Cellular Microbiology

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.

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.

Yersinia pseudotuberculosis (Yptb) replicates in immune cell-encompassed microcolonies within tissues. Bacterial replication is controlled by protection against neutrophil attack and by macrophage-released antimicrobial factors, such as nitric oxide (NO). During these attacks, bacteria located on the microcolony periphery encounter extracellular signals that differ from those in the interior. To dissect individual microbial populations, {gamma} interferon-activated macrophages were used to challenge microdroplet-grown Yptb harboring an NO-responsive mCherry reporter. Subsequently, bacterial subpopulations that hyperactivated the reporter were isolated from droplets composed of a reversible polymer matrix. RNA-seq analysis indicated that induction of nitrosative stress-associated genes was the primary determinant distinguishing peripheral bacteria from the remaining population. In addition, a secondary stress response that induced prophage-associated genes was detected, which could not be traced to either DNA damage or nitrosative stress responses. Activated macrophages also induced the expression of the Yptb itaconate degradation enzyme-encoding transcript throughout the entire colony. To determine if itaconate production by the interferon-activated Irg1 protein played a role in restricting Yptb, bacteria harboring an itaconate-responsive reporter and Yptb mutants defective for itaconate degradation were analyzed during bacterial colonization of the murine spleen. Only a subset of colonies appeared to be exposed to itaconate, which may explain the very small defects exhibited by mutants unable to degrade the interferon-induced macrophage product. These results indicate that the primary response of bacteria to macrophage-elicited factors is likely associated with protection against NO-derived metabolites.

Human malaria infections begin with the injection of Plasmodium sporozoites via mosquito saliva. Whole sporozoite immunizations have been used as a model to study immune responses to malaria parasites, having culminated in circumsporozoite protein (CSP)-targeting vaccines and monoclonal antibodies (mAbs). However, antibody responses targeting non-CSP antigens on the sporozoite surface remain poorly characterized. Here, we isolated single B cells from a human volunteer immunized by Plasmodium falciparum-infected mosquito bites, who had acquired non-CSP-specific antibodies that recognize sporozoites. We identified two mAbs that recognize the surface of P. falciparum sporozoites, but do not bind to CSP. Using immunoprecipitation followed by mass-spectrometry, we found that the target of these mAbs is not a P. falciparum protein but the mosquito salivary protein SG1L3. We observed that recombinant SG1L3 binds to P. falciparum sporozoites. However, the SG1L3-specific mAbs and SG1L3-specific polyclonal antibodies from this volunteer, as well as polyclonal antibodies raised against recombinant SG1L3 in rabbits, fail to block liver stage infection in vitro, making this an unlikely target for functional antibodies. We observed that inhabitants from an area with intense Anopheles exposure in Burkina Faso can have antibodies against SG1L3, and that antibody titers increase with age. In conclusion, we identified the first human mAbs against a mosquito saliva protein that binds to the surface of sporozoites. Future work should assess whether naturally acquired antibodies against this protein may be used as a serological marker of mosquito exposure.

Background & AimsAntiviral therapies targeting hepatitis B virus (HBV) suppress viral replication, but rarely achieve functional cure. Understanding HBV-host cell interaction is crucial for developing novel therapeutic approaches. Here, we report host cell proteins associated with HBV virions and filamentous subviral particles (fSVPs) and characterize one of them, apolipoprotein C1 (ApoC1), mechanistically. MethodsHighly purified HBV virions and fSVPs were obtained by sequential use of several biophysical methods. Particles were analyzed by mass spectrometry and associated proteins were evaluated phenotypically using an HBV infection model. The top hit, ApoC1 was characterized in detail. ResultsAssociated with virions and fSVPs, we identified in addition to known chaperones such as HSP90AB1 and HSC70, several apolipoprotein-related factors. RNAi-based phenotypic validation identified strongest effects for ApoC1, likely due to two complementary effects. First, ApoC1 depletion reduced intracellular cholesterol level impairing HBV infection and SVP production, which was compensated by exogenous cholesterol substitution. Second, ApoC1 that is mainly enriched in high-density lipoprotein (HDL), associates with HBV virions and fSVPs and increases HBV infectivity. The same was found for hepatitis D virus (HDV), a satellite virus utilizing HBV envelopes. Supplementation of exogenous HDL enhanced infection most likely via scavenger receptor class B type 1 (SR-B1), the natural HDL receptor. Consistently, inhibition of SR-B1 suppressed HBV and HDV infection. ConclusionsWe established a method for obtaining highly purified HBV virions and fSVPs and identified the HDL component ApoC1 to associate with both particle types. ApoC1 promotes HBV and HDV infection most likely via SR-B1 facilitating viral entry.

