Protein abundance in the midgut of wild tsetse flies (Glossina palpalis palpalis) naturally infected by Trypanosoma congolense s.l.

Tsetse flies (Glossina spp.) are major vectors of African trypanosomes, causing either Human or Animal African Trypanosomiasis (HAT or AAT). Several approaches have been developed to control the disease, among which is the anti-vector Sterile Insect Technique. Another approach to anti-vector strategies could consist of controlling the fly's vector competence through hitherto unidentified regulatory factors (genes, proteins, biological pathways, etc.). The present work aims to evaluate the protein abundance in the midgut of wild tsetse flies (Glossina palpalis palpalis) naturally infected by Trypanosoma congolense s.l. Infected and non-infected flies were sampled in two HAT/AAT foci in Southern Cameroon. After dissection, the proteomes from the guts of parasite-infected flies were compared to that of uninfected flies to identify quantitative and/or qualitative changes associated with infection. Among the proteins with increased abundance were fructose-1,6-biphosphatase, membrane trafficking proteins, death proteins (or apoptosis proteins) and SERPINs (inhibitor of serine proteases, enzymes considered as trypanosome virulence factors) that displayed the highest increased abundance. The present study, together with previous proteomic and transcriptomic studies on the secretome of trypanosomes from tsetse fly gut extracts, provides data to be explored in further investigations on, for example, mammal host immunisation or on fly vector competence modification via para-transgenic approaches.

Antimalarial proteasome inhibitor reveals collateral sensitivity from intersubunit interactions and fitness cost of resistance.

We describe noncovalent, reversible asparagine ethylenediamine (AsnEDA) inhibitors of the Plasmodium falciparum proteasome (Pf20S) ß5 subunit that spare all active subunits of human constitutive and immuno-proteasomes. The compounds are active against erythrocytic, sexual, and liver-stage parasites, against parasites resistant to current antimalarials, and against P. falciparum strains from patients in Africa. The ß5 inhibitors synergize with a ß2 inhibitor in vitro and in mice and with artemisinin. P. falciparum selected for resistance to an AsnEDA ß5 inhibitor surprisingly harbored a point mutation in the noncatalytic ß6 subunit. The ß6 mutant was resistant to the species-selective Pf20S ß5 inhibitor but remained sensitive to the species-nonselective ß5 inhibitors bortezomib and carfilzomib. Moreover, resistance to the Pf20S ß5 inhibitor was accompanied by increased sensitivity to a Pf20S ß2 inhibitor. Finally, the ß5 inhibitor-resistant mutant had a fitness cost that was exacerbated by irradiation. Thus, used in combination, multistage-active inhibitors of the Pf20S ß5 and ß2 subunits afford synergistic antimalarial activity with a potential to delay the emergence of resistance to artemisinins and each other.

A Histone Methyltransferase Inhibitor Can Reverse Epigenetically Acquired Drug Resistance in the Malaria Parasite Plasmodium falciparum.

Malaria parasites invade and replicate within red blood cells (RBCs), extensively modifying their structure and gaining access to the extracellular environment by placing the plasmodial surface anion channel (PSAC) into the RBC membrane. Expression of members of the cytoadherence linked antigen gene 3 (clag3) family is required for PSAC activity, a process that is regulated epigenetically. PSAC is a well-established route of uptake for large, hydrophilic antimalarial compounds, and parasites can acquire resistance by silencing clag3 gene expression, thereby reducing drug uptake. We found that exposure to sub-IC50 concentrations of the histone methyltransferase inhibitor chaetocin caused substantial changes in both clag3 gene expression and RBC permeability, and reversed acquired resistance to the antimalarial compound blasticidin S that is transported through PSACs. Chaetocin treatment also altered progression of parasites through their replicative cycle, presumably by changing their ability to modify chromatin appropriately to enable DNA replication. These results indicate that targeting histone modifiers could represent a novel tool for reversing epigenetically acquired drug resistance in P. falciparum.

