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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.

Introduction: Tumor-associated macrophages may act to either limit or promote tumor growth, yet the molecular basis for either path is poorly characterized. Methods: We use a larval Drosophila model that expresses a dominant-active version of the Ras-oncogene (RasV12) to study dysplastic growth during early tumor progression. We performed single-cell RNA-sequencing of macrophage-like hemocytes to characterize these cells in tumor- compared to wild-type larvae. Hemocytes included manually extracted tumor-associated- and circulating cells. Results and discussion: We identified five distinct hemocyte clusters. In addition to RasV12 larvae, we included a tumor model where the activation of effector caspases was inhibited, mimicking an apoptosis-resistant setting. Circulating hemocytes from both tumor models differ qualitatively from control wild-type cells—they display an enrichment for genes involved in cell division, which was confirmed using proliferation assays. Split analysis of the tumor models further reveals that proliferation is strongest in the caspase-deficient setting. Similarly, depending on the tumor model, hemocytes that attach to tumors activate different sets of immune effectors—antimicrobial peptides dominate the response against the tumor alone, while caspase inhibition induces a shift toward members of proteolytic cascades. Finally, we provide evidence for transcript transfer between hemocytes and possibly other tissues. Taken together, our data support the usefulness of Drosophila to study the response against tumors at the organismic level.

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.

The liver is a major entry point and gatekeeper for invasive pathogens. However, high-resolution, spatiotemporal transcriptomic analysis of host-pathogen interactions has remained challenging. Afriat et al. have deconvoluted Plasmodium berghei liver-stage maturation at an unprecedented scale and discovered molecular signatures of heterogeneity during pre-erythrocytic development of malarial parasites.

The coronavirus disease 2019, COVID-19, is a complex disease with a wide range of symptoms from asymptomatic infections to severe acute respiratory syndrome with lethal outcome. Individual factors such as age, sex, and comorbidities increase the risk for severe infections, but other aspects, such as genetic variations, are also likely to affect the susceptibility to SARS-CoV-2 infection and disease severity. Here, we used a human 3D lung cell model based on primary cells derived from multiple donors to identity host factors that regulate SARS-CoV-2 infection. With a transcriptomics-based approach, we found that less susceptible donors show a higher expression level of serine protease inhibitors SERPINA1, SERPINE1, and SERPINE2, identifying variation in cellular serpin levels as restricting host factors for SARS-CoV-2 infection. We pinpoint their antiviral mechanism of action to inhibition of the cellular serine protease, TMPRSS2, thereby preventing cleavage of the viral spike protein and TMPRSS2-mediated entry into the target cells. By means of single-cell RNA sequencing, we further locate the expression of the individual serpins to basal, ciliated, club, and goblet cells. Our results add to the importance of genetic variations as determinants for SARS-CoV-2 susceptibility and suggest that genetic deficiencies of cellular serpins might represent risk factors for severe COVID-19. Our study further highlights TMPRSS2 as a promising target for antiviral intervention and opens the door for the usage of locally administered serpins as a treatment against COVID-19.

Mosquito immune cells, known as hemocytes, are integral to cellular and humoral responses that limit pathogen survival and mediate immune priming. However, without reliable cell markers and genetic tools, studies of mosquito immune cells have been limited to morphological observations, leaving several aspects of their biology uncharacterized. Here, we use single-cell RNA sequencing (scRNA-seq) to characterize mosquito immune cells, demonstrating an increased complexity to previously defined prohemocyte, oenocytoid, and granulocyte subtypes. Through functional assays relying on phagocytosis, phagocyte depletion, and RNA-FISH experiments, we define markers to accurately distinguish immune cell subtypes and provide evidence for immune cell maturation and differentiation. In addition, gene-silencing experiments demonstrate the importance of lozenge in defining the mosquito oenocytoid cell fate. Together, our scRNA-seq analysis provides an important foundation for future studies of mosquito immune cell biology and a valuable resource for comparative invertebrate immunology.

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.