Cellular Microbiology

Malaria, of which the most virulent form is caused by Plasmodium falciparum parasites, remains a major global health burden. The appearance of resistance to first line treatments artemisinin-based therapies, emphasizes the need to identify new parasite vulnerabilities to develop new therapeutics. Phosphoinositides are central regulators of membrane identity, vesicular trafficking, and signaling, and their synthesis depends on tightly controlled phosphatidylinositol transfer by Sec14-like phosphatidylinositol transfer proteins in many eukaryotes, yet their roles in P. falciparum remain poorly defined. Here, we analyzed six P. falciparum Sec14 domain-containing proteins: PfSec14-1 (PF3D7_0626400), PfSec14-2 (PF3D7_0629900), PfSec14-3 (PF3D7_0717100), PfSec14-4 (PF3D7_0920700), PfSec14-5 (PF3D7_1007200), and PfSec14-6 (PF3D7_1127600). Domain organization segregates these proteins into a BNIP-2 and Cdc42GAP homology (BCH) subfamily (PfSec14-3, PfSec14-5) and a canonical Sec14 subfamily (PfSec14-1, PfSec14-2, PfSec14-4, PfSec14-6). Yeast complementation assays showed that PfSec14-1, PfSec14-4, and PfSec14-6 partially rescue growth of a temperature-sensitive sec14 mutant, suggesting phosphatidylinositol and phosphatidylcholine transfer activity. Gene disruption revealed that PfSec14-1 is important for asexual blood-stage proliferation, whereas PfSec14-2 is dispensable under standard culture conditions. In contrast, mislocalization of PfSec14-1 and PfSec14-4 using a knock-sideways approach did not impair asexual growth. Subcellular localization indicates distinct distributions for PfSec14-1, PfSec14-2, and PfSec14-4. Together, these findings reveal functional and spatial diversification of Sec14-like phosphatidylinositol transfer proteins in P. falciparum.

Background & AimsHepatitis B virus (HBV) drives hepatocellular carcinoma in part through the activity of its X protein (HBx), yet the mechanisms by which HBx alters hepatocyte function remain incompletely understood. Progress has been limited by the lack of relevant human models that support controlled HBx expression in mature hepatocytes. Here, we use an improved hepatocyte-like organoid (HLO) platform that supports enhanced hepatocyte maturation to investigate HBx function in a differentiated hepatocyte context. MethodsAdult stem cell-derived HLOs were differentiated using an optimized protocol to generate hepatocyte-like cells with enhanced maturation and transcriptional similarity to primary liver tissue. HBx function was interrogated using both cognate promoter-driven expression and doxycycline-inducible systems across multiple donor-derived organoid lines. Transcriptomic, pathway, and single-cell imaging analyses were performed to assess the impact of HBx expression on hepatocytes. ResultsHBx expression consistently suppressed apoptosis-associated transcripts and reduced expression of core hepatocyte identity genes, including CYP3A4. Pathway analysis revealed downregulation of liver-specific functions, including metabolism, detoxification, complement, and coagulation. At the single-cell level, higher HBx expression was associated with reduced caspase 3/7 activation following apoptotic challenge and decreased hepatocyte marker expression. Functionally, HBx expression increased resistance to apoptosis and enhanced the ability of differentiated hepatocyte-like cells to revert to a proliferative, less differentiated state. ConclusionsHBx expression in differentiated human liver organoids reduces apoptosis and impairs hepatocyte identity, consistently across donors and expression systems. These findings support a model in which HBx promotes a survival-permissive less differentiated state that may contribute to early HBV-driven tumorigenesis. This HLO platform provides a relevant system to dissect HBV-host interactions and reveals a mechanism by which HBV may prime the liver for malignant transformation. Impact and implicationsUnderstanding how HBV promotes hepatocellular carcinoma remains a critical challenge, partly due to the lack of physiologically relevant human derived model systems to study HBx function. Using a differentiated adult human liver organoid system, we show that HBx simultaneously suppresses apoptosis and disrupts hepatocyte identity, providing a mechanistic framework for how HBV may prime hepatocytes for malignant transformation. These findings are particularly relevant for researchers studying HBV pathogenesis and liver cancer, as well as for clinicians aiming to better understand early disease progression. While further validation in more complex multicellular systems is needed, this platform can support the identification of HBx-targeted therapeutic strategies and guide the development of improved adult human derived models for virus-host interaction studies.

