Kinetic tracking of Plasmodium falciparum antigen presentation reveals determinants of protein export and membrane insertion

Intracellular malaria parasites export many proteins into their host cell, inserting several into the erythrocyte plasma membrane to enable interactions with their external environment. While static techniques have identified some surface-exposed proteins, other candidates have eluded definitive localization and membrane topology determination. Moreover, both export kinetics and the mechanisms of membrane insertion remain largely unexplored. We introduce Reporter of Insertion and Surface Exposure (RISE), a method for continuous nondestructive tracking of antigen exposure on infected cells. RISE utilizes a small 11 aa NanoLuc fragment inserted into a target protein and detects surface exposure through high-affinity complementation. We tracked insertion of CLAG3, a malaria parasite protein linked to nutrient uptake, throughout the P. falciparum cycle in human erythrocytes. Our approach also revealed key determinants of trafficking and surface exposure. Removal of a C-terminal transmembrane domain aborted export. Unexpectedly, certain increases in the exposed reporter size improved surface exposure by up to 50-fold, revealing that both size and charge of the extracellular epitope influence membrane insertion. Insertion of parasite proteins at the host cell surface and antigen accessibility is regulated by multiple factors, enabling intracellular parasite survival and immune evasion under a broad range of conditions.

Lectin-Mediated Bacterial Modulation by the Intestinal Nematode Ascaris suum

Ascariasis is a global health problem for humans and animals. Adult Ascaris nematodes are long-lived in the host intestine where they interact with host cells as well as members of the microbiota resulting in chronic infections. Nematode interactions with host cells and the microbial environment are prominently mediated by parasite-secreted proteins and peptides possessing immunomodulatory and antimicrobial activities. Previously, we discovered the C-type lectin protein AsCTL-42 in the secreted products of adult Ascaris worms. Here we tested recombinant AsCTL-42 for its ability to interact with bacterial and host cells. We found that AsCTL-42 lacks bactericidal activity but neutralized bacterial cells without killing them. Treatment of bacterial cells with AsCTL-42 reduced invasion of intestinal epithelial cells by Salmonella. Furthermore, AsCTL-42 interacted with host myeloid C-type lectin receptors. Thus, AsCTL-42 is a parasite protein involved in the triad relationship between Ascaris, host cells, and the microbiota.

Opinion: What do rescue experiments with heterologous proteins tell us and what not?

The recent progress in sequencing technology allowed the compilation of gene lists for a large number of organisms, though many of these organisms are hardly experimentally tractable when compared with well-established model organisms. One popular approach to further characterize genes identified in a poorly tractable organism is to express these genes in a model organism, and then ask what the protein does in this system or if the gene is capable of replacing the homologous endogenous one when the latter is mutated. While this is a valid approach for certain questions, I argue that the results of such experiments are frequently wrongly interpreted. If, for example, a gene from a parasitic nematode is capable of replacing its homologous gene in the model nematode Caenorhabditis elegans, it is often concluded that the gene is most likely involved in the same biological process in its own organism as the C. elegans gene is in C. elegans. This conclusion is not valid. All this experiment tells us is that the chemical properties of the parasite protein are similar enough to the ones of the C. elegans protein that it can perform the function of the C. elegans protein in C. elegans. Here I discuss this misconception and illustrate it using the analog of similar electric switches (components) controlling various devices (processes).

Molecular Characterization of a Functional Membrane-Associated Progesterone Receptor Component 2 (PGRMC-2) in Maturing Oocytes of the Human Parasite Nematode Trichinella spiralis: Implications to the Host-Parasite Relationship

We explored the hypothesis that progesterone direct effect on Trichinella spiralis might be mediated indeed by a new steroid-binding parasite protein. Our first results showed that Progesterone decreases the parasite molting rate. We amplify, isolated, cloned and sequenced the PGRMC2 sequence using specific primers from known species. Furthermore, we expressed the protein and developed an antibody to performance immunofluorescent confocal microscopy, where detected that parasite cells showed expression of a P4-binding protein exclusively located at the oocyte and the parasite´s cuticle. Presence of the PGRMC2 protein in these cells was also confirmed by western blot and flow cytometry. Molecular modeling studies accompanied by computer docking using the sequenced protein showed that PGRMC2 is potentially able to bind steroid hormones such as progesterone, estradiol, testosterone, and dihydrodrotestosterone with different affinities. Phylogenetic analysis and sequence alignment clearly demonstrated that Trichinella spiralis PGRMC2 is related to a steroid-binding protein of another platyhelminths. Progesterone may probably act upon Trichinella spiralis oocytes probably by binding to PGRMC2. This research has implications in the field of host-parasite co-evolution as well as the sex-associated susceptibility to this infection. In a more practical matter, present results may contribute to the molecular design of new drugs with anti-parasite actions.

