PLOS Pathogens

Like most herpesviruses, KSHV encodes multiple microRNAs (miRNAs). Collectively, they comprise an important mechanism through which the virus maintains latency and persists in cells. At the same time, individual miRNAs can also play distinct, nonredundant roles. Past experiments with single miRNA knockout viruses showed that miR-K12-9, in particular, filled a unique niche. Endothelial cells latently infected with the miR-K12-9 knockout grew to be many times larger than WT-infected cells and proliferated at a significantly slower rate. Their ability to migrate was slowed as well. RNA-seq identified nearly 8,500 differentially expressed genes between miR-K12-9 knockout- and WT-infected cells. To further study miR-K12-9, we generated Telomerase-Immortalized Microvascular Endothelial (TIME) cells expressing either miR-K12-9 or a control miRNA from a lentivirus. Unexpectedly, after approximately one month in culture, unmistakable morphological changes began to occur in two of the three miR-K12-9-expressing cell lines. These smaller, more rounded cells proliferated rapidly and swiftly took over the two cultures. Given this result, we proceeded to characterize all the lentivirus-transduced cell lines in various assays focused on oncogenesis. When looking at colony formation in soft agar, only those two miR-K12-9-expressing cell lines produced colonies, indicating a loss of contact inhibition. NOD/SCID mice injected with the two cell miR-K12-9-expressing cell lines developed tumors while those receiving other cell lines did not. To confirm reproducibility of these results, we transduced both TIME and primary endothelial cells (HUVECs) with the miR-K12-9 and control lentiviruses. Once again, approximately half of the cell lines expressing miR-K12-9 showed hallmark phenotypes of transformation. We are currently characterizing the miR-K12-9 targetome in the transduced cell lines and mouse tumors using bulk and single-cell RNA-seq. This should yield insights into the underlying mechanism and required cofactors of miR-K12-9-induced transformation. To our knowledge, this is the first description of transformation of endothelial cells by a viral miRNA.

Cryptosporidium is a protozoan that infects epithelial cells of the small intestine and is a cause of diarrhea and death in immunocompromised individuals and malnourished children. Immunity to this parasite is mediated by an intestinal T cell response, which is generated in gut-associated lymphoid tissues and dependent on type 1 conventional dendritic cells (cDC1s). The initial priming of T cells is accompanied by changes in integrin expression and subsequent trafficking to the site of infection. The role of specific integrins in trafficking to the ileum during cryptosporidiosis is largely unknown. The development of a transgenic Cryptosporidium strain that expresses MHCI and MHCII-restricted model antigens provides the ability to track T cell responses to this parasite. Our studies in this system revealed marked changes in the integrin profile of parasite-specific T cells as they are activated and traffic to the gut, and demonstrate that cDC1s contribute to the expression of the integrins 4, {beta}7, {beta}1, and L. Surprisingly, blockade of the canonical gut-homing integrin 4{beta}7 does not impact the ability of parasite-specific T cells to access the gut. However, blockade of integrin L decreases the parasite-specific T cell frequency at the site of infection and delays control of parasite burden. These datasets highlight an 4{beta}7-independent mechanism of T cell trafficking to the small intestine and indicate that L is an integrin required for T cell-mediated resistance to Cryptosporidium.

Influenza A viruses (IAV) are clinically important pathogens that cause seasonal epidemics and pandemics in humans. IAV produce pleomorphic, enveloped virions, which can range from a spherical or bacilliform morphology, the predominant form in the most commonly studied laboratory strains, to long filamentous virions which are characteristic of clinical and veterinary isolates. Understanding the structure and function of filamentous virions is crucial for clarifying their role in viral persistence and immune evasion, and for informing the development of therapeutics that target their entry and/or egress pathways. Structural characterisation of influenza virions is challenging however owing to their fragility, heterogeneity and compared to most virus particles, unusually large size. Here, we combined structural and compositional approaches with integrative modelling to define the complete molecular architecture of influenza virions. In doing so we provide the first description of distinctive structural features of IAV filaments, including the selective incorporation of lipids, specific enrichment of viral and host proteins, and a viral cytoskeleton including a secondary helical layer within the viral capsid and extended fibrils of cofilactin. Collectively our findings suggest an important regulatory role for cofilactin in driving filament morphogenesis and provide important insights into the organisation and composition of IAV filamentous virions.

