Cellular Microbiology

Interleukin-15 (IL-15) trans-presentation (TP) by B cells is an important mechanism of T-cell activation; however, the spatial organisation of interleukin-15 receptor (IL-15R) relative to major histocompatibility complex (MHC) molecules on B-cell microvilli remains poorly understood. As microvilli protrude from the B-cell surface and may serve as sites of initial B cell-T-cell contact, the distribution of IL-15R and MHC molecules within these structures may be important during the earliest stages of T-cell recognition and activation. Here, we investigated the spatial association and molecular proximity of IL-15R with MHC class I and class II molecules on B-cell microvilli before immunological synapse formation, using confocal microscopy, stimulated emission depletion (STED) microscopy, stochastic optical reconstruction microscopy (STORM), and fluorescence lifetime imaging microscopy-based Forster resonance energy transfer (FLIM-FRET). Both MHC class I and class II molecules showed significant spatial association with IL-15R; however, the extent of colocalisation decreased as spatial resolution increased. STED microscopy revealed significant colocalisation between IL-15R and MHC class I, whereas STORM did not detect this association. In contrast, IL-15R and MHC class II remained significantly colocalised at both resolutions. FLIM-FRET further demonstrated molecular proximity between IL-15R and both MHC class I and class II molecules, with higher FRET efficiency observed for MHC class II. Collectively, these findings indicate that IL-15R is spatially organised in proximity to both MHC class I and class II molecules on B-cell microvilli before immunological synapse formation. This arrangement at potential sites of initial B-cell-T-cell contact may facilitate the coordination of IL-15 trans-presentation and antigen presentation during the earliest stages of B-cell-T-cell interactions.

Replication and transcription of Mononegavirales (MNV) occur in cytoplasmic viral factories. In rabies virus (RABV) infected cells, these membrane-less, liquid-liquid phase-separated organelles are called Negri bodies (NBs). Their accumulation of viral mRNAs suggests that, in addition to supporting viral RNA synthesis, they may also serve as sites of viral protein translation. Using immunofluorescence and confocal microscopy, we demonstrate that ribosomal subunit proteins and translation initiation factors are excluded from NBs, including the ribosomal protein RPL40, a component of the large ribosomal subunit involved in the unconventional translation initiation mechanism of RABV. To investigate the spatiotemporal dynamics of protein synthesis during RABV infection, we performed pulse-labeling experiments with the methionine analogue homopropargylglycine (HPG). HPG labeling revealed a rapid accumulation of newly synthesized proteins within NBs, occurring independently of both microtubule and actin cytoskeletons. RABV infection also triggered the formation of stress granules (SGs), in which some ribosomal proteins and translation initiation factors are concentrated. Notably, a marked inhibition of protein synthesis was observed in SG-positive cells. Together, these findings indicate that in RABV infected cells translation occurs outside NBs, followed by the targeting of nascent viral proteins to these compartments.

Sporozoites, the infectious stage of Plasmodium malaria parasites, must invade two different tissues in two very different hosts to ensure transmission: the mosquito salivary glands and the human liver. Whether infectivity to hepatocytes is affected by physiological components, such as the availability of nutrients within the Anopheles female, is not well understood. Here we show that Plasmodium falciparum sporozoite infectivity to hepatocytes is increased if mosquitoes take an additional blood meal while oocysts are developing. Infection rates of HC04.J7 hepatocytes at 24 h were 1.8-fold higher for P. falciparum salivary gland sporozoites isolated from Anopheles gambiae females fed twice (2BF) compared to those which only received the infectious blood meal (1BF). This increase in infectivity was observed at an early infection timepoint (day 11) and was accompanied by a marked shift in the transcriptional profiles of 2BF salivary gland sporozoites, identifying genes likely involved in infection and development in the mammalian host. Overall, our data reveal new factors potentially involved in sporozoite infectivity, and have broad implications for understanding transmission of malaria in endemic areas where multiple blood meals commonly shape available resources for adult female mosquitoes.