Developing a multivariate prediction model of antibody features associated with protection of malaria-infected pregnant women from placental malaria

Background:Plasmodium falciparum causes placental malaria, which results in adverse outcomes for mother and child. P. falciparum-infected erythrocytes that express the parasite protein VAR2CSA on their surface can bind to placental chondroitin sulfate A. It has been hypothesized that naturally acquired antibodies towards VAR2CSA protect against placental infection, but it has proven difficult to identify robust antibody correlates of protection from disease. The objective of this study was to develop a prediction model using antibody features that could identify women protected from placental malaria.Methods:We used a systems serology approach with elastic net-regularized logistic regression, partial least squares discriminant analysis, and a case-control study design to identify naturally acquired antibody features mid-pregnancy that were associated with protection from placental malaria at delivery in a cohort of 77 pregnant women from Madang, Papua New Guinea.Results:The machine learning techniques selected 6 out of 169 measured antibody features towards VAR2CSA that could predict (with 86% accuracy) whether a woman would subsequently have active placental malaria infection at delivery. Selected features included previously described associations with inhibition of placental binding and/or opsonic phagocytosis of infected erythrocytes, and network analysis indicated that there are not one but multiple pathways to protection from placental malaria.Conclusions:We have identified candidate antibody features that could accurately identify malaria-infected women as protected from placental infection. It is likely that there are multiple pathways to protection against placental malaria.Funding:This study was supported by the National Health and Medical Research Council (Nos. APP1143946, GNT1145303, APP1092789, APP1140509, and APP1104975).

#23: Investigation of Phosphomannomutase as an Antimalarial Drug Target

Background Malaria continues to pose an enormous economic and global health threat, killing over 200,000 people annually, primarily children under the age of 5. With the constant barrier of antimalarial resistance and the rise of delayed clearance by artemisinin, it is especially important to identify drug/target pairs that rapidly kill parasites. We study targetable metabolic pathways in the malaria parasite, Plasmodium falciparum, to guide such future drug development against this disease. In recent years, we have discovered that a large family of hydrolases, the Haloacid Dehalogenase (HAD) Superfamily of proteins, are implicated in regulating a variety of P. falciparum metabolic pathways, which can lead to dramatic changes in central carbon metabolism and drug resistance. We now turn our attention to a related HAD protein, the putative phosphomannomutase in these parasites, HAD5, responsible for the interconversion of mannose-6-phosphate and mannose-1-phosphate. This is an essential process for all stages of the parasite, and thus has potential as a broad antimalarial target. We examined the role of HAD5 in these parasites, and its potential to be chemically inhibited. Methods Recombinant protein was generated and purified for enzymatic assays to determine HAD5 activity and test inhibitor potency against HAD5 compared to recombinant human orthologs, PMM1 and PMM2. In parallel, CRISPR/Cas9 was used to generate inducible knockdown parasite strains to demonstrate this gene’s essentiality and its role in parasite biology. Parasite growth was measured by flow cytometry and light microscopy. Immunofluorescence analysis (IFA) was used to track the parasite development on a molecular scale. Results Inhibition of HAD5 was achieved in biochemical assays, with an IC50 of 68µM in our most potent compound, representing roughly 10-fold increased potency against the parasite protein compared to human orthologs. In culture, knockdown of HAD5 leads to interrupted egress from and reinvasion into red blood cells, culminating in parasite death. In IFA-visualized parasites, reinvasion-facilitating proteins were no longer anchored to parasite surfaces, accounting for the inhibition of the parasite life cycle. Conclusion In the search for new antimalarial targets, identifying proteins that are essential across multiple parasite life-stages while being distinct from human orthologs is necessary to block parasite transmission, cure symptomatic infection, and minimize off-target effects. HAD5 is an essential protein in malaria parasites that is expressed throughout the parasite’s life cycle, and can be specifically targeted by inhibitors, giving it promise as a future drug target.

Prediction of Human-Plasmodium vivax Protein Associations From Heterogeneous Network Structures Based on Machine-Learning Approach

Malaria caused by Plasmodium vivax can lead to severe morbidity and death. In addition, resistance has been reported to existing drugs in treating this malaria. Therefore, the identification of new human proteins associated with malaria is urgently needed for the development of additional drugs. In this study, we established an analysis framework to predict human- P. vivax protein associations using network topological profiles from a heterogeneous network structure of human and P. vivax, machine-learning techniques and statistical analysis. Novel associations were predicted and ranked to determine the importance of human proteins associated with malaria. With the best-ranking score, 411 human proteins were identified as promising proteins. Their regulations and functions were statistically analyzed, which led to the identification of proteins involved in the regulation of membrane and vesicle formation, and proteasome complexes as potential targets for the treatment of P. vivax malaria. In conclusion, by integrating related data, our analysis was efficient in identifying potential targets providing an insight into human-parasite protein associations. Furthermore, generalizing this model could allow researchers to gain further insights into other diseases and enhance the field of biomedical science.