A common form of Wolbachia-induced manipulation of host reproduction is Cytoplasmic Incompatibility (CI). In CI, Wolbachia modification of sperm results in pronounced defects in paternal chromosome condensation, replication, and segregation during the first mitotic division. Recent studies in D. simulans demonstrate that CI also induces independent and distinct later developmental defects resulting in high rates of mitotic errors during the mid-blastula transition and larval lethality. Here we show that in D. melanogaster, embryos derived from CI crosses experienced significant mitotic defects during gastrulation and increased larval lethality, both of which were eliminated in the progeny of Rescue crosses (both sexes infected). Examination of CI using females from 13 genetically distinct wild-type lines of the Drosophila Genetic Reference Panel (DGRP) revealed significant variation in the strength of the CI-induced lethality. Early embryonic pre-hatching and late larval lethal phases were uncorrelated, suggesting distinct factors influence the extent of the two lethal phases. Additionally, 3rd instar larvae and adults derived from D. melanogaster CI crosses exhibited locomotor defects that were also eliminated in Rescue crosses. These studies support a model in which Wolbachia effects on the sperm chromatin produce delayed developmental and locomotor defects, suggesting the involvement of epigenetic mechanisms. Support for this idea comes from our finding that levels of the heritable chromatin mark H3K27me1 are significantly elevated in CI-derived embryos. We conclude that the full measure of CI strength should take into account pre- and post-hatching lethality as well as locomotor defects. Together our findings suggest that the strength of these CI-induced phenotypes is governed at least in part by epigenetics and the maternal genetic background. AUTHOR SUMMARYSince the discovery of the antiviral properties of the bacteria Wolbachia, numerous strategies using this insect endosymbiont have been developed to combat vector-borne disease. While the success of these strategies relies on the rapid spread of Wolbachia through a naturally uninfected insect population, the molecular mechanisms by which Wolbachia promote their spread remain poorly defined. Current research on the primary mechanism behind Wolbachia spread, cytoplasmic incompatibility (CI), focuses on understanding the dramatic decrease in egg hatch rates that occurs when uninfected females mate with infected males. Here, we demonstrate that CI also induces substantial post-hatching larva and adult locomotor defects and lethality. In accord with these developmentally delayed defects, we show Wolbachia dramatically alter an epigenetic chromatin mark. Finally, we show that host maternal factors contribute to CI strength. Taken together, these results demonstrate that CI induces a much more expansive and developmentally delayed suite of phenotypes than previously reported.

Actin superfamily members are critical for the biology of eukaryotes and archaea. Actin-related proteins (Arps) are a subgroup within the actin superfamily and play essential roles in trafficking, replication and motility. The genome of the malaria parasite Plasmodium contains a set of Arps unique to apicomplexans, termed actin-like proteins (Alps). However, the importance and specific roles of many of these Alps in Plasmodium progression are not yet understood. Here, we determined the functional contribution of Plasmodium berghei Alp3 and Alp5a (recently relabelled as Arp3) by generation of knock-out (KO) lines and their subsequent characterisation across different life cycle stages. Deletion of either Alp did not affect blood stage growth, gametogenesis and ookinete gliding motility. However, deletion of Alp5a lead to smaller and fewer oocysts as well as severely impaired sporozoite formation. The Alp3KO line had highly reduced oocyst loads compared to wild-type parasites. This striking decrease was due to impaired ookinete penetration of the mosquito midgut epithelium. Our study shows that both Alp3 and Alp5a are indispensable for Plasmodium transmission at different steps of initial mosquito infection, provides insights into the role of specific unique members of the actin superfamily during parasite progression and the requirements for efficient midgut penetration.