The phosphodiesterase (PDE) NbdA (NO-induced biofilm dispersion locus A) consists of a membrane-integrated MHYT domain, a degenerated diguanylate cyclase (DGC) AGDEF domain and an EAL domain. The integral membrane domain MHYT is proposed to sense a so far unknown extracellular signal and transfers the information to the cytosolic enzyme domains to modulate cellular c-di-GMP level. Here, we show that full length NbdA from Pseudomonas aeruginosa PAO1 is an active PDE in vivo. In line with its PDE activity, overexpression leads to slightly reduced global c-di-GMP levels, and reduced twitching motility. Surprisingly, overexpression of truncated cytosolic NbdA variants exhibited increased c-diGMP levels, suggesting previously uncharacterized DGC activity despite lacking a canonical GGDEF motif. While full-length NbdA overexpression resulted in only slight c-di-GMP reduction, cytosolic variants induced a significant increase, indicating a potential for nonenzymatic effects like protein-protein interactions. Further investigation revealed a connection between NbdA and type IV pilus (T4P) function. Overexpression of NbdA conferred resistance to the T4P-dependent phage DMS3vir, suggesting interference with T4P assembly or function. Microscopic analysis demonstrated dynamic localization of NbdA, partially co-localizing with T4P components, supporting a role in T4P regulation. However, no clear link was re-established with flagellar motor switching or chemotaxis signaling. These findings position NbdA in the complex signaling network of c-diGMP and T4P-mediated surface behavior in P. aeruginosa. Future work will focus on elucidating the precise mechanisms of NbdAs PDE activity and its interplay with other DGC/PDE networks. ImportanceIn this work, we show the in vivo activity of the membrane-bound phosphodiesterase NbdA of Pseudomonas aeruginosa, its role in c-di-GMP homeostasis, cellular localization and implications in surface behavior. Using strains overexpressing NbdA and truncated protein variants, we detected a strong defect in growth on solid surfaces and an altered phage susceptibility. Co-localization experiments supported further the hypothesis of interaction with the type IV pilus apparatus. We propose for NbdA to be part of the protein network responsible for c-di-GMP level modulation at the cell pole and thereby regulating the function of type IV pilus apparatus.

Chronic co-infections by HBV and its satellite virus HDV are associated with a high risk of progression to cirrhosis and liver cancer, and therapeutic options for achieving a cure are still unsatisfactory. HBs is the main surface glycoprotein of both viruses, and is also massively secreted by infected hepatocytes in the form of empty subviral particles which suppress the host immune responses. This makes HBs an attractive target to develop therapeutic strategies. Here, we took advantage of the known interaction between the Large form HDV antigen (HDAg-L) and the small form of HBs (S-HBs) to develop a non-infectious, minimalistic reporter assay for the assembly and secretion of HDV particles. By screening the existing pharmacopeia for drugs that could interfere with S-HBs and HDAg-L co-secretion, we found that ritonavir and other Cytochrome P450 inhibitors affect the biogenesis of HBs and impair the production of infectious HDV virions. Mechanistically, we established that these drugs induce oxidative stress which dysregulates disulfide bond formation in the endoplasmic reticulum. As a consequence, the production of HBs, which depends on a dense network of disulfide bonds, is markedly affected as evidenced by an abnormal glycosylation profile, altered antigenic properties, and a poor expression of the largest form of HBs (L-HBs) which is essential to virus entry into target cells. This is associated with induction of the unfolded protein response, with the upregulation of CHOP/DDIT3 and key enzymes involved in the synthesis of the reducing metabolite glutathione (PHGDH, SHMT2, MTHFD2). Overall, our results indicate that alterations in redox homeostasis significantly impact HBs biogenesis, and reveal a druggable pathway that could be exploited to eliminate HDV in chronically infected patients. IMPACT AND IMPLICATIONSMore effective therapies are still needed to achieve a functional cure in patients chronically co-infected by HBV and HDV. In this study, we discovered that ritonavir, along with other cytochrome P450 inhibitors, can affect the production of infectious HDV particles in human hepatocyte cultures. Mechanistically, ritonavir induces oxidative stress and the unfolded protein response in the endoplasmic reticulum, thereby altering the biogenesis of HBs, the surface glycoprotein of both viruses. This work highlights the potential benefit and mechanism of action of ritonavir and related molecules in the treatment of co-infected patients.