Subcellular organelles must undergo periodic fission to be evenly distributed during cell division. These division events are mediated by protein members of the dynamin family, including dynamin-related proteins. Protozoan parasites, including trypanosomatids such as Trypanosoma brucei, have several single-copy organelles, suggesting tightly regulated systems for organelle fission and segregation. However, trypanosomatid genomes typically encode only one dynamin-like protein (DLP), which in T. brucei has multiple roles including endocytosis and mitochondrial fission. How DLPs are recruited to different membranes, and how their fission activity is regulated, are unknown. We used tandem-affinity purification in the related trypanosomatid Crithidia fasciculata to identify interacting partners of DLP. Surprisingly, we found that CfDLP co-purified with multiple proteins predicted to localize to glycosomes, peroxisome-related glycolytic organelles. Using expansion microscopy, we confirmed the localization of CfDLP to glycosomes, specifically those that appear to be undergoing division. To see if changes in the levels of DLP could alter glycosome morphology, we conducted RNAi-mediated knockdown and inducible overexpression experiments in T. brucei. TbDLP knockdown causes subtle changes in glycosome size, while overexpression of TbDLP1 causes an increase cytoplasmic vesicles and altered permeability of glycosomal membranes. These results suggest that the multifunctional DLP of trypanosomatids plays a role in glycosome maintenance. Author SummaryTrypanosomatids are eukaryotic parasites that cause devastating diseases in humans and animals. Like all eukaryotic cells, they must maintain their subcellular compartments through organelle division and other membrane remodeling events. Dynamin-like proteins are enzymes that work with other proteins to apply mechanical force to membranes. The dynamin-like proteins of Trypanosoma brucei, the causative agent of human African trypanosomiasis, have been implicated in endocytosis and mitochondrial division, although how these activities are regulated is not known. We have used a model trypanosomatid, the mosquito parasite Crithidia fasciculata, to look for dynamin-interacting proteins. In addition to proteins of unknown function, we show that dynamin-like protein associates with proteins found on glycosomes, trypanosomatid-specific organelles that contain enzymes required for breakdown of sugars. Knockdown and overexpression of dynamin-like proteins in T. brucei causes changes in glycosomes, supporting a role in organelle maintenance. Dynamin-like proteins likely regulate organelle structure and function, allowing parasites to adapt to different energetic requirements during their life cycle.

The apicoplast of malaria parasites retains a reduced genome encoding a small set of genes with unknown functions. Among these genes is a putative ClpM chaperone, which unlike other apicoplast Clp-family members is not nuclear but plastid-encoded. In this study, we used ClpM as a model case to investigate evolutionary and molecular basis for plastid genome retention. Phylogenetic analyses across plastid-containing eukaryotes revealed that ClpM orthologues are broadly conserved and consistently plastid-encoded in all organisms with a red alga-derived plastid, irrespective of parasitism, photosynthesis or physiology. This broad phenomenon suggested gene-specific evolutionary constraints that were subsequently tested experimentally. To test whether clpM can be functionally expressed from the nucleus, we generated transgenic parasites carrying a nuclear ClpM copy fused to an apicoplast-targeting transit peptide. Unexpectedly, standard transgenesis resulted in transcriptional silencing, and we therefore forced transcription using integration into an endogenous essential locus. This led to robust clpM mRNA, however no detectable ClpM protein was observed. Multiple analyses ruled out apicoplast-dependent instability, ER-associated degradation, misfolding or membrane sequestration. Attempts to express clpM or other plastid-derived genes using endogenous sequences were found to be toxic, suggesting nucleotide-sequence incompatibility. In contrast, a transgene carrying a second copy of the nuclear ClpC ortholog was readily expressed. Comparative analysis of ClpM and ClpC domain architecture showed that their ATPase domains form distinct evolutionary clusters, suggesting conserved but functionally divergent roles. Subsequently, domain-swap experiments between ClpC and ClpM rescued partial expression and identified specific domains as contributors to the nuclear-expression barrier. Together, these findings demonstrate that clpM retention in the apicoplast genome is enforced by multilayered constraints involving evolutionary conservation, nucleotide-sequence incompatibility, transcriptional block and protein-intrinsic translational barriers. This work provides experimental evidence for mechanisms that restrict organelle-to-nucleus gene transfer and contribute to organelle genome retention.