The dengue virus (DENV) non-structural protein 1 (NS1) is a glycoprotein highly conserved among mosquito-borne orthoflaviviruses. NS1 is typically localized in the lumen of the endoplasmic reticulum, where it forms part of the replication complexes, and is also exposed at the plasma membrane. In addition, NS1 is secreted as a lipoprotein. Here, using a combination of approaches, including confocal microscopy with deconvolution, in situ analysis, and biochemical cell fractionation, we show that a substantial fraction of NS1 (up to 30%) translocates to the nucleus during infection. We identified a conserved, structurally exposed bipartite nuclear localization signal (NLS) within NS1. Pharmacological inhibition with ivermectin and site-directed mutagenesis of the NLS in recombinant confirmed that nuclear import of NS1 is an active process, dependent on the classical importin /{beta} pathway. Notably, both dimeric and multimeric forms of NS1 were detected in the nucleus in association with nuclear lamin. Introduction of the NLS mutations into DENV2 infectious clones resulted in a non-viable virus. Production of virus progeny and completion of the replicative cycle by the mutant genomes could be rescued by trans-complementation with wild-type NS1, but not with an NLS-mutated NS1, indicating that an NS1 nuclear phase is required for a productive infection. Transcriptomic analysis by RNA-seq further revealed that NS1 functions depend on its subcellular location. Nuclear NS1 induced the overexpression of genes associated with DNA-binding transcription factors, whereas NLS-mutated NS1, retained in the cytoplasm, failed to induce these genes and instead triggered pro-inflammatory and metabolic responses. Together, these findings reveal a previously unrecognized nuclear phase of NS1 that is required for an efficient viral life cycle, redefining NS1 as a modulator of the host transcriptional environment. These findings also suggest new avenues for antiviral and vaccine development. Authors summaryDengue virus NS1 is a glycoprotein of approximately 45-50 kDa that rapidly dimerizes after proteolytic maturation. Dimeric NS1 is located in the lumen of the endoplasmic reticulum where it acts as a scaffold component of the viral replication complexes. In addition, NS1 is secreted from infected cells as a tetramer or hexamer and circulates in the serum of infected individuals during the acute phase of dengue disease. Circulating NS1 is widely used as a diagnostic marker and has also been associated with dengue pathogenesis through several mechanisms. Here, we expand the current understanding of DENV NS1 by identifying a previously unrecognized and essential nuclear location of this protein. We show that NS1 contains a conserved nuclear localization signal that mediates import into the nucleus via the classical import pathway. Using wild-type and NLS-mutated infectious clones, we demonstrated that nuclear localization of NS1 is required for completion of the DENV replicative cycle. Transcriptomic analysis further revealed that nuclear NS1 promotes the expression of host genes involved in nucleic acid metabolism, whereas retention of NS1 in the cytoplasm triggers an antiviral and inflammatory response. Together, these findings identify the nucleus as an important site of dengue virus-host interactions and redefine NS1 as a regulator of the host transcriptional environment during infection.

Persistence - characterized by the transient ability of subpopulations of drug-susceptible parasites to survive exposure to drug - is a major driver of treatment failure and clinical relapses in leishmaniasis. Persisters are characterized by non-dividing or slow-growing state and increased drug tolerance. However, the molecular mechanisms governing formation of persisters remain poorly understood in Leishmania parasites. Here, we developed a model to explore persistence in Leishmania mexicana. Viable promastigote persister-like subpopulations were enriched using Ficoll density gradient centrifugation following lethal exposure to antimonial drugs that killed 80% of parasites. The surviving parasites exhibited delayed growth in drug-free medium that is characteristic for persisters, and significantly higher tolerance to drug upon rechallenge. Transcriptomic profiling across acute stress, drug-free recovery, and rechallenge phases revealed a global remodeling in persisters under all tested conditions. Induction phase in Leishmania persisters was characterized by downregulation of several biological processes and a robust upregulation of nucleolar pathways, supporting epitranscriptomic changes during formation of persisters. Upon drug removal, this profile rapidly reverted, initiating ribosomal biogenesis to exit latency and resume cellular proliferation. Resuscitation phase exhibited activated protein synthesis and upregulation in many biological processes associated with metabolic and mitochondrial functions. Furthermore, comparative analysis of drug responses in rechallenged drug-tolerant persisters and parental parasites exposed to the drug for the first time, revealed that persisters exhibit distinct drug response profiles compared to parental parasites by rapidly implementing a highly conserved, coordinated survival reprogramming, where 316 genes were uniquely downregulated, and 241 genes were upregulated. The distinct features of the drug response in rechallenged persister cells were characterized by the downregulation of mitochondrial function and protein synthesis machinery to induce a dormant, idling state, and the upregulation of drug-response and stress-tolerance genes to survive immediate toxicity. In contrast, parental parasites displayed a broad and disorganized drug response. Additionally, rechallenged persisters exhibited a distinct transcriptomic memory that transiently phenocopies stable genetic resistance. This pre-adapted state is characterized by the targeted upregulation of epigenetic modulators, heavy metal transporters, and catabolic enzymes to maintain viability. These findings demonstrate that drug persistence in Leishmania is not merely a metabolic collapse, but rather a sophisticated survival strategy involving active transcriptome remodeling, downregulation of translation and epigenetic adaptations. This transient state constitutes an initial evolutionary step toward permanent drug resistance and highlights new molecular vulnerabilities for therapeutic interventions aimed at preventing clinical relapse. Author summaryThe efficacy of leishmaniasis chemotherapy is often fails because Leishmania parasites survive the drugs by entering a dormant state, causing relapses in patients. In this study, however, we discovered that these parasites are not simply quiescent. On the contrary, they orchestrate a highly active survival response: they shut down energy-consuming functions while keeping their cellular alert mechanisms active. Remarkably, upon re-exposure to antileishmanial drugs, the parasites recall previous stress events and transiently activate defensive pathways characteristic of stable drug resistance. Understanding how Leishmania employs this temporary adaptive strategy provides critical targets for developing therapies designed to eradicate the infection and avert relapses.