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.

Chlamydia trachomatis infection is the most common bacterial sexually transmitted infection worldwide and a leading cause of inflammatory reproductive tract disease and infertility in women. Much of the tissue damage associated with genital chlamydial infection arises from host inflammatory responses rather than direct bacterial cytotoxicity. Epithelial cells lining the female reproductive tract represent the primary host cells infected during chlamydial infection and play key roles in initiating innate immune responses. Among the cytokines produced by infected epithelial cells, type-I interferons have emerged as important regulators of host defense and inflammatory signaling; however, the specific contribution of interferon-{beta} (IFN-{beta}) to epithelial transcriptional responses during chlamydial infection remains incompletely defined. In the present study, we investigated the role of IFN-{beta} in coordinating epithelial immune signaling networks during infection with Chlamydia muridarum. Using wild-type murine oviduct epithelial cells (OE-WT) and IFN-{beta}-deficient epithelial cells (OE-IFN{beta}-KO), we performed pathway-focused RT{superscript 2} Profiler PCR array analyses examining transcriptional responses across four biological pathways: (1) innate and adaptive immune responses, (2) type-I interferon signaling, (3) inflammatory and autoimmune responses, and (4) fibrosis-associated pathways. Infection of OE-WT cells resulted in coordinated induction of cytokines, chemokines, and interferon-stimulated genes associated with antimicrobial defense and immune cell recruitment. In contrast, IFN-{beta} deficiency resulted in widespread dysregulation of these transcriptional programs, including reduced induction of interferon-responsive chemokines such as CCL5 and CXCL10, altered inflammatory cytokine expression, and transcriptional signatures consistent with enhanced tissue remodeling responses. Notably, IFN-{beta} deficiency resulted in increased TNF expression accompanied by reduced IL-6 induction, suggesting disruption of balanced inflammatory signaling networks. Pathway analyses further revealed dysregulated expression of fibrosis-associated genes including Serpine1, Ctgf, and Eng in IFN-{beta}-deficient epithelial cells, indicating potential mechanisms linking interferon signaling to tissue remodeling during infection. Collectively, these findings identify IFN-{beta} as a central regulator of epithelial immune networks during chlamydial infection and suggest that disruption of IFN-{beta} signaling may promote inflammatory and fibrotic pathology within the female reproductive tract. Author SummarySexually transmitted infections caused by Chlamydia trachomatis are a major cause of infertility worldwide. Although antibiotic treatment can eliminate the bacteria, damage to the reproductive tract often results from the bodys own immune response to infection. The epithelial cells lining the reproductive tract are the first cells infected and play an important role in initiating immune responses. In this study, we investigated how a specific immune signaling molecule, interferon-{beta} (IFN-{beta}), regulates the gene expression programs activated in epithelial cells during chlamydial infection. Using pathway-focused gene expression arrays, we found that IFN-{beta} coordinates multiple immune pathways, including interferon signaling, inflammatory cytokine networks, and genes associated with tissue remodeling. When IFN-{beta} was absent, many of these pathways became dysregulated, resulting in altered inflammatory signaling and gene expression patterns linked to fibrosis. These findings suggest that IFN-{beta} functions as a key regulator that helps balance protective immune responses with inflammatory processes that can damage reproductive tissues during infection.