Placental malaria, due to the sequestration of Plasmodium falciparum-infected erythrocytes (IE), causes adverse pregnancy outcomes. The sequestration is mediated by VAR2CSA, a protein that binds to placental chondroitin sulfate A (CSA). VAR2CSA antibodies protect against adverse pregnancy outcomes; however, no licensed VAR2CSA-based vaccine or therapeutic exists to date. We identified and expressed VAR2CSA-specific IgG1 monoclonal antibodies (mAbs) using B cells of malaria-exposed Papua New Guinean women. VAR2CSA mAbs were characterised by their ability to recognise eight heterologous CSA-binding P. falciparum strains, to neutralise CSA binding and/or to induce phagocytosis of IEs by THP-1 monocytes. We identified 16 mAbs, all of which targeted just two of the six domains of VAR2CSA, DBL3X and DBL5{varepsilon}. Cross-reactivity varied between mAbs, but was highest among mAbs to DBL5{varepsilon}, with four of eight of these mAbs binding to all eight strains. Although individual mAbs did not promote phagocytosis, combinations of mAbs recognising distinct epitopes either on the same domain or over different domains did. None of the mAbs inhibited IEs from binding to CSA. Our findings suggest that a combination of mAbs recognising more than one epitope would be needed for a therapeutic aiming to promote parasite clearance by phagocytosis; that DBL5{varepsilon} could be considered for a VAR2CSA vaccine that aims to elicit cross-reactive antibodies that promote phagocytosis; and that identification of binding-inhibitory mAbs requires thoughtful B-cell bait design.

The Golgi is an essential organelle that serves as a central hub for endomembrane trafficking. In the protozoan parasite Toxoplasma gondii, a single Golgi stack is essential for parasite survival; however, the molecular determinants governing Golgi structure and function remain poorly understood. Here, we characterize a Golgi-associated protein that is required for Golgi integrity and function, which we named Toxoplasma Golgi Integrity Factor (TGIF). Loss of TGIF disrupts parasite replication and natural egress and is lethal to the parasite. To investigate the impact of TGIF depletion on secretory protein trafficking, we adapted a fluorescence-based pulse-chase assay to monitor the synthesis and trafficking of microneme and rhoptry proteins. We found that loss of TGIF significantly impaired the synthesis and trafficking of microneme and rhoptry neck proteins, whereas trafficking of rhoptry bulb proteins was minimally affected. These findings suggest that rhoptry bulb proteins may traffic independently of canonical Golgi-dependent pathways. Collectively, our study provides new insight into the mechanisms of Golgi-mediated trafficking in T. gondii and identifies TGIF as a critical regulator of parasite secretory pathway organization and function.

The lack of effective anti-hepatitis B virus (HBV) therapies highlights the need for a new type of treatment that targets different stages of the viral life cycle. The HBV core protein (HBc) is a critical component of this cycle. Various capsid assembly modulators (CAMs) have been developed to target the HBc and inhibit HBV replication. We recently described a subset of capsid assembly modulators (CAMs) that induce the formation of aberrant structures from the HBc in the nucleus, leading to cell death via annexin A1 (ANXA1)-driven apoptosis. Thus, we further elucidated the mechanism of HBc aggregation in the nucleus, with a particular focus on the interplay between nuclear HBc aggregates and PML nuclear bodies. We found that long-term treatment with CAM-A induced the formation of enlarged PML bodies, approximately 1-2 m in diameter, that accumulated aggregated HBc. PML silencing in HBc-overexpressing HepG2-NTCP cells led to a dramatic increase in apoptosis following CAM-A-induced HBc aggregation, which was associated with elevated ANXA1. Next, we showed that PML nuclear bodies orchestrate proteasomal degradation of nuclear HBc aggregates via sumoylation-dependent recruitment of RNF4. Collectively, our results suggest that PML nuclear bodies act as storage compartments for aggregated HBc proteins in the nucleus, thereby counteracting the apoptotic elimination of cells. Further study of PML function and the targeting of PML nuclear bodies in HBV-infected hepatocytes could reveal new ways to enhance the effectiveness of CAMs.