Background and aims: Cyclin E1 is a canonical regulator of cell cycle progression known to promote liver fibrosis and hepatocarcinogenesis. However, its precise contribution to hepatic immune responses remains poorly understood. We investigated whether Cyclin E1 regulates macrophage-associated inflammation and acute immune-mediated liver injury. Methods: Hepatic immune populations were analyzed in untreated and concanavalin A-treated wildtype and constitutive Ccne1-/- mice by flow cytometry. Liver injury was assessed by survival, histopathology, and caspase activation. Inflammatory mediators were quantified by qPCR and ELISA. Bone marrow-derived macrophages were examined during differentiation and pro-inflammatory polarization using flow cytometry, cytokine measurements, and RNA-sequencing. Lipid nanoparticle-mediated delivery of Ccne1-specific siRNA was evaluated in J774A.1 macrophage-like cells. Results: Under homeostatic conditions, Ccne1-/- mice exhibited reduced hepatic immune cell numbers, including Kupffer cells and CD4+ and CD8+ lymphoid cells. Following concanavalin A administration, Ccne1-/- mice showed improved survival, reduced hepatic necrosis, and diminished caspase-3 and caspase-8 activation. Protection was associated with reduced Kupffer and CD8+ lymphoid cell abundance and attenuated inflammatory mediator expression. Cyclin E1 deficiency did not substantially alter bone marrow-derived macrophage differentiation or cell cycle distribution. However, pro-inflammatory Ccne1-/- macrophages showed reduced interleukin-6 expression, attenuated inflammatory gene programs, and lower inferred IRF1 and NF-{kappa}B1 activity. Ccne1 knockdown similarly reduced interleukin-6 expression in pro-inflammatory J774A.1 cells. Conclusions: Cyclin E1 contributes to hepatic immune homeostasis and promotes macrophage-associated inflammatory signaling during immune-mediated hepatitis. Its inflammatory function is uncoupled from overt changes in macrophage cell cycle progression and may involve regulation of IRF1- and NF-{kappa}B-associated transcriptional programs. Targeting Cyclin E1 may therefore provide a strategy to attenuate selected inflammatory responses in liver disease.

Background. The cholinergic anti-inflammatory pathway regulates inflammation through vagus signaling that activates the 7 nicotinic acetylcholine receptor (7nAChR) on leukocytes. Suppression of macrophage inflammatory cytokine production through 7nAChR-mediated inhibition of NF-{kappa}B activation is considered a central mechanism of this pathway. However, the effects of cholinergic activation of 7nAChR on neutrophils remain poorly understood. Here, we investigated whether and how activation of 7nAChR in neutrophils regulates neutrophil migration, defined the underlying molecular mechanism, and evaluated its contribution to anti-inflammatory protection during endotoxemia. Results. The ex vivo swarming assays revealed that activation of 7nAChR with the selective agonists PNU-282987 and AR-R17779 significantly inhibited human and mouse neutrophil migration. The inhibitory effect of PNU-282987 was abolished in 7nAChR-deficient neutrophils, confirming receptor specificity. Mechanistically, 7nAChR activation reduced integrin M{beta}2 activation, decreased F-actin polymerization, and impaired neutrophil polarization. These effects required M{beta}2, as PNU-282987 failed to inhibit actin remodeling in M-deficient neutrophils. Modulation of M{beta}2 activation with an activating antibody (MEM48) demonstrated that 7nAChR regulates neutrophil migration by limiting excessive M{beta}2 activation and maintaining an optimal adhesive state. In vivo, adoptively transferred fluorescently labeled PNU-282987-treated neutrophils exhibited reduced recruitment to the lungs compared with vehicle-treated neutrophils during LPS-induced endotoxemia. Furthermore, neutrophil depletion markedly diminished the protective effect of PNU-282987 on survival, demonstrating that neutrophils contribute to 7nAChR-mediated protection during systemic inflammation. Conclusions. Our findings identify neutrophils as an important cellular target of the cholinergic anti-inflammatory pathway and demonstrate that 7nAChR activation limits neutrophil migration by suppressing integrin M{beta}2 activation and actin cytoskeletal remodeling. Together, these findings establish M{beta}2 as a critical downstream effector of 7nAChR signaling in neutrophils and identify regulation of integrin activation as a previously unrecognized mechanism of the cholinergic anti-inflammatory pathway.