The matrix protein VP40 of orthoebolaviruses coordinates virion release and downregulates viral RNA synthesis through distinct oligomeric states, including dimers, octamers, and filamentous assemblies. To dissect the contributions of two oligomeric interface residues, L117 and W95, in the Sudan virus (SUDV) VP40 (sVP40), we created variants carrying alanine substitutions and assessed their structural and functional properties. sVP40 L117A failed to form dimers and was predominantly monomeric showing increased structural flexibility, reduced thermal stability together with loss of plasma membrane transport, budding activity, and the ability to regulate viral RNA synthesis. VP40 W95A preserved dimerization but also exhibited increased structural flexibility and reduced thermal stability. Functionally, sVP40 W95A more strongly inhibited viral RNA synthesis and markedly enhanced budding. However, in a transcription- and replication-competent virus-like particle (trVLP) assay, trVLPs produced with sVP40 W95A induced substantially reduced reporter activity in target cells, indicating impaired particle infectivity or functionality and suggesting possible defects in minigenome packaging, entry, or early post-entry steps. These results demonstrate that mutations at key oligomerization interfaces exert distinct structural and functional effects and highlight the requirement for precise oligomerization in coordinating sVP40s dual roles in genome regulation and virion release. By defining the contributions of L117 and W95, this study advances mechanistic understanding of sVP40 function and identifies processes that may serve as targets for antiviral intervention. ImportanceSudan virus (SUDV) causes regular outbreaks in Sub-Sahara Africa with unusually high lethality rates. However, in contrast to the more often occurring Zaire ebolavirus (EBOV), no monoclonal antibodies or vaccines are available and SUDV is generally understudied. The matrix protein VP40 is responsible for the downregulation of viral genome replication and transcription as well as budding. Here, we present structural and functional characterization of the SUDV VP40 interface residues L117 and W95 and show that while both amino acids are crucial for VP40s structural integrity, their functional effects are dramatically different ranging from complete abolishment to improving regulatory and budding activities.

Kaposis Sarcoma Herpesvirus (KSHV), an enveloped double-stranded DNA virus, is the etiological agent of Kaposis sarcoma (KS), an endothelial cell-based tumor. KSHV is a leading cause of infection-related cancers in sub-Saharan Africa and immunocompromised individuals worldwide. Therefore, it is vital to identify the underlying mechanisms of viral infection and transmission to effectively identify specific therapeutic strategies and combat the disease. Here, we demonstrate that KSHV rewires the host cell lipidome during lytic infection. Bulk lipidomic analysis shows significant changes in the abundance of neutral lipids and phospholipids during lytic infection. We further investigated fatty acid-binding proteins (FABPs) to understand the underlying mechanisms that support KSHV pathogenesis. Using the doxycyclin-inducible iSLK.BAC16 cell line, we find that FABP genes are differentially regulated by lytic KSHV infection compared to latent infection. We report that FABP4 is significantly upregulated during lytic infection. Loss of FABP4 during lytic infection does not impact viral gene transcription however, lytic protein translation is reduced. Moreover, our intracellular and extracellular viral titers indicate that FABP4 affects maximal infectious virion production. This study highlights the role of FABP4 and its therapeutic potential as a target that facilitates KSHV infection and pathogenesis.

Plasmodium falciparum invasion of human erythrocytes is a complex and tightly coordinated process, involving host cell attachment, moving junction formation and engagement of the parasites actomyosin motor. The temporal precision of these events is mediated by distinct ligand-receptor interactions and the sequential release of the merozoites apical organelles. What remains unclear is how these molecular and biophysical interactions enable Plasmodium to bypass the stable erythrocyte membrane-cytoskeletal complex. Here, several P. falciparum lines expressing different fluorescently tagged apical organelle proteins, were imaged with lattice light sheet microscopy (LLSM) to determine the timing of cytoskeletal disassembly and apical organelle release. Blocking the AMA1-RON2 interaction has no effect on the PfRh5-basigin Ca2+ flux but prevents host cytoskeleton disassembly. In contrast, the inhibition of parasite actin polymerisation had no effect on cytoskeletal clearance but caused a sustained Ca2+ response. We further demonstrate that establishment of the moving junction is temporally linked to clearance of the host cytoskeleton. Collectively, our findings support the existence of an association between the RON complex and components of the host cytoskeleton, which mediates the localised disruption of the erythrocyte-membrane cytoskeletal complex during invasion.