Repressor-of-differentiation kinase 1 (RDK1) is one of two kinases expressed in bloodstream form Trypanosoma brucei parasites that were found to repress premature and spontaneous differentiation into the insect procyclic form. However, the effect of RDK1 RNAi was previously limited to the expression of a single surface coat protein, EP1 procyclin. Thus, there remains a significant gap in knowledge on the impact of RDK1 expression in bloodstream form T. brucei parasites. Here, we employ a systems biology approach and performed several proteomics analyses to identify RDK1 protein interactions and to determine the impact of loss of RDK1 expression on the bloodstream form proteome and phosphoproteome to uncover clues about potential mechanisms for RDK1 function. We found that RDK1 is dual localized to the cell membrane and the mitochondrial inner membrane with the kinase domain oriented towards the cytoplasm and mitochondrial inner membrane. Unexpectedly, the most enriched RDK1-proximal proteins were mitochondrial proteins. Furthermore, RDK1 depletion causes bloodstream form parasites to significantly upregulate many mitochondrial proteins and glycosomal proteins, several of which are upregulated in procyclic form parasites. Surprisingly, the mitochondrial phosphoproteome is largely unaffected by RDK1 depletion, while RDK1-dependent phosphoregulation is restricted to the cell membrane localization of RDK1. Lastly, we determined that RDK1 does not possess adenyl cyclase activity or alter intracellular cAMP levels; however, the dysregulated phosphoproteins correlate with functions in cyclic nucleotide signaling. In conclusion, RDK1 exhibits localization-specific kinase activity to regulate cyclic nucleotide signaling and mitochondrial proteomic maintenance in bloodstream form parasites. IMPORTANCETrypanosoma brucei is the unicellular parasite that causes African sleeping sickness and nagana disease in livestock across 36 sub-Saharan African countries. The parasite encounters different environmental niches as it is transmitted from an infected human to the tsetse fly vector as the fly takes a blood meal. T. brucei must sense environmental cues to initiate intracellular signaling pathways to promote effective differentiation and cellular remodeling from the mammalian bloodstream forms to the insect procyclic form. RDK1 is one of two kinases shown to repress premature differentiation to procyclic form, which would be detrimental for parasite survival in the human host. Therefore, it is essential to uncover mechanisms of RDK1 function to better understand how T. brucei maintains homeostasis in the human host and signals for effective cellular remodeling during parasite transmission.

Effector translocation during host-pathogen interactions is a prerequisite for the entry of Salmonella into non-phagocytic cells and establishment of a replication permissive intracellular niche. Deciphering the dynamics and kinetics of translocation and subcellular localization demands live-cell imaging and tagging approaches that do not introduce detection delays or perturb the translocation process via the type III secretion system (T3SS). Effector fusions with self-labelling enzymes (SLE), such as HaloTag, allow localization and tracking at high temporal and spatial resolution. However, interference with T3SS-dependent translocation has hampered analyses of the process of translocation and early subcellular distribution and dynamics. Herein, we report that amino acid substitutions of the HaloTag can reduce the thermodynamic stability, resulting in less steric hindrance during translocation of effector-HaloTag fusions by the T3SS in mammalian cells. The top variant, HT-SP5, showed reduced retention in Salmonella, enabling more sensitive and earlier detection of translocated effector proteins of the SPI1 and SPI2 T3SS of Salmonella and of the T3SS effector Map of enteropathogenic Escherichia coli (EPEC). We applied the improved HaloTag HT-SP5 to single molecule tracking, and to follow effector protein dynamics in living host cells early after translocation by invading and intracellular bacteria. Taken together, the improved HaloTag variant HT-SP5 represents a robust and versatile SLE tag for dynamic real-time analyses of delivery and fate of T3SS-translocated effector proteins in living cells host. Application of HT-SP5 will facilitate research on effectors throughout the entire infection process at native effector levels to understand host-pathogen interactions.

