasites, and responses to conditions that affect this. We compared two different methods of measuring the rates in a Plasmodium falciparum line with engineered sexual commitment variation, demonstrating higher sensitivity and precision by detection of an early differentiation marker, and this method was then used to survey diverse P. falciparum lines in culture with extensive biological replicate measurements. Recent clinical isolates from Ghana showed mean sexual commitment rates per cycle ranging from 3% to 12%, with significant differences among isolates as well as variation among biological replicates. Long-term laboratory-adapted lines of diverse origins had a wide range, most having rates similar to those of the clinical isolates, while a minority consistently had much lower or zero rates. There was quantitative variation among lines in the effects of adding choline to suppress commitment, although most assays of lines that had relatively high rates showed significant effects. Performing multiple biological replicates and comparisons of recent parasite isolates is vital to understand intra-specific variation in this important reproductive trait, and move towards investigating direct effects on disease transmission.

microbiology↗

bioRxiv2019

Plasmodium condensin core subunits (SMC2/SMC4) mediate atypical mitosis and are essential for parasite proliferation and transmission

Condensin is a multi-subunit protein complex regulating chromosome condensation and segregation during cell division. In Plasmodium spp., the causative agent of malaria, cell division is atypical and the role of condensin is unclear. Here we examine the role of SMC2 and SMC4, the core subunits of condensin, during endomitosis in schizogony and endoreduplication in male gametogenesis. During early schizogony SMC2/SMC4 localize to a distinct focus, identified as the centromeres by NDC80 fluorescence and ChIP-seq analyses, but do not form condensin I or II complexes. In mature schizonts and during male gametogenesis, there is a diffuse SMC2/SMC4 distribution on chromosomes and in the nucleus, and both condensin I and II complexes form at these stages. Knockdown of smc2 and smc4 gene expression revealed essential roles in parasite proliferation and transmission. The condensin core subunits (SMC2/SMC4) form different complexes and may have distinct functions at various stages of the parasite life cycle.

cell biology↗