Understanding the consequences of global change for our ecosystems requires reliable, accurate, and scalable tools for detecting ecological change. To this end, recent efforts have focused on scaling up the quantification of demographic processes from individual organisms (the fundamental ecological unit) to the larger spatial scales they inhabit to yield more precise and informative estimations of ecological change, especially in response to disturbance. Here, we describe a transferable workflow that combines robust underwater photogrammetry techniques with machine-learning-assisted image analysis to quantify demographic changes in coral colonies (growth, stasis, partial mortality, and complete mortality) through time on the reef landscape. We then demonstrate several analytical applications of this workflow that can be broadly applied to sessile and/or clonal organisms in other ecosystems. First, we tracked the fates of 3,162 coral colonies through a marine heatwave that caused substantial mortality. Second, our approach enabled detection of size- and taxon-dependent patterns in demographic changes from before to after the heatwave. Finally, for a subset (425) of these colonies, we further demonstrated how our method can be used to track fates across multiple years, including a year of change before and after the heatwave took place, revealing nuanced patterns of survival and mortality, as well as changes in size structure that occurred before and after the disturbance. Our workflow offers an important advance for increasing the accuracy of change detection on coral reefs and other ecosystems by linking demographic changes in individual organisms with spatially extensive image-based surveys through time.

Chlamydia trachomatis is an obligate intracellular, developmentally regulated pathogen with a reduced genome as a consequence of adapting to the host cell environment. Despite possessing only eight transcription factors, its developmental cycle is tightly regulated. Hence, we hypothesize that factors like post-transcriptional and post-translational modifications play significant roles in controlling developmental progression. Chlamydia encodes serine/threonine kinases, including an ortholog of Pkn1. In this study, we characterized the function of Pkn1 in chlamydial biology and revealed its role as a cell division regulator. We designed an anhydrotetracycline (aTc)-inducible construct encoding Pkn1 tagged with six histidines at the C-terminus (Pkn1_6xH). Wild-type Pkn1 localized primarily at the division septum whereas a kinase-null Pkn1 isoform localized in the cytosol. To further explore the association of Pkn1 with the divisome, we designed a plasmid encoding an aTc-inducible CRISPRi system targeting pkn1(pkn1KD). Intriguingly, decreased pkn1 transcript levels resulted in enlarged chlamydial cells with irregular coccoid shapes akin to penicillin-treated cells, indicating a block in cell division when pkn1 transcripts are reduced. These effects were complemented by expressing an additional wild-type copy, but not a kinase-null copy, of pkn1 during knockdown. Using a bacterial two hybrid system, we identified an interaction between Pkn1 and MreB, a cytoskeletal protein forming a ring at the septum in Chlamydia. We showed the co-localization of these proteins in Chlamydia and that inhibition of MreB function disrupts Pkn1 localization. Overall, our data indicate an essential function for Pkn1 activity in regulating cell division in Chlamydia.

Pre-prolipoprotein phosphatidylglycerol diacylglyceryl transferase (Lgt) catalyzes the first step in the lipoprotein modification pathway and is essential for growth and viability of Helicobacter pylori. We explored the fate of H. pylori cells under Lgt depletion conditions to obtain insight into Lgt function on morphology and physiology. Downregulation of lgt expression led to growth arrest and a heterogenous population of small rod-shaped cells and small round cells with spiky protrusions, a loose outer membrane and disintegrated flagella. H. pylori overcomes a defect in lipoprotein modification through alteration of its gene expression profile resulting in so-called revertants. Outer membrane protein synthesis, carbon metabolism and nutrient uptake was reduced, and protein translation and flagellar biogenesis increased. Lgt specific regulation was observed in phospholipid biosynthesis resulting in an initial increase in cardiolipin followed by an increase in lysophospholipids at the time of adaptation. Suppressors of {Delta}lgt that were selected on solid media under restrictive conditions either lacked chromosomal mutations or had mutations in the cagUT-tetR regulon controlling the expression of lgt. A suppressor without mutations had an altered expression profile that led to downregulation of a DNA methyltransferase and upregulation of HP_0014, a motility associated glycosyltransferase family protein. Transmission electron microscopy and single cell video microscopy showed that a subpopulation of cells is motile in the revertant while the suppressor regained full motility with higher average speed compared to the wild type strain. Overall, our data show that lipoprotein modification by Lgt is important for membrane and flagellar integrity and that the heterogenous character of H. pylori cultures allows individual cells to overcome this cell envelope stress and survive. Our findings are important for antibacterial development strategies targeting essential integral membrane enzymes involved in lipoprotein modification and stimulate exploration of synergetic activity between known antibiotics and novel antibacterial agents targeting novel cellular pathways.