African trypanosomes employ specialised mechanisms of membrane trafficking as a key strategy to persist in both the mammalian host and insect vector. Their survival and pathogenicity rely on the continuous synthesis and surface delivery of extremely abundant surface coat proteins, imposing an extraordinary biosynthetic burden on the secretory pathway. Despite this, the luminal proteome of the T. brucei endoplasmic reticulum (ER) and Golgi apparatus remains incompletely characterised. Here, we exploit TurboID proximity biotinylation, using the abundant ER chaperone BiP (Binding-immunoglobulin protein) as luminal bait to map the ER proteome in bloodstream and procyclic lifecycle stages of Trypanosoma brucei. Comparison with BiPN, a truncated secretory form of BiP that transits the Golgi, provides differential compartmental labelling, together identifying 366 (BiP) and 428 (BiPN) proximity partners respectively and encompassing established ER quality control machinery, secretory cargo, and Golgi proteins. Quantitative ranking of BiP labelling intensity identifies a cohort of candidate BiP interactors: the most strongly enriched is Tb927.5.1160, a protein sharing structural homology with the mammalian BiP nucleotide exchange inhibitor MANF (mesencephalic astrocyte-derived neurotrophic factor). Endogenous mNeonGreen tagging confirms ER localisation of TbMANF in both life cycle stages, and reciprocal manipulation of its abundance by RNAi and inducible expression produces opposing shifts in cellular sensitivity to ER stress. These data are consistent with a role in regulating BiP ATPase cycling in an organism that, unlike yeast and mammals, lacks a canonical unfolded protein response, making TbMANF the first candidate regulator of BiP activity identified in kinetoplastids. Finally, TurboID proximity labelling anchored at the inner face of the nuclear pore via NUP65 extends our endomembrane map to the inner nuclear membrane, identifying candidate proteins of this specialised ER-continuous domain. AUTHOR SUMMARYAfrican sleeping sickness is caused by Trypanosoma brucei, a parasite that survives in the mammalian bloodstream by constantly renewing its protective protein coat. To synthesise and export this surface coat, the parasite relies on two intracellular compartments, the endoplasmic reticulum (ER) and the Golgi apparatus, which function as a quality control and sorting factory for proteins entering the secretory pathway. However, the identity of the proteins that populate these compartments in blood-stage parasites, and that maintain functioning under stress conditions, has remained poorly mapped. Here, we used an enzyme-based proximity labelling strategy that identifies neighbouring proteins in live cells without disturbing their targeting signals, generating a comprehensive protein inventory of both compartments across the two main T. brucei lifecycle stages. Among the most strongly labelled proteins was Tb927.5.1160, a protein structurally related to a mammalian regulator of the master ER chaperone BiP. Reducing or increasing the abundance of Tb927.5.1160 in parasites produced opposite changes in ER stress tolerance, identifying it as a candidate modulator of ER homeostasis in a lineage that regulates protein quality control through mechanisms distinct from those operating in yeast or human cells. Together, our findings provide a new molecular resource for understanding how T. brucei sustains secretory pathway function under the biosynthetic demands of mammalian and insect host infection.