Toxoplasma gondii is a single-celled parasite belonging to the Apicomplexa phylum. Toxoplasmas single mitochondrion is highly dynamic, changing its morphology as the parasite undergoes egress and invasion. Recently, we have demonstrated that mitochondrial morphology is driven by a protein named Lasso Maintenance Factor 1 (LMF1). This protein interacts with IMC10, a protein present at the parasites inner membrane complex (IMC), mediating a unique membrane contact site between the IMC and mitochondrion. Interestingly, parasites lacking either LMF1 or IMC10 have abnormal mitochondrial morphology, cell division defects, and delayed propagation in tissue culture. Although both components of the tether were identified, the functions of this contact site remain unknown. In this work, we show that {Delta}lmf1 parasites exhibit upregulation of egress signaling and downregulation in folate metabolism and pantothenate biosynthesis. {Delta}lmf1 parasites exhibit increased intracellular calcium levels, leading to greater sensitivity to ionophore-induced egress and microneme secretion. We have confirmed that parasites have decreased levels of tetrahydrofolate and coenzyme A, showing a limitation in cofactor production. Interestingly, the {Delta}lmf1 parasites prefer glutamine instead of glucose as a catabolic substrate. Accordingly, we demonstrate for the first time that proper mitochondrial positioning is crucial for folate and Coenzyme A metabolism as well as egress signaling. IMPORTANCEToxoplasma gondii is the causative agent of Toxoplasmosis, a disease that affects a third of the worlds population. This parasite has a single, highly dynamic mitochondrion. The parasites mitochondrion changes shape depending on environmental conditions (inside or outside the host cell) or on stressors, such as drugs. Our laboratory characterized the proteins involved in regulating mitochondrial dynamics in the parasite, but the functional importance of these mitochondrial changes has not yet been described. Here, we show that the shape of Toxoplasmas mitochondrion is important for the synthesis of key cofactors, such as folates and coenzyme A. We show that mitochondrial shape in this parasite is important for signaling the parasites exit from the host cell, a critical process in its life cycle. These findings review a previously unknown function of a parasite-specific organelle contact site, providing new insights into the importance of mitochondria for these parasites.

Apicomplexan parasites such as Toxoplasma and Plasmodium spp. rely on the sequential secretion of parasite apical organelles, called micronemes and rhoptries, to invade host cells. The claudin-like apicomplexan microneme protein (CLAMP) is a conserved protein that plays an essential role during host cell invasion in Toxoplasma and Plasmodium zoites. Previous studies have shown that CLAMP is essential in Plasmodium merozoites for erythrocyte invasion and also in sporozoites for the invasion of the mosquito vector salivary glands and of mammalian host hepatocytes. In Toxoplasma gondii tachyzoites, CLAMP forms a complex with two other microneme proteins, the Secreted Protein with an Altered Thrombospondin Repeat (SPATR) and the CLAMP-Linked Invasion Protein (CLIP). Both SPATR and CLIP are also expressed in Plasmodium sporozoites, and downregulation of SPATR impacts sporozoite infectivity in P. berghei. In contrast, the role of CLIP in sporozoites remains unknown. To study the function of CLIP, we used a CRISPR-assisted conditional genome editing strategy based on the dimerisable Cre recombinase in the rodent malaria model parasite P. berghei. Deletion of clip in P. berghei blood stages impaired parasite growth and prevented erythrocyte invasion by merozoites. Upon deletion of clip gene in P. berghei transmission stages, sporozoite development in mosquitoes was not affected, but invasion of the mosquito salivary glands was dramatically reduced. In addition, CLIP-deficient sporozoites were impaired in cell traversal and productive invasion of mammalian hepatocytes, associated with a defect in gliding motility, recapitulating the phenotype of CLAMP-deficient parasites. Collectively, our data demonstrate that CLIP plays an essential role in host cell invasion by P. berghei merozoites and sporozoites, and support a conserved role of the CLAMP-CLIP-SPATR complex in invasive stages of apicomplexan parasites.