Mycobacterium tuberculosis (Mtb) virulence relies in part on its ability to induce phagosomal membrane rupture, enabling bacterial access to the host cell cytosol. This process is largely mediated by the ESX-1 secretion system, which is present in Mtb but absent from the vaccine strain BCG. Galectin-3 (Gal3), a {beta}-galactoside-binding lectin, is recruited to damaged endomembranes and functions as a cytosolic sensor of membrane disruption. However, the kinetics and quantitative features of Gal3 recruitment to Mtb-containing vacuoles have remained poorly characterized. Here, we performed a longitudinal quantitative imaging study of Gal3 recruitment in human macrophages over a five-day infection period. Gal3 was recruited to mycobacteria-containing vacuoles shortly after infection with both live and heat-killed Mtb. Differences between the two conditions emerged from day 1 post-infection and persisted until macrophage death. A similar kinetic profile was observed with recombinant BCG::ESX-1, whereas parental BCG failed to induce Gal3 recruitment, confirming the requirement for ESX-1-dependent membrane damage. Cytoplasmic Gal3 levels were higher in bystander macrophages than in infected cells and were comparable to non-infected controls, suggesting that diffuse cytoplasmic Gal3 is associated with cells lacking intracellular mycobacteria. Functional studies revealed that Gal3 silencing enhanced long-term intracellular Mtb replication, demonstrating a role for Gal3 in restricting bacterial growth. Importantly, Gal3 recruitment was not observed in alveolar epithelial cells. This cell-type specificity was confirmed in a microfluidic alveolus-on-chip model. Together, these findings identify sustained Gal3 recruitment to the mycobacteria-containing vacuole as a robust quantitative marker of ESX-1-dependent phagosomal rupture and Mtb virulence.

Raf kinase inhibitor protein (RKIP) plays a key role in regulating critical signaling pathways in higher eukaryotes. We previously demonstrated that Plasmodium falciparum RKIP (PfRKIP) modulates the activity of PfCDPK1, a key regulator of red blood cell invasion. Here, we have used a pharmacological approach to investigate the function of PfRKIP. Locostatin, a mammalian RKIP inhibitor, shows a dose-dependent decrease in RBC invasion. Mechanistically, locostatin increases the interaction between PfRKIP and PfCDPK1, thereby sequestering PfCDPK1 in a complex preventing substrate phosphorylation. Surprisingly, PfRKIP could be knocked out from the parasite without any perceptible growth defect. Interestingly, the PfRKIP-deficient parasites show an increase in the import of host RKIP. Like the parasite RKIP, the host RKIP interacts with PfCDPK1 and is associated with high-molecular-weight complexes comprising PfCDPK1 suggesting functional complementation by the host RKIP in the PfRKIP null parasites. As expected, locostatin shows similar inhibitory effect on the knock-out parasites as the WT. Our study reveals a novel host-parasite interaction wherein the parasite co-opts host proteins to maintain critical signaling pathways. Targeting the PfRKIP signaling axis, along with the host proteins, represents a potential strategy for anti-malarial drug development that is conceptually less prone to parasite-driven resistance development.