Listeria monocytogenes relies on a tightly controlled set of surface-associated and secreted proteins to mediate host interaction and infection. The correct localization and exposure of these proteins at the bacterial surface are critical for virulence, yet the role of cell wall components in organizing this process remains incompletely understood. In particular, wall teichoic acid (WTA) glycosylation has been implicated in anchoring and function of selected surface proteins, but its global impact on protein distribution across the bacterial cell envelope is unclear. Here, we performed a comprehensive proteomic analysis to investigate how WTA glycosylation influences protein distribution in L. monocytogenes. Using isogenic mutants lacking rhamnose ({Delta}rmlT) or GlcNAc ({Delta}lmo1079) WTA glycosylation, we compared the exoproteome, the surface-accessible proteome and the surface-exposed proteome. Loss of WTA glycosylation did not result in a global disruption of the surface proteome but instead induced a redistribution of proteins across extracellular and surface-associated fractions. This effect was dependent on protein anchoring mechanisms, with limited changes observed for LPXTG-anchored proteins, moderate effects on non-covalently associated proteins, and a marked enrichment of lipoproteins in the surface-exposed proteome, particularly in the {Delta}lmo1079 mutant. In parallel, virulence-associated proteins displayed altered accessibility and exposure, with a progressive shift towards increased surface localization and a combination of shared and mutant-specific responses. This global effect was supported by functional annotation, which revealed that the affected proteins were associated with similar biological processes across fractions, highlighting a broad rather than pathway-specific impact of WTA glycosylation loss Together, these findings indicate that WTA glycosylation plays a key role in organizing the bacterial surface by modulating protein retention, exposure and release. Rather than affecting specific proteins, WTA glycosylation broadly shapes the spatial distribution of proteins across the cell envelope, with potential consequences for host- pathogen interactions.

Leishmaniases remain a significant global public health threat, with Leishmania amazonensis and Leishmania infantum representing the etiological agents of the cutaneous and visceral forms in the Americas, respectively. Building on our previous identification of Phospholipase A1 (PLA1) in Leishmania braziliensis, this study provides a comprehensive molecular, immunological, and biochemical characterization of PLA1 in L. amazonensis and L. infantum promastigotes. We analyzed PLA1 activity and expression, purified the recombinant enzyme from L. amazonensis, and validated protein expression using a specific anti-PLA1 serum. The major contribution of this research is the first description of the subcellular localization of a PLA1 within the Leishmania genus. Moreover, our results reveal an unprecedented association between PLA1 and lipid droplets within the parasites. This discovery is of particular interest as it provides the first evidence linking this enzyme to lipid storage organelles in Leishmania. Given that PLA1 is an established virulence factor in other trypanosomatids, these findings suggest a specialized role for the enzyme in parasite lipid metabolism and potentially in its pathogenic mechanisms, opening new perspectives for understanding Leishmania biology.

Type I interferon (IFN) induction is a central component of the innate immune response to viral infection, and the cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS) has been identified as a key mediator of IFN production during human cytomegalovirus (HCMV) infection. However, how cGAS detects HCMV remains unresolved, as the viral genome is encapsidated and trafficked directly to the nucleus, limiting cytoplasmic exposure. Here, we show that IFN induction during HCMV infection of primary fibroblast cells is predominantly driven by cGAS recognition of exogenous DNA present in standard laboratory virus preparations rather than the encapsidated viral genome. DNase treatment of AD169 and low-passage TB40/E-GFP viral stocks substantially reduced total DNA content without affecting infectivity, yet markedly abrogated IFN induction, IFN-stimulated gene expression and IRF3 nuclear translocation. Immunofluorescence analysis further revealed cytoplasmic accumulation of DNA in cells infected with untreated virus stocks, which was absent following DNase treatment. Together, these findings demonstrate that contaminating DNA in viral preparations is sufficient to activate cGAS and drive IFN responses during HCMV infection in vitro, highlighting a critical confounding factor in studies of innate immune sensing. Author SummaryHuman cytomegalovirus (HCMV) is a common herpesvirus that establishes lifelong infection and can cause serious disease in immunocompromised individuals and newborns. When cells detect viral infection, they produce type I interferons (IFNs), antiviral molecules that help limit virus spread. Previous studies have suggested that HCMV is sensed by a cellular DNA sensor called cGAS, which detects viral DNA in the cytoplasm and triggers IFN production. However, how cGAS gains access to the HCMV genome has remained unclear, because the viral DNA is enclosed within a protective capsid and transported directly to the nucleus during infection. In this study, we show that most IFN production observed during HCMV infection of fibroblast cells in vitro is driven not by sensing of the viral genome itself, but by contaminating DNA present in standard laboratory virus preparations. Treating virus stocks with DNase to remove this exogenous DNA abolished IFN induction without affecting viral infectivity. These findings highlight the importance of controlling for exogenous nucleic acids when interpreting how host cells detect viral infection.