Microsporidia are divergent fungal pathogens that infect a wide range of hosts. Microsporidia have evolved highly reduced genomes, resulting in the loss of many metabolic pathways. Encephalitozoon intestinalis is a human-infecting microsporidian species with the smallest known eukaryotic genome (2.3 Mbp). As an obligate intracellular parasite, E. intestinalis is dependent on its host for replication. Our current understanding of E. intestinalis cell biology remains limited. This is largely due to genetic intractability, which has made live-cell imaging challenging, and its small size (~2 m), which is within the diffraction limit of conventional microscopy. Here we develop a live-cell imaging technique, "silhouette microscopy", that does not rely on genetic manipulation of microsporidia, and enables us to study the live cell dynamics of E. intestinalis replication in host cells, from entry to exit. We report the timing of cell division, kinetics of the parasite life cycle, and dynamics of the parasitophorous vacuole within which E. intestinalis resides. The parasitophorous vacuole undergoes frequent fission and fusion, and ultimately ruptures to facilitate parasite release from the host cell. To gain a higher resolution understanding into the cell biology of E. intestinalis development, we performed ultrastructure expansion microscopy (U-ExM) on infected cells. U-ExM reveals the organization of the E. intestinalis cytoskeleton and development of the specialized invasion organelle, the polar tube. The combination of live-cell imaging and U-ExM provide a platform to study the E. intestinalis life cycle, and sheds light on the dynamics and cell biology of E. intestinalis in host cells.

Protein kinase R (PKR) is an antiviral sensor that induces translational shutdown and stress granule (SG) formation through the phosphorylation of eIF2. Although previous studies have proposed that hepatitis C virus (HCV) non-structural protein 5A (NS5A) inhibits PKR dimerization, other studies consistently observe PKR activation and SG formation during HCV infection. To address this discrepancy, we interrogated the ability of NS5A from different genotypes and strains to regulate canonical PKR-eIF2 activation in a variety of cell culture models. Regardless of the genotype or strain tested, HCV NS5A failed to inhibit PKR dimerization or dsRNA-induced PKR activation, nor did it rescue downstream steps in the PKR-eIF2 pathway, such as translation halt and SG formation, in contrast to known viral PKR antagonists. Similarly, NS5A did not antagonise PKR activation when expressed in the context of other HCV non-structural proteins. We also tested the role of the host factor cyclophilin A (CypA), which interacts with both NS5A and PKR. However, the absence of CypA did not affect downstream PKR signaling or the activity of viral PKR antagonists or NS5A. Collectively, these findings indicate that NS5A does not effectively antagonise the PKR-eIF2 pathway and further support previous findings that HCV replication occurs in the presence of an active PKR response.

Hepatic stellate cells (HSCs) are the key cellular drivers of liver fibrosis. During liver injury and chronic inflammation HSCs adopt an activated phenotype and secrete fibrotic extracellular matrix (ECM) components such as collagen 1. Mechanical cues derived from the fibrotic ECM drive and support the activation of HSCs, via mechanisms that involve integrins and the mechano-sensitive transcriptional regulator YAP1. It is not yet well understood how external mechanical cues are translated into a molecular response that alters YAP1 nuclear shuttling. There is evidence that suggests the PDZ and LIM domain protein (PDLIM) 5 can regulate YAP1 shuttling in human epithelial cells. We therefore investigated whether PDLIM5 is expressed in HSCs and contributes to YAP1 associated HSC mechano-activation. PDLIM5 protein was detected in HSCs in fibrotic human and mouse liver. PDLIM5 transcript and protein were expressed by primary human and mouse HSCs and by the immortalised HSC LX-2 cell line. PDLIM5 localised with actin stress fibres suggesting a role in HSC adhesion. Co-immunoprecipitation and proximity ligation in LX-2 cells support an association between PDLIM5 and YAP1. We used pharmacological (paclitaxel) and genetic (siRNA and CRISPRi) approaches to inhibit PDLIM5 in HSCs. Inhibiting PDLIM5 reduced YAP1 nuclear localisation and fibrotic gene (COL1A1, ACTA2) expression in LX-2 cells. Overall, these data support a role for PDLIM5 in regulating YAP1 localisation and fibrogenic gene expression in HSCs.