Cilia are assembled by intraflagellar transport (IFT), which relies on two protein complexes: IFT-A and IFT-B. It is generally assumed that IFT-B and IFT-A are critical for anterograde and retrograde transport, respectively. However, full deletion of IFT-A genes in several organisms suggests a possible contribution to anterograde transport. In many species, cilia collapse when IFT is altered, hindering functional studies. Here, we investigated the role of IFT-A in the protist Trypanosoma brucei, where IFT is not required for cilium maintenance. Following the inducible knockdown of IFT88 (an IFT-B member) or IFT140 (an IFT-A member), we monitored the fate of several IFT proteins in preassembled cilia using live imaging and evaluated the consequences on train formation by volumetric electron microscopy. Surprisingly, both IFT88 and IFT140 turned out to be essential for anterograde train assembly. Their depletion initially led to the formation of shorter trains and subsequently to an inhibition of train injection. We propose a model to reconcile the diverging phenotypes reported in the literature.

Heterotrimeric kinesin 2 is the canonical motor protein for anterograde intraflagellar transport (IFT), driving movement of protein complexes towards the tip of cilia and flagella. Here, we show that all members of the Euglenozoa group lack genes for heterotrimeric kinesins and instead possess a variable number of genes for two homodimeric kinesins termed KIN2A and KIN2B. When expressed in vitro, both Trypanosoma brucei kinesins form homodimers and move processively along brain microtubules, KIN2A being faster than KIN2B. Studies in T. brucei and Leishmania mexicana show anterograde and retrograde IFT of both kinesins, with KIN2A travelling throughout the whole length of the flagellum, while KIN2B is concentrated at its base. In the proximal portion of the flagellum, most KIN2B molecules travel without IFT proteins, except for a few particles that are associated with IFT proteins and reach the tip. Surprisingly, the absence of KIN2A has mild effects on IFT and flagellum assembly, whereas KIN2B is essential for both. Investigation of trypanosome flagella deprived of KIN2B revealed that IFT proteins do not access these flagella but that KIN2A can still circulate. These results support a division-of-labour model where KIN2B is responsible for the import of IFT proteins while KIN2A is responsible for most of the anterograde transport.

Plasmodium falciparum ATP4 mutations A211V and G223R allow parasites to survive the lethal effects of antimalarials PA21A092 (PA92) and cipargamin (CIP), respectively. An A211V mutant line (Dd2A211V) treated with PA92 showed enhanced levels of lipid production, which prompted the idea that components of the phospholipid biosynthesis pathway could be involved in the survival mechanism of PfATP4 mutant parasites. As phosphatidylserine synthase (PfPSS) is the only enzyme that produces phosopholipid phosphatidylserine (PS) in P. falciparum parasites, we hypothesized that PfPSS is both essential for parasite survival and that reduced PfPSS expression would cause resistant PfATP4 mutant parasites to become susceptible to PA92 or CIP. We created a CIP-resistant G223R mutant line (Dd2G223R) via CRISPR-Cas9 and integrated a conditional PfPSS knockdown construct into a Dd2A211V ({downarrow}PSS-Dd2A211V) and our Dd2G223R line ({downarrow}PSS-Dd2G223R). We treated these knockdown lines with PA92 or CIP to determine the half-maximal effective concentration (EC50) of each antimalarial with normal or reduced PfPSS levels. While we found that PfPSS is essential for parasite survival, we did not find any significant alterations to the EC50 values of PA92 or CIP based on the reduced levels of PfPSS in our mutant lines. Although PfPSS does not appear to be involved, other components of the phospholipid production pathway could still affect the resistance mechanism of PfATP4 mutations. Identification of novel targets to counteract the mechanism by which PfATP4 mutant parasites resist lethal drug effects is crucial for the successful application of antimalarials in endemic countries where resistance is on the rise.