Clinical and Molecular Evidence of Atovaquone and Azithromycin Resistance in Relapsed Babesia microti Infection Associated With Rituximab and Chronic Lymphocytic Leukemia.

Babesiosis treatment failures with standard therapy have been reported, but the molecular mechanisms are not well understood. We describe the emergence of atovaquone and azithromycin resistance associated with mutations in the binding regions of the target proteins of both drugs during treatment of an immunosuppressed patient with relapsing babesiosis.

scDual-Seq of Toxoplasma gondii-infected mouse BMDCs reveals heterogeneity and differential infection dynamics.

Dendritic cells and macrophages are integral parts of the innate immune system and gatekeepers against infection. The protozoan pathogen, Toxoplasma gondii, is known to hijack host immune cells and modulate their immune response, making it a compelling model to study host-pathogen interactions. Here we utilize single cell Dual RNA-seq to parse out heterogeneous transcription of mouse bone marrow-derived dendritic cells (BMDCs) infected with two distinct genotypes of T. gondii parasites, over multiple time points post infection. We show that the BMDCs elicit differential responses towards T. gondii infection and that the two parasite lineages distinctly manipulate subpopulations of infected BMDCs. Co-expression networks define host and parasite genes, with implications for modulation of host immunity. Integrative analysis validates previously established immune pathways and additionally, suggests novel candidate genes involved in host-pathogen interactions. Altogether, this study provides a comprehensive resource for characterizing host-pathogen interplay at high-resolution.

Single-Cell Transcriptomics To Define Plasmodium falciparum Stage Transition in the Mosquito Midgut.

Malaria inflicts the highest rate of morbidity and mortality among the vector-borne diseases. The dramatic bottleneck of parasite numbers that occurs in the gut of the obligatory mosquito vector provides a promising target for novel control strategies. Using single-cell transcriptomics, we analyzed Plasmodium falciparum development in the mosquito gut, from unfertilized female gametes through the first 20 h after blood feeding, including the zygote and ookinete stages. This study revealed the temporal gene expression of the ApiAP2 family of transcription factors and of parasite stress genes in response to the harsh environment of the mosquito midgut. Further, employing structural protein prediction analyses, we found several upregulated genes predicted to encode intrinsically disordered proteins (IDPs), a category of proteins known for their importance in regulation of transcription, translation, and protein-protein interactions. IDPs are known for their antigenic properties and may serve as suitable targets for antibody- or peptide-based transmission suppression strategies. In total, this study uncovers the P. falciparum transcriptome from early to late parasite development in the mosquito midgut, inside its natural vector, which provides an important resource for future malaria transmission-blocking initiatives. IMPORTANCE The malaria parasite Plasmodium falciparum causes more than half a million deaths per year. The current treatment regimen targets the symptom-causing blood stage inside the human host. However, recent incentives in the field call for novel interventions to block parasite transmission from humans to the mosquito vector. Therefore, we need to better understand the parasite biology during its development inside the mosquito, including a deeper understanding of the expression of genes controlling parasite progression during these stages. Here, we have generated single-cell transcriptome data, covering P. falciparum's development, from gamete to ookinete inside the mosquito midgut, uncovering previously untapped parasite biology, including a repertoire of novel biomarkers to be explored in future transmission-blocking efforts. We anticipate that our study provides an important resource, which can be further explored to improve our understanding of the parasite biology as well as aid in guiding future malaria intervention strategies.

Special Issue: Insects, Nematodes, and Their Symbiotic Bacteria.

This special issue contains articles that add to the ever-expanding toolbox of insect pathogenic nematodes (entomopathogenic nematodes; EPNs) as well articles that provide new insights into the mutualistic interaction between EPNs and their hosts. The study of natural infection models such as EPNs allows detailed insight into micro- and macro-evolutionary dynamics of innate immune reactions, including known but also emerging branches of innate immunity. Additional new insights into the kinetics of EPN infections are gained by increased spatiotemporal resolution of advanced transcriptome studies and live imaging.

High-Resolution Infection Kinetics of Entomopathogenic Nematodes Entering Drosophila melanogaster.