Cell-type-specific molecular responses are obscured in bulk RNA sequencing, whereas generating matched single-cell datasets across environmental conditions remains costly and technically challenging, particularly in non-model organisms. Here, we present a workflow that integrates existing bulk and single-cell RNA-seq resources to prioritize cell-type-associated environmental-response genes. We demonstrate the workflow by examining heat-stress-associated neuronal genes in the reef-building coral Stylophora pistillata. Neuronal genes were first identified based on cell type-specific expression in single-cell transcriptomic data and then intersected with differentially expressed genes from bulk RNA-seq datasets under heat-stress. In parallel, we applied a deconvolution approach to infer cell type-specific expression changes directly from bulk data, independently capturing neuronal transcriptional responses. This integrated pipeline reduced thousands of heat-responsive genes previously identified in bulk RNA-seq studies to nine heat-associated, neuron-enriched candidates. Eight of the nine candidates showed their highest mean expression in the same neuronal subcluster. Targeted sequence-based characterization supported the identification of a TRPA-family ion channel, two calmodulin/centrin-family Ca2+-binding proteins, and a Class A/rhodopsin-like GPCR of unresolved ligand specificity. Two additional candidates were predicted secreted proteins belonging to conserved coral protein families, while the molecular functions of the remaining candidates remained unresolved. Their coordinated regulation suggests heat-associated remodeling of neuronal sensory and intracellular signaling processes. Our workflow provides a scalable framework for extracting cell type-associated molecular responses from existing transcriptomic resources and prioritizing candidates for further investigation. More broadly, it offers a generalizable strategy for investigating cell type-specific gene expression responses across organisms, cell types, and environmental conditions without requiring new single-cell datasets.

Respiratory syncytial virus (RSV) is an enveloped virus that assembles and buds at the plasma membrane, producing filamentous infectious particles. Unlike many other enveloped viruses, a substantial fraction of RSV virions remains associated with the surface of infected cells, limiting our understanding of the membrane-scission mechanisms responsible for particle release. Here, we identified the actin-binding protein drebrin as a cellular interactor of the RSV matrix (M) protein, both in vitro and in infected cells. We demonstrated that RSV infection induces the relocalization of drebrin to actin-rich structures at the cell periphery associated with viral filaments. Using a Transwell-based assay to quantify infectious particles release without mechanically perturbing cells, we confirmed that RSV release is independent of the endosomal sorting complex required for transport (ESCRT) pathway and showed that drebrin depletion reduced RSV release, whereas drebrin overexpression enhanced it. Since drebrin depletion did not significantly affect overall infectious virus production, our results suggest that drebrin may act during the final step leading to membrane scission and RSV particle release. Finally, proteomics analysis identified Transmembrane 9 superfamily member 2 (TM9SF2) as a drebrin-associated protein specifically during infection. TM9SF2 depletion reduced RSV particle release and impaired the RSV-induced relocalization of drebrin to the plasma membrane. Together, these findings identify drebrin and TM9SF2 as host factors regulating RSV release and support a model in which TM9SF2 promotes the recruitment of drebrin to viral budding sites.

The endonuclear symbiont Holospora obtusa invades the macronucleus of Paramecium caudatum using an infectious-form invasion-tip containing a major 89-kDa periplasmic protein implicated in escape from the host digestive vacuole and macronuclear invasion. Sequence-based analyses placed this protein within a Holospora-specific family and predicted an N-terminal membrane-anchoring module and a long alpha-helical C-terminal region. AlphaFold2 modeling followed by molecular dynamics indicated that the C-terminal region forms an antiparallel, spectrin-repeat-like helical solenoid containing a localized three-helix coiled-coil. The protein exhibits a large pH-dependent charge difference, becoming 55 units more positive at pH 4 than at pH 7. Independent simulations at these pH values differed mainly in the relative orientation of the N-terminal core and C-terminal rod, described by DynDom as an approximately 35 degrees rigid-body rotation about a hinge centered on Gly291. However, none of the nearby glutamates (Glu273, Glu275, and Glu302) formed a pH-dependent salt bridge, and no specific protonation-dependent interaction responsible for the conformational difference was identified. We nonetheless propose, as a testable hypothesis, that acidification of the digestive vacuole may promote this reorientation, thereby repositioning the N-terminal membrane-anchoring module at the bacterial surface as an early step toward escape from the vacuole.