The human malaria parasite Plasmodium falciparum (Pf) expresses ten different thrombospondin type 1 repeat (TSR) domain-bearing proteins at different stages throughout its life cycle. TSRs can be modified by two types of glycosylation: O-fucosylation at conserved serine (S) or threonine (T) residues and C-mannosylation at conserved tryptophan (W) residues. PfTRAP, which is expressed in mosquito-stage sporozoites, has one TSR domain that is O-fucosylated at Thr256 and C-mannosylated at Trp250. We employed site-directed mutagenesis by CRISPR/Cas9 gene editing to generate two PfTRAP glyco-null mutant parasites, PfTRAP_T256A and PfTRAP_W250F, and assessed the fitness of these mutant parasites across the life cycle compared to the wild-type NF54 line as well as a PfTRAP knockout line. The PfTRAP glyco-null parasites exhibited major fitness defects comparable to knockout: sporozoites were unable to productively colonize the salivary glands and were highly impaired in gliding motility and the ability to invade cultured human hepatocytes. PfTRAP abundance in these mutants was significantly decreased despite normal transcript levels. Biophysical assays with recombinant proteins confirmed that glycosylation of the PfTRAP TSR stabilizes the domain and is likely required for its folding and secretion. These findings demonstrate that glycosylation of PfTRAPs TSR is critical for its proper expression and function, and underscore the importance of TSR glycosylation in the mosquito stage of the life cycle. IMPORTANCEMalaria is a mosquito-borne disease caused by Plasmodium parasites, of which P. falciparum is the deadliest. Plasmodium has ten proteins bearing thrombospondin type 1 repeats (TSRs), protein folds that aid cell-cell recognition and binding. Each of Plasmodiums ten TSR-bearing proteins is important for invading tissues in the mosquito vector and human host. TSRs are decorated with sugar molecules, a modification termed glycosylation. To better understand the importance of TSR glycosylation in Plasmodium, we investigated the P. falciparum protein TRAP, which is only expressed in mosquito-stage parasite forms called sporozoites. When PfTRAP was mutated to prevent glycosylation, abundance of the protein significantly decreased and parasites were unable to colonize the mosquito salivary glands. Furthermore, these mutant sporozoites were unable to move or to invade human liver cells. Our study reveals how TSR glycosylation can support the function of proteins that are required for parasite virulence.

Severe malaria disproportionately affects children during their earliest Plasmodium falciparum infections, when immunopathology rather than parasite burden often drives clinical deterioration. Because direct investigation of host-parasite interactions during severe disease is ethically impossible, we developed a two-dimensional ex vivo co-culture system that recapitulates key physiological features of malaria pathogenesis. Using PBMCs from malaria-naive and malaria-exposed adults co-cultured with a freshly adapted P. falciparum isolate, we modelled the combined effects of febrile temperature, pipecolic acid (PA), and lysophosphatidylcholine (LPC) depletion on IL-6 secretion. We also integrated clinical data from children with severe malaria in Anambra State, Nigeria. Across conditions, IL-6 output was not driven by temperature alone but by a metabolically gated interaction: febrile temperature amplified IL-6 only when PA was present, and LPC was not limiting. LPC depletion suppressed IL-6 to near-baseline levels regardless of temperature or PA, indicating that lipid availability constrains inflammatory signalling. Clinical data showed that adverse outcomes clustered with markers of multi-organ dysfunction. Together, these findings support a model in which IL-6 is a context-dependent mediator - participating in inflammatory pathways but not acting as a singular causal driver - and in which metabolic stress, febrile cues, and host tolerance mechanisms jointly shape cytokine production. Ongoing bioinformatics analysis will define the transcriptional responses of both parasite and host cells under these malaria-relevant conditions.

Entamoeba histolytica is a protozoan parasite that can cause severe liver disease known as amoebic liver abscess. However, only a subset of infected individuals develops invasive disease, indicating that host-parasite interactions are critical determinants of disease outcome. In this study, we investigated the clone-specific modulation of hepatic immune responses using non-pathogenic A1np and pathogenic B2p E. histolytica clones. Time-resolved transcriptome analyses (6, 12, 24 hours post-infection) in a murine model revealed distinct immune trajectories. Both clones activated innate immune pathways early after infection, but their responses differed markedly in magnitude and composition. A1np infection induced a rapid and controlled inflammatory response associated with antimicrobial activity and resolution-promoting signalling. In contrast, B2p infection triggered a stronger and more complex immune response characterised by pronounced cytokine and chemokine expression, activation of stress and redox pathways, and tissue remodelling processes. The B2p induced response exhibited features of excessive immune activation, accompanied by the upregulation of counter-regulation genes such as Ackr2. These findings indicate that liver pathology is not solely determined by parasite presence, but rather may also be influenced by the nature and regulation of the host immune response. Overall, the observed differences between A1np and B2p infections suggest that parasite-specific properties shape hepatic immune activation and may influence disease progression. Author summaryAlthough infection with the parasite Entamoeba histolytica can lead to severe liver disease, most infected individuals remain asymptomatic. This suggests that the outcome of the disease is not determined solely by the parasite, but also by how the host responds to the infection. In this study, we used a mouse model to compare how the liver reacts to infection with two E. histolytica clones that differ in their ability to cause amoebic liver abscesses. Using this model and time-resolved transcriptome analysis, we found that both clones trigger an early immune response; however, the nature of this response differs markedly. The non-pathogenic clone induced a rapid and controlled reaction associated with antimicrobial defence and tissue protection. In contrast, the pathogenic clone provoked a stronger and more prolonged inflammatory response accompanied by cellular stress and tissue remodelling processes. Notably, this heightened response also activated regulatory mechanisms that attempted to limit excessive inflammation. Our findings demonstrate that differences in disease severity are linked to the activation and regulation of the host immune system, rather than simply to the presence of the parasite.