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

Plasmodium spp., the parasites that are the causative agents of malaria, encode a repertoire of divergent protein kinases that coordinate essential processes including cell division and host cell invasion, yet the functions of many kinases are poorly defined. Plasmodium Protein Kinase 2 (PK2) is essential for asexual blood-stage proliferation and has been implicated in P. falciparum merozoite invasion of red blood cells. However, its role in the sexual stages of the Plasmodium life cycle responsible for transmission is unknown. Here, using live cell imaging, functional analyses, ultrastructure microscopy and phosphoproteomics, we demonstrate that PK2 has a significant role in the Plasmodium berghei life cycle in the mosquito. We show that PK2 is expressed in merozoites, ookinetes and sporozoites - the invasive stages of the parasite life cycle. A conditional knockdown approach revealed that PK2 is required for the ookinete to oocyst transition in the mosquito midgut, potentially associated with altered microneme positioning. Using haemocoel injection to bypass the midgut barrier revealed that PK2 is also required for sporozoite development after midgut invasion. Following PK2 knockdown, global proteome abundance was largely unaffected at 24 h post activation, whereas phosphoproteomics identified changes in phosphorylation of proteins linked to midgut traversal, parasite architecture, and gene regulation. These studies provide insight into the importance of PK2 function in Plasmodium sexual stages and parasite transmission through the mosquito, highlighting its essential function during the three invasive stages of the parasites life cycle.

We used transposon sequencing (Tn-seq) to define genetic requirements for intracellular survival of Brucella neotomae, a rodent-associated species. A near-saturating mutant library was subjected to selection during infection of J774A.1 macrophages, identifying 54 genes required for intracellular fitness. These included core components of the VirB type IV secretion system, multiple regulatory factors, an aquaporin gene with a strong fitness defect, and a set of metabolic genes involved in amino acid biosynthesis. Targeted mutagenesis revealed that methionine and histidine biosynthesis are indispensable for intracellular growth, whereas tryptophan biosynthesis was required for full intracellular fitness, with mutants exhibiting significant but incomplete attenuation. Notably, these auxotrophs grew normally in minimal medium under axenic conditions, indicating that their requirement is specific to the intracellular environment. Amino acid supplementation rescued intracellular growth in a concentration- and time-dependent manner, consistent with increased metabolic demand during intracellular replication. Disruption of the aquaporin gene similarly impaired intracellular survival, suggesting a role for water homeostasis during adaptation to the macrophage vacuolar environment. Beyond metabolic and osmotic adaptation, we identify OmpR1 as an upstream regulator of B. neotomae virulence. Biochemical, genetic, and transcriptional analyses establish a hierarchical regulatory cascade in which OmpR1 activates the BvrR/BvrS system, which in turn controls VjbR and downstream VirB expression. Under the conditions examined, OmpR1 is required for activation of this cascade. Consistent with this, OmpR1 loss is not rescued by VjbR and requires BvrR activity for restoration of intracellular growth. Phylogenetic analysis places OmpR1 in a distinct lineage relative to other well-characterized Brucella transcriptional regulators, suggesting that this regulatory pathway has been underappreciated across the genus. Together, these findings reveal that intracellular fitness in Brucella depends on metabolic capacity, osmotic homeostasis, and a hierarchical regulatory cascade centered on OmpR1. Author SummaryBrucella species are bacteria that survive and replicate inside immune cells called macrophages, where they cause persistent infection. To live within these cells, the bacteria must carefully balance their metabolism with the expression of genes required for virulence. We used a genome-wide genetic approach to determine which genes are specifically required for intracellular survival of Brucella neotomae, a rodent-associated species. We found that several amino acid biosynthesis pathways, including those required to produce methionine and histidine, are essential for replication inside macrophages but are not required during growth in laboratory media. This indicates that the intracellular environment imposes nutrient limitations not apparent in culture. We also discovered that a gene encoding an aquaporin, which regulates water movement across the bacterial membrane, is critical for intracellular survival, highlighting the importance of maintaining water balance within the host cell vacuole. In addition, we identify OmpR1 as an upstream regulator that controls a hierarchical virulence cascade required for intracellular growth. Our findings show that successful infection depends on metabolic capacity, virulence regulation and water homeostasis, and provide new insight into how Brucella adapts to its host environment.