Chronic hepatitis B cure requires the inactivation and/or elimination of covalently closed circular DNA (cccDNA), together with silencing of integrated viral genomes and restoration of HBV-specific immune responses. The TLR1/2 agonist Pam3CSK4 has previously been identified as a potent direct anti-HBV agent in vitro. In the present study, we engineered a liver-targeting polymeric nanoparticle formulation of Pam3CSK4 to enhance its in vivo immunostimulatory and antiviral activity. We evaluated the antiviral efficacy of this novel nanoformulation carrying the TLR1/2 agonist (NP-Pam3CSK4) in monotherapy and started to investigate its mechanism of action through immunological correlates in an immune-competent AAV-HBV mouse model. AAV-HBV-infected mice received intravenous administrations of NP-Pam3CSK4 at doses of 5 or 20 g twice per treatment cycle over four cycles, followed by a 2-week follow-up period. Soluble Pam3CSK4 was administered at substantially higher doses (100 g). Serial blood samples were regularly collected to monitor virological and host immune parameters. At study completion, liver tissues were harvested for intrahepatic quantification of viral and immunological markers using immunoassays, quantitative PCR, and histological analyses. The most pronounced antiviral effects were observed in mice treated with NP-Pam3CSK4 formulations, which achieved greater viral suppression than free Pam3CSK4 despite markedly lower administered doses. Histological examination of liver biopsies from treated animals revealed prominent immune cell infiltration, including macrophages, monocytes, and T cells, organized in dense cluster-like structures. These findings support the induction of coordinated innate and adaptive immune responses contributing to HBV control and clearance. Collectively, our results demonstrate that nanoparticle-based delivery of TLR1/2 agonist represents a promising therapeutic strategy for chronic HBV infection and may improve the likelihood of achieving functional cure. Further mechanistic and translational studies (combination) are warranted to support clinical development.

Coxiella burnetii is a Gram-negative, obligate intracellular pathogen and the causative agent of the zoonotic disease Q fever. Resident alveolar macrophages are the first target cells, but C. burnetii spreads to other cell types. While we have information about C. burnetii uptake and the establishment of the replication-competent phagolysosomal-like C. burnetii-containing vacuole (CCV), it is not well studied how C. burnetii exits its host cell. Here, we show that an infection with C. burnetii also triggers the activation of TFEB, a master regulator of autophagy and lysosomal development. The activation occurs in a time-dependent manner and depends on the size of the CCV. Importantly, TFEB activation during C. burnetii infection depend on MCOLN1, which channels Ca2+ across the lysosomal membrane into the cytosol. Knock-down of MCOLN1 resulted in reduced TFEB activation and smaller CCVs, while MCOLN1 activation boosted bacterial egress. Indeed, peripheral CCVs are positive for LAMP1/2 and release bacteria, without inducing host cell death. Importantly, LAMP1/2 and C. burnetii were stainable in non-permeabilized cells at sites of bacterial release, demonstrating fusion of the lysosome with the plasma membrane. Importantly, while replication of C. burnetii is not inhibited in cells lacking LAMP1/2, egress is impaired. Taken together, our data indicates that with increasing CCV size, TFEB is activated by the release of Ca2+ from lysosomes via the MCOLN1 channel, which in turn enables further CCV development and damage of the CCV membrane. This triggers lysosomal exocytosis and egress of C. burnetii without cell death induction.

The fungal pathobiont Candida albicans acquires heme from host proteins via a set of soluble and anchored extracellular CFEM-type hemophores that can capture the bound heme and exchange it, eventually delivering it to the cell membrane for endocytosis into the yeast cell. Yet the molecular mechanism by which the heme is transferred through this protein cascade across the cell envelope remains unclear. To address this mechanism, we developed a set a fusions of three C. albicans hemophores with fluorescent proteins. Fluorescence of these fusion proteins is strongly quenched when heme is bound to the hemophore moiety, enabling to measure heme transfer instantaneously. Kinetic analysis of the different transfer reactions reveals that heme transfer from the host protein to the CFEM hemophores and between the CFEM hemophores are governed by different regimes. Kinetics of heme transfer from hemoglobin or serum albumin to the CFEM hemophores is mainly first-order, suggesting that heme stochastically released from host proteins is captured by the hemophores. In contrast, transfer of heme between the hemophores is near second-order, consistent with a mechanism requiring protein-protein interactions. To confirm this, we show that CFEM hemophores can interact in homodimeric and heterodimeric complexes. Furthermore, while dimerization-defective mutants of the soluble hemophore Csa2 are proficient in heme binding and extraction, they are defective in heme transfer. This supports a model of heme transfer by direct interaction between the members of the fungal hemophore cascade. SIGNIFICANCEAcquisition of extracellular heme as iron or heme sources is common in microorganisms, and particularly prevalent among pathogenic organisms that must contend with an iron-poor host environment. To extract heme from host proteins, microorganisms deploy various systems that include extracellular soluble and cell-anchored hemophores. Here we describe a new approach for monitoring heme binding and transfer in real time, based on the development of fluorescent derivatives of fungal hemophores. These novel reagents open a new window on the study of a common virulence factor of microbial pathogens.