Hundreds of plant viruses are transmitted by aphid vectors, among which non-circulative ones are acquired and inoculated from one host to another within seconds. These viruses are retained on receptors located at the surface of the cuticle of aphid mouthparts. Members of the Potyviridae family are the most abundant RNA viruses infecting plants, and they cause significant economic losses. Among them, viruses of the Potyvirus genus are transmitted by aphids in a stylet-borne manner. Their receptors in aphid stylets remain poorly characterized. Using turnip mosaic virus (TuMV, Potyvirus rapae) as a model, we developed complementary approaches to investigate potyvirus-aphid interactions in three vector species. Immunofluorescence detection and transmission electron microscopy revealed the presence of TuMV in the distal part of aphid maxillary stylets, both in the food canal and on the acrostyle. This cuticular organ houses Stylin proteins, including Stylin-01, the receptor for the cauliflower mosaic virus (Caulimovirus tessolobrassicae). Using CRISPR-Cas9-edited Stylin-01 mutant lines in the pea aphid, we demonstrated that this protein plays an important role in TuMV transmission. Complementary RNA interference silencing experiments revealed that Stylin-04/04bis also mediate TuMV transmission. Furthermore, our findings reveal that targeting simultaneously Stylin-01 and Stylin-04/04bis more strongly impaired the aphids ability to transmit TuMV, suggesting that virus transmission relies on a multi-component stylin interface rather than a single receptor. In conclusion, these results highlight that in complex interactions between potyviruses and their aphid vectors, Stylin proteins are key actors, underscoring their importance in the transmission of stylet-borne viruses.

MicroRNAs (miRNAs) are small noncoding RNAs that play critical roles in regulating immune responses and have emerged as potential biomarkers and therapeutic targets in complex diseases. Leishmaniasis is a neglected disease that compromises host immunity and is associated with challenging treatments regimens. Leishmania amazonensis (L. amazonensis), an intracellular protozoan parasite, causes cutaneous leishmaniasis by replicating inside mammalian macrophages to establish infection. In this context, miRNAs have emerged as vital post-transcriptional factors that regulate the inflammatory landscape during infection. In this study, we aimed to analyze the function of miR-721 in macrophages during L. amazonensis infection by integrating in silico miR-721 target prediction with RNAseq data from macrophages of two distinct mouse genotypes, resistant C57BL/6 and susceptible BALB/c. We found that miR-721 is induced in macrophages infected with L. amazonensis, but is not in LPS-stimulated macrophages, suggesting a TLR4-independent activation. Integrating miR-721 target prediction with comparative transcriptomic analyses in resistant C57BL/6 and susceptible BALB/c models revealed the TNF-IRF1 axis as a primary miR-721-associated regulatory network. Specifically, miR-721 is predicted to target the 3UTRs of Tnf and Irf1 to suppress the inflammatory response. Functional inhibition of miR-721 successfully restored Tnf and Irf1 expression and reduced the amastigote burden over 24 hours. Furthermore, we showed that the miR-721/TNF-IRF1 axis regulates downstream genes associated with macrophage response, such as Serpine1, Csf1, Cd69 and Maf. Our work demonstrated that Leishmania induces miR-721, which negatively modulates the TNF-IRF1 axis, thereby suppressing the immune response and favoring parasite persistence. While C57BL/6 macrophages exhibit a robust activation of the TNF-IRF1 network, promoting inflammatory response, BALB/c macrophage showed a breakdown of this network. This was associated with post-transcriptional suppression of inflammatory responses, thereby favoring parasite persistence. These findings link miR-721 to the establishment of macrophage polarization, providing relevant insights into the mechanisms of parasite subversion of the host immune response.