Epigenetic processes play important roles in the biology of the malaria parasite Plasmodium falciparum. We recently showed that Plasmodium histones are not only acetylated and methylated but also lactylated. This new epigenetic mark could form a particularly important axis of host-parasite signalling because severe malaria is often characterised by hyperlactataemia. In the human host this can cause respiratory distress, which is potentially fatal. For the parasite, it could be advantageous to sense this state of pathology and respond by modulating virulence. Virulence processes known to be under epigenetic control include antigenic variation, invasion switching and conversion to sexual gametocytes, i.e. gametocytogenesis. Here, we investigated the influence of lactate on gametocytogenesis. Hyperlactataemia in malaria patients is defined as [≥] 5mM blood lactate and we confirmed that in laboratory culture, adding 5mM L-lactate was sufficient to boost gametocytogenesis. Expression of the GDV-1 gene increased within one cell cycle of asexual parasites being exposed to L-lactate, suggesting that the canonical epigenetic switch for gametocytogenesis was involved: GDV-1 regulates epigenetic de-repression of the gene encoding the master transcription factor for gametocytogenesis, AP2-G. Indeed, parasite histones became rapidly lactylated after L-lactate exposure and chromatin profiling by CUT&Tag detected inducible lactylation of chromatin specifically upstream of the AP2-G gene, and also of the antisense RNA that regulates GDV-1. Thus L-lactate joins S-adenosylmethionine as a metabolite that can epigenetically regulate gametocytogenesis in P. falciparum.

Background: Hepatitis B virus (HBV) basal core promoter (BCP) and precore (PC) mutations occur during chronic HBV infection with the BCP-mutant being associated with increased hepatocellular carcinoma (HCC) risk. Objectives: The effects of these mutants on viral replication, hepatocyte biology, and carcinogenesis remain poorly defined. To address this, we characterized BCP- and PC-mutants using human hepatocyte chimeric mice and asked whether the resulting infection-induced transcriptomes correspond to subsets of human HBV-related hepatocellular carcinomas (HBV-HCCs). Design: Isogenic wild-type (WT), BCP- and PC-mutants of HBV genotypes A, C, and D (HBV-A, -C, -D) were generated from recombinant covalently-closed circular DNA to infect chimeric mice. HBV-HCC transcriptomic datasets from The Cancer Genome Atlas (TCGA) were used to stratify association of HBV variants with HCC subtypes. Results: WT, BCP-mutant, and PC-mutant HBV sequences remained genetically stable. The BCP mutation, but not the PC mutation, accelerated the rise in serum viremia in HBV-D- and HBV-A-infected chimeras; in HBV-C, acceleration required both (PC+BCP) mutations. Further comparisons of HBV-D variants revealed that the BCP-mutant increased intrahepatic viral DNA, viral protein expression, and upregulated cancer-related pathways, including transcripts associated with a subset of HBV-HCCs. Analysis of HBV-HCC samples from the TCGA revealed that tumors often harbor a mixture of WT and mutant transcripts, and that WT- and BCP-mutant-associated HCCs exhibit distinct transcriptional profiles. Conclusions: The HBV BCP-mutant directly perturbs hepatocyte homeostasis via virus-intrinsic mechanisms, selectively activating cancer-related pathways and defining a molecularly distinct subset of HBV-HCC. These findings suggest that HBV variants form distinct subcategories of HBV-HCCs.

Bacillus cereus biovar anthracis (Bcbva) strains, which cause fatal anthrax-like disease, have been isolated from animals in Central and West Africa. Like B. anthracis, Bcbva produces a poly-{gamma}-D-glutamic acid (PDGA) capsule, as well as edema factor (EF), lethal factor (LF) and protective antigen (PA). Bcbva also possesses a hyaluronic acid capsule that is not expressed by B. anthracis. The pathogenesis of Bcbva has recently been characterized in inhalational infection models, in rabbits and guinea pigs. Here, we characterized the ki-netics of Bcbva infection and toxin production in a murine model of cutaneous infection using the Bcbva CA strain isolated in Cameroon. We found that spore germination oc-curred rapidly at the inoculation site, followed by bacterial dissemination to the draining cervical lymph nodes. Germination was associated with rapid production and diffusion of LF and EF. Bacteria rapidly disseminated to lymphoid organs and other compartments, with distal germination observed 18 hours post-infection. Infected mice developed septi-cemia within 24 hours, accompanied by high circulating LF levels and signs of cardiac and renal injury. These findings may contribute to the fulminant course of Bcbva infection observed in animals and highlight its potential risk to humans.