Adhesion interactions between Plasmodium falciparum infected erythrocytes (IEs) and human cells bring about microvascular sequestration and contribute to severe malaria pathology. Parasite adhesion molecules on the IE surface are members of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family, encoded by var genes, which interact with receptors on human cells. Progress in understanding PfEMP1-host receptor interaction is hindered by the lack of genetic tools for PfEMP1 functional studies in live parasites and the spontaneous switching of var gene transcription in culture leading to change in adhesion phenotype. We developed a CRISPR/Cas9 genome editing strategy that takes advantage of var gene mutually exclusive expression to generate single variant P. falciparum lines and enable reverse genetic studies of PfEMP1 function. A drug resistance gene and 2A peptide enabling bi-cistronic transcription were inserted between the promoter and exon I of the it4var60 gene encoding a PfEMP1 variant that mediates the virulence-associated rosetting phenotype. After genome editing and drug selection, only it4var60-transcribing parasites survived, and >90% of IEs expressed IT4VAR60-PfEMP1 on their surface and formed rosettes. When drug pressure was removed, switching to other variants occurred. The approach was adapted to generate epitope tagged-PfEMP1 allowing immunofluorescent detection with commercial antibodies, and modifications of the homology directed repair template enabled investigation of PfEMP1 function including point mutations and a gene knockout that abolished adhesion. These methods can be applied to any var gene in any P. falciparum genotype and are potentially transformative for functional studies of multi-gene family members in live parasites.

Liver sinusoidal endothelial cells (LSEC) rapidly dedifferentiate in 2D-monoculture, losing their high endocytic activity and characteristic morphology, limiting their use in mechanistic studies. We established and validated culture conditions that preserve LSEC endocytic capacity for at least 10 days, enabling efficient in vitro siRNA-mediated gene silencing. Mouse LSEC were cultured in 5% oxygen, growth media partially exchanged daily and assessed for cell viability, endocytic capacity, morphology and ultrastructure. Despite typical culture-induced defenestration, the cells showed high viability and efficient endocytosis via scavenger-receptors. This allowed for siRNA-mediated mannose receptor knockdown exemplified by 96% and 76% reduction in Mrc1 mRNA and protein expression at 72h (validated by qPCR and Western blot), with functional assays confirming decreased mannose-receptor-mediated endocytosis. Extended maintenance of LSEC viability and functions, previously restricted to complex co-culture systems, provide a practical platform for investigating LSEC-specific molecular mechanisms and hepatic sinusoid physiology.

Toxoplasma gondii relies on tightly regulated protein synthesis to adapt to diverse host environments and to progress through its developmental stages. Here, we investigated the role of the ATP-binding cassette protein ABCE1, a broadly-conserved factor involved in ribosome recycling and translational control. Using a conditional knockdown approach, we demonstrate that depletion of TgABCE1 severely impairs parasite growth and disrupts global protein synthesis, confirming its essential role in maintaining translational capacity. TgABCE1 function depends on the incorporation of iron-sulfur (Fe-S) clusters, likely mediated by the cytosolic iron-sulfur assembly (CIA) pathway component HCF101. Depletion of TgABCE1 phenocopies the defects observed in TgHCF101-depleted parasites, supporting a functional link between these proteins. Notably, loss of TgABCE1 also disrupts lipid homeostasis, resulting in the accumulation of lipid droplets. Together, these findings uncover a critical link between translational regulation, Fe-S cluster biogenesis, and lipid homeostasis, highlighting the central role of proteostasis in parasite survival and development.