Entomopathogenic nematodes (EPNs) have been a useful model for studying wound healing in insects due to their natural mechanism of entering an insect host either through the cuticle or an orifice. While many experiments have shed light on nematode and host behavior, as well as the host immune response, details regarding early nematode entry and proliferative events have been limited. Using high-resolution microscopy, we provide data on the early infection kinetics of Heterorhabditis bacteriophora and its symbiotic bacteria, Photorhabdus luminescens. EPNs appendage themselves to the host and enter through the host cuticle with a drill-like mechanism while leaving their outer sheath behind. EPNs immediately release their symbiotic bacteria in the host which leads to changes in host behavior and septicemia within 6 h while EPNs travel through the host in a predictable manner, congregating in the anterior end of the host. This paper sheds light on the entry and proliferative events of EPN infection, which will further aid in our understanding of wound healing and host immune activation at a high spatiotemporal resolution.

Drosophila melanogaster Responses against Entomopathogenic Nematodes: Focus on Hemolymph Clots.

Several insect innate immune mechanisms are activated in response to infection by entomopathogenic nematodes (EPNs). In this review, we focus on the coagulation of hemolymph, which acts to stop bleeding after injury and prevent access of pathogens to the body cavity. After providing a general overview of invertebrate coagulation systems, we discuss recent findings in Drosophila melanogaster which demonstrate that clots protect against EPN infections. Detailed analysis at the cellular level provided insight into the kinetics of the secretion of Drosophila coagulation factors, including non-classical modes of secretion. Roughly, clot formation can be divided into a primary phase in which crosslinking of clot components depends on the activity of Drosophila transglutaminase and a secondary, phenoloxidase (PO)-dependent phase, characterized by further hardening and melanization of the clot matrix. These two phases appear to play distinct roles in two commonly used EPN infection models, namely Heterorhabditis bacteriophora and Steinernema carpocapsae. Finally, we discuss the implications of the coevolution between parasites such as EPNs and their hosts for the dynamics of coagulation factor evolution.

Data on Drosophila clots and hemocyte morphologies using GFP-tagged secretory proteins: Prophenoloxidase and transglutaminase.

Insect hemolymph coagulation: Kinetics of classically and non-classically secreted clotting factors Schmid et al., 2019. The linked article demonstrates the localization of two secretory proteins in Drosophila melanogaster, Prophenoloxidase (PPO2) and Transglutaminase-A (Tg) in hemocytes as well the clot with different tissue-specific drivers. Here we provide further data for the usefulness of the GFP-tagged version of the two crosslinking enzymes that are involved in clot hardening. The morphology of crystal cells is described using GFP-tagged PPO2 rather than with the use of antibodies in ex vivo hemolymph preparations. The use of the GFP-tagged proteins PPO2 and Tg is shown in additional contexts.

Insect hemolymph coagulation: Kinetics of classically and non-classically secreted clotting factors.

In most insects, hemolymph coagulation, which is analogous to mammalian blood clotting, involves close collaboration between humoral and cellular components. To gain insights into the secretion of cellular clotting factors, we created tagged versions of three different clotting factors. Our focus was on factors which are released in a non-classical manner and to characterize them in comparison to a protein that is classically released, namely Glutactin (Glt). Transglutaminase-A (Tg) and Prophenoloxidase 2 (PPO2), both of which lack signal peptide sequences, have been previously demonstrated to be released from plasmatocytes and crystal cells (CCs) respectively, the two hemocyte classes in naïve larvae. We found that at the molecular level, Tg secretion resembles the release of tissue transglutaminase in mammals. Specifically, Drosophila Tg is associated with vesicular membranes and remains membrane-bound after release, in contrast to Glt, which we found localizes to a different class of vesicles and is integrated into clot fibers. PPO2 on the other hand, is set free from CCs through cytolysis. We confirm that PPO2 is a central component of the cytosolic crystals and find that the distribution of PPO2 appears to vary across crystals and cells. We propose a tentative scheme for the secretory events during early and late hemolymph coagulation.