Severe dengue is characterized by systemic inflammation and vascular leakage. Recent evidence indicates that Toll-like receptor 2 (TLR2) supports early steps of dengue virus (DENV) infection in monocytes, whereas intracellular viral RNA sensing drives much of the subsequent inflammatory response. However, whether this entry-associated function of TLR2 differs among human monocyte subsets remains unknown. Here, we investigated how the TLR2 axis regulates DENV serotype 2 (DENV2) infection and inflammatory output in purified primary human monocytes and sorted monocyte subsets. Blocking TLR2 at the cell surface or targeting its intracellular TIR domain reduced virus-cell association, productive infection, and the release of inflammatory and antiviral mediators. Among TLR2-associated receptors, CD14 and, to a lesser extent, CD36 contributed to efficient infection, whereas TLR1, TLR6, and TLR10 were dispensable under the conditions tested. Notably, TLR2 blockade reduced viral production and cytokine release in CD16- classical monocytes but not in pooled CD16+ monocytes, identifying a subset-specific dependence on the TLR2 axis. TLR3 contributed to inflammatory and antiviral responses without affecting viral replication, consistent with post-entry recognition of viral RNA. Finally, blocking the TLR2 axis during monocyte infection prevented the loss of endothelial barrier integrity induced by monocyte-derived soluble mediators. Together, these findings identify a TLR2-associated entry route that preferentially supports productive DENV2 infection in classical monocytes and thereby enables RNA-sensor-dependent inflammatory responses that compromise endothelial function.

Leishmania species are transmitted between mammals by sand flies, within which they undergo a complex developmental programme involving transitions between replicative and non-replicative life cycle forms. Recent work using single-cell transcriptomics has revealed the developmental pathways of Leishmania major in the sand fly, but it is unclear how faithfully such development is recapitulated during Leishmania culture in vitro. In addition, no work has revealed how gene expression changes across the cell cycle of replicative Leishmania forms. Using single-cell transcriptomics on unsynchronised cultures of L. major and L. mexicana, we addressed both questions. We show that in vitro L. mexicana promastigotes display the same two developmental endpoints seen in L. major cells recovered from sand flies, metacyclics and haptomonads. In contrast, L. major promastigote development in vitro does not predict the presence of leptomonads or haptomonads, although non-dividing late metacyclics form through an early metacyclic stage as parasites enter stationary phase. Using computational reconstruction of the cell cycle in procyclic promastigote forms of both Leishmania species identified hundreds of genes whose transcript levels vary as the cell cycle progresses, revealing core conserved processes between the two species and with the closely related Trypanosoma brucei. These genes allowed us to examine connections between life cycle and cell cycle progression, revealing that some developmental transitions are strongly linked to a cell cycle phase, whereas others occur asynchronously. This work provides a comprehensive atlas and marker gene list for the interlinked cell and life cycles of two laboratory-cultured Leishmania species.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a human tumor virus. Common marmosets, a potential non-human primate model for vaccine development and infection studies, have previously been shown to support KSHV infection. We established infection of marmosets with BAC-derived, recombinant KSHV to analyze the role of individual glycoproteins (GPs) in tissue tropism. Three groups of four animals were infected intravenously with BAC-derived KSHV wild type (wt), EphA2-receptor-binding-deficient KSHV gHASAELAAN, or KSHV{triangleup}K8.1, lacking glycoprotein K8.1. Viral DNA was detected in multiple tissues of all animals, with the highest loads in spleen, liver, heart, and bone marrow. In most tissues, viral DNA loads did not significantly differ between groups. However, KSHV{triangleup}K8.1-infected animals exhibited significantly lower KSHV DNA levels in liver and bone marrow. KSHV wt infection induced activation of CD4+ and CD8+ T-cells. In animals infected with KSHV wt, KSHV gHASAELAAN or KSHV{triangleup}K8.1, different B cell subpopulations expanded after infection, which suggests that the glycoproteins themselves or the cell tropism imparted by their presence or absence might shape the host response to KSHV. In summary, our findings demonstrate that neither the Eph receptor interaction nor GP K8.1 is essential for intravenous infection, but K8.1 critically contributes to KSHV tissue tropism in vivo.