Cyanobacteria utilize type IV pili for many behavioural responses, such as phototaxis, aggregation, floating, and DNA uptake. Type IV pilus-dependent functions are regulated by the nucleotide second messengers, c-di-GMP and cAMP. In this study, we investigated the role of a recently identified c-di-GMP receptor (CdgR) in cyanobacteria that harbours a ComFB domain. ComFB-domain proteins are widespread in cyanobacteria and are also present in heterotrophic bacteria. We demonstrated that the CdgR homolog from the cyanobacterium Synechocystis sp. PCC 6803, a model organism for studying type IV pilus-dependent functions, specifically binds to c-di-GMP. Genetic and phenotypic analyses revealed that Synechocystis CdgR is involved in phototactic motility and natural competence. Inactivation of cdgR resulted in altered expression of specific sets of minor pilins, which are essential for motility or natural competence. We identified interactions between CdgR and the CRP-family transcription factors, SyCRP1 and SyCRP2. Disruption of these CdgR-SyCRP1 and CdgR/SyCRP2 complexes is initiated by elevated c-di-GMP levels. Moreover, the assembly and stability of these complexes are influenced by other cyclic nucleotides, such as cAMP and c-di-AMP. These observed interactions imply a complex regulatory mechanism by which CdgR influences gene expression in response to cyclic nucleotide messenger signalling, particularly c-di-GMP. The present findings highlight the importance of CdgR in c-di-GMP signalling and its role in regulating type IV pilus-dependent functions in Synechocystis. The modulation of the expression of specific minor pilin genes by CdgR, through interactions with the transcription factors SyCRP1 and SyCRP2, contributes to the establishment of multiple type IV pilus functions and adaptive behaviours of cyanobacteria.

Campylobacter jejuni is a leading global cause of acute foodborne gastroenteritis however, C. jejuni lacks some of the classic virulence determinants associated with other common enteric bacterial pathogens. In recent years an increasing number of C. jejuni isolates have been identified to encode Type Six Secretion System (T6SS), an apparatus utilised by Gram-negative bacteria to secrete toxic bacterial effectors into neighbouring cells. Despite the prevalence of the T6SS and previous investigations, the roles of the C. jejuni T6SS are still not well characterised especially when compared to our knowledge of other clinically relevant T6SS-positive bacterial species. Additionally, as of yet, no C. jejuni T6SS cargo effectors have been characterised. In this study, we show the C. jejuni 488 strain T6SS displays contact-dependent antagonistic behaviour towards T6SS-negative C. jejuni, Campylobacter coli, Escherichia coli and Enterococcus faecium strains suggesting the presence of the T6SS contributes to the competitive capacity of this C. jejuni T6SS-positive strain. Moreover, this antagonistic activity is linked to the functionality of CJ488_0980 and CJ488_0982, two novel putative Tox-REase-7 domain-containing effectors, which were identified through bioinformatical analysis of the C. jejuni 488 strain genome. Additionally, our investigations propose the C. jejuni 488 T6SS contributes to interaction, invasion and intracellular survival in human intestinal epithelial cells (IEC). Collectively, these initial findings are the first examples of in vitro investigation of putative cargo effectors in Campylobacter spp. and provide valuable insights into the roles of C. jejuni T6SS effectors in bacterial competition and pathogenesis. This study highlights the importance of T6SS as an emerging virulence determinant in Campylobacter spp. warranting further investigation.

Cell-mediated immune responses are crucial for protecting the host against Toxoplasma gondii infection. However, impaired immunity, such as T-cell exhaustion, is a common phenomenon during chronic infection. This may represent a strategy employed by T. gondii to evade host defenses. T-cell immunoglobulin and mucin-domain containing 3 (Tim-3) is an important regulatory molecule involved in cell-mediated immunity. This study examined the expression of Tim-3 and the effects of its blockade in a mouse model of toxoplasmosis. In mice with chronic T. gondii infection, we found that Tim-3 is highly expressed in both cyst-bearing and non-cyst-bearing tissues, and its expression correlates with the parasite burden. Blocking the Tim-3 pathway with an anti-Tim-3 antibody enhances the immune response, resulting in elevated levels of cytokines (IFN-{gamma}, IL-12p70, IL-2, IL-9) and the chemokine CXCL1 in the serum, increased leukocyte infiltration (CD3+, CD14+ cells) in the brain, and downregulation of Tim-3 expression in microglial cells. As a result, the anti-Tim-3 treatment resulted in a 62% reduction in the number of tissue cysts and a trend towards an increase in the homeostatic signature, P2RY12, in microglia. Our study provides proof of concept for an anti-Tim-3 approach in treating chronic T. gondii infection and potentially other brain-residing pathogens.

Plasmodium vivax transmission from humans to mosquitoes depends on the density of gametocytes that in turn depends on asexual parasite replication and gametocyte commitment. These processes are often analyzed separately, despite being biologically linked and measured with substantial uncertainty. We used a joint Bayesian latent-variable model to simultaneously analyze parasite density, gametocyte density, and mosquito infectivity while accounting for measurement error and propagating uncertainty across linked processes. The model was applied to individual-level data from three P. vivax transmission studies conducted in Ethiopia (n = 455). A tenfold increase in gametocyte density was associated with more than a twofold increase in the odds of mosquito infection (odds ratio [OR] = 2.32, 95% credible interval [CrI]: 2.12-2.54). Asexual parasite density was also positively associated with infectivity after accounting for gametocyte density (OR = 1.74, 95% CrI: 1.60-1.90), and inclusion of parasite density improved predictive performance. Pathway decomposition within the joint model indicated that approximately 41% of the parasite-infectivity association operated through gametocyte density. Increasing age was associated with lower asexual parasite density but higher gametocyte density, resulting in minimal overall association with infectivity. Predicted infection probability increased sigmoidally with gametocyte density, remaining low at lower densities before increasing sharply and approaching a plateau at higher densities. Gametocyte density produced the largest predicted changes in the proportion of infected mosquitoes, while asexual parasite density added predictive information not fully captured by measured gametocyte density alone. This approach links molecular parasite measurements with mosquito infection risk while accounting for measurement uncertainty and provides an interpretable framework for studying the P. vivax infectious reservoir. Author SummaryMalaria transmission occurs when mosquitoes ingest sexual-stage parasites, called gametocytes, during a blood meal. In Plasmodium vivax infections, human-to-mosquito transmission depends on linked biological stages, including asexual parasite replication, gametocyte production, and mosquito infection. These processes are closely connected and often measured with uncertainty, making them difficult to study using standard approaches that analyze them separately. In this study, we applied a joint Bayesian model that analyzes parasite density, gametocyte density, and mosquito infectivity together while accounting for uncertainty in laboratory measurements. Using data from three studies in Ethiopia, we quantified how parasite density, gametocyte density, and host characteristics relate to mosquito infection. The analysis showed that measured gametocyte density alone did not fully explain variation in infectivity, and that asexual parasite density provided additional predictive information. We also found that age was associated differently with asexual parasite and gametocyte densities, resulting in little overall association with infectivity. This approach helps link molecular parasite measurements with transmission outcomes and improves understanding of the P. vivax infectious reservoir in endemic settings.