Retrovirus replication requires coordinated interplay between viral proteins and host cellular machinery, including reverse transcription of the viral RNA genome into DNA and its subsequent integration into the host genomes. SOX2-dependent retrotransposon dynamics have been reported for endogenous retrovirus HERV-K; however, whether a similar intracellular pathway exists for exogenous retroviruses remains unclear. To address whether infection-independent intracellular reverse transcription and partial integration can occur, that is, whether retrovirus can exhibit retrotransposon-like activity, we utilized HeLa and 293T cells, which express no HIV-1 entry receptors. We engineered an Env-deficient HIV-1 NL4-3-based reporter encoding a reverse-oriented, intron-disrupted nanoluciferase cassette that becomes expressible only after splicing followed by reverse transcription. We found that reporter activation depends on reverse transcriptase and protease activities. While integrase is dispensable for early expression, it is essential for long-term maintenance of the nanoluciferase signal. Integration site mapping using next-generation sequencing further confirmed that stable reporter activity requires integrase-dependent proviral insertion. Functional analysis of Gag revealed that membrane binding, multimerization, and budding are prerequisite steps for reporter activation. Concentrated virus preparations from culture supernatants failed to activate the reporter in 293T cells, ruling out a role for reinfection. Electron and confocal microscopy suggested that Gag or viral particles traffic through endosomal compartments. Furthermore, inhibition of dynamin- and clathrin-dependent endocytic pathways reduced reporter activity, indicating that these pathways contribute to efficient reporter activity. Collectively, these finding support the conclusion that HIV-1 can undergo intracellular reverse transcription and partial integration in an infection-independent manner, prompting a reconsideration of the boundary between exogenous retroviruses and endogenous retroelements. Author summaryEnv-independent, infection-independent intracellular reverse transcription and integration in HIV-1 may contribute to integration-site diversity within the same cell. More broadly, this phenomenon suggests continuity between the retroviral life cycle and retrotransposition dynamics, therefore informing our understanding of host-virus coevolution, mechanisms of long-term persistence, and the redesign of therapeutic strategies targeting pre-integration steps.

Toxoplasma gondii is an obligate intracellular parasite that infects virtually all warm-blooded animals, progressing through acute and chronic stages. The Akt/mTOR signaling axis plays critical roles in cell survival, proliferation, and metabolism, making it a key target for intracellular pathogens. This study investigated how T. gondii infection modulates this pathway during both infections. Outbred CD-1 mice were infected intraperitoneally with the virulent GT1 strain of T. gondii. Mice for acute studies were sacrificed five days post-infection, while those for chronic studies were treated with sulfadiazine and sacrificed five months post-infection. Phosphoprotein expression of eight Akt/mTOR pathway components was measured in liver tissues using a multiplexed bead-based immunoassay. Acute T. gondii infection caused broad suppression of Akt/mTOR signaling, with 6 of 8 markers significantly downregulated, including pS6RPSer235/236, pAKTS473, pBADSer136, pIRS1S636/639, pPTENSer380, and pGSK-3/{beta}Ser21/9. In contrast, chronic infection selectively activates specific nodes of the pathway in a cyst burden-dependent manner, including pBADSer136, pmTORSer2448, and pGSK-3/{beta}Ser21/9. There are strong correlations in signaling changes between inter-components, which reflect coherent and coordinated pathway-level reprogramming rather than random perturbation. These findings show that acute and chronic T. gondii infections have opposing effects on host Akt/mTOR signaling for their own benefit, which may present new therapeutic targets. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=157 SRC="FIGDIR/small/716682v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@8c5021org.highwire.dtl.DTLVardef@1e0cdcaorg.highwire.dtl.DTLVardef@1e690eaorg.highwire.dtl.DTLVardef@342c0b_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIAcute T. gondii infection broadly suppresses hepatic Akt/mTOR signaling C_LIO_LIChronic infection exerts cyst burden-dependent activation of specific Akt/mTOR nodes C_LIO_LIT. gondii has distinct strategies to manipulate host survival based on its life stages. C_LIO_LIThe Akt/mTOR pathway may serve as a therapeutic target for the treatment of T. gondii. C_LI

Brucella spp. are widespread intracellular animal pathogens that cause brucellosis, a significant zoonosis. Despite the global impact of brucellosis on animal and human health, the host genes that support Brucella infection remain incompletely defined. To address this knowledge gap, we developed a flow cytometry-based infection assay with fluorescent Brucella and performed a genome-wide CRISPR-Cas9 loss-of-function screen in human macrophage-like cells. Disruption of >150 host genes significantly reduced intracellular B. abortus burden at 3 h post-infection. In addition to recovering known host factors, the screen revealed previously unappreciated genes linked to endosomal trafficking, cytoskeletal remodeling, and lipid homeostasis. The screen was robust, as validation within these functional categories confirmed that the small GTPase RAB14, the Src-family kinase regulator CSK, and the phospholipid flippase subunit TMEM30A support early infection by B. abortus and B. ovis without impairing general phagocytosis. Gene set enrichment analysis further revealed positive regulators of mTORC1 signaling as key host factors; this result was validated through targeted disruption of LAMTOR2 and AKT1, and pharmacologic inhibition of AKT1. Thus, the AKT-Ragulator-mTORC1 signaling axis contributes to the establishment of a permissive intracellular niche during early Brucella infection. Finally, to assess whether these host requirements extend beyond Brucella, we examined infection by the unrelated intracellular pathogen Mycobacterium abscessus. CSK, AKT1, and LAMTOR2 were required for efficient M. abscessus infection, whereas RAB14 was dispensable. Together, these results define host genes that support early Brucella infection and distinguish shared versus pathogen-specific host dependencies exploited by intracellular bacteria.