Cellular Microbiology

Mycobacterium tuberculosis (Mtb) primarily infects human lung macrophages, which serve as its major replication niche. Mtb can manipulate host macrophage cell death pathways to its advantage by inhibiting apoptosis and inducing necrotic cell death. However, the specific necrotic cell death pathway activated in human macrophages after Mtb infection remains unclear. Here, we used the THP-1 cell line and primary human monocyte-derived macrophage (hMDM) to analyze multiple programmed cell death pathways during days 1-3 after Mtb infection. Confocal microscopic analysis demonstrates that Mtb-infected THP-1 cells or hMDMs rarely exhibited apoptosis. Immunoblotting shows that Mtb induces significant CASP3 and GSDME activation in THP-1 cells, but not in hMDMs. We show that Mtb, in THP-1 cells but not hMDM, induces a significant increase in GSDMD cleavage, a hallmark of pyroptosis. MLKL phosphorylation was not observed in THP-1 cells or hMDMs during Mtb infections, indicating an absence of necroptosis. No changes in ferroptosis markers such as GPX4 expression or lipid peroxidation levels were detected. Time-lapse live-cell imaging revealed no lysosomal membrane permeabilization prior to plasma membrane rupture (PMR). However, we observed DNA release from Mtb-infected THP-1 cells and hMDMs after PMR. The DNA released from THP-1 cells exhibits low levels of myeloperoxidase and histone H3 citrullination. High-resolution confocal imaging shows that Mtb is associated with the released DNA. We demonstrate that pyroptosis induction in THP-1 cells is dispensable for the DNA release and cell death induction. In conclusion, our results reveal that Mtb-triggered cell death in hMDMs bypasses canonical cell death pathways like apoptosis, pyroptosis, necroptosis, and ferroptosis. Instead, cell death in both THP-1 cells and hMDMs correlates with DNA release, potentially through a pathway similar to NETosis in neutrophils.

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.

MmpL proteins play an important role in the various mechanisms associated with mycobacterial virulence. Identification of interacting protein partners required for a detailed understanding of their role remains hampered because of their large size (> 100 kDa) and the presence of twelve transmembrane domains by classical methods. In this study, we used two independent biotin proximity labelling assays (APEX2 and BioID) to define the proxisome of MmpL11 in M. smegmatis. Indeed, these techniques are performed directly in the organism of interest, allowing the detection of potentially transient or weak interactions in multiprotein complexes and preserving the subcellular structures and the presence of cofactors or post-translational modifications that can also impact protein-protein interactions. BioID leads to the biotinylation of lysine residues, whereas APEX2 leads to the biotinylation of mainly tyrosine residues; they have also been shown to have different effective labelling radii. On one hand, an interaction was detected between the cytoplasmic C-terminal domain of MmpL11 and MSMEG_0240, a protein of unknown function, using BioID. This interaction was confirmed using both MmpL11 and MSMEG_0240 as fusions with BirA and was corroborated by AlphaFold3 prediction. On the other hand, APEX2 failed to detect an interaction between MmpL11 and MSMEG_0240, probably due to the absence of accessible tyrosines. However, both approaches identified MSMEG_0940 as an additional interactant with MmpL11 that also depends on the C-terminal domain. Overall, this study demonstrates that APEX2 and BioID as complementary tools for defining the proxisome of mycobacterial proteins.

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.

BackgroundLeishmania donovani (LD) is an obligate intracellular parasite that survives and replicates within macrophages. Leptomonas seymouri (LS), a traditionally monoxenous trypanosomatid, has been repeatedly co-isolated with LD from visceral leishmaniasis (VL) and post-kala-azar dermal leishmaniasis (PKDL) patients in India, often together with Leptomonas seymouri narna-like virus 1 (Lepsey NLV1). Whether LS can survive and replicate within mammalian macrophages, and how co-infection influences parasite and viral dynamics, remains unresolved. Methods and FindingsUsing murine (RAW 264.7) and human (THP-1) macrophages, we systematically evaluated intracellular survival, replication, and revival of LS alone and during co-infection with LD. Quantitative ITS1 PCR demonstrated significant increases in intracellular parasite DNA over 48-168 h post-infection, with mean intracellular loads rising up to [~]7.6-fold, indicating active replication rather than persistence. Reduced extracellular parasite load suggested restricted cell lysis and enhanced macrophage survival in co-infection compared to mono-infections. In co-infection scenario, generally LS displayed higher persistence compared to LD. Giemsa staining confirmed intracellular localization of LS. Parasites recovered from infected macrophages remained viable and revived as motile promastigotes, whereas extracellular parasites failed to survive beyond 48 h, confirming macrophages as the exclusive niche for prolonged viability. Interestingly, co-infection dampened macrophage IL-12 production, suggesting altered host immune activation. Lepsey NLV1 RNA accumulated predominantly within macrophages and persisted up to 168 h post-infection. Virus load within LS in co-infection state was [~]2.5-4 and [~]7.5-23 fold higher (for RAW 264.7 and THP-1 respectively) compared to LS mono-infection. Purified virus alone failed to enter macrophages, indicating LS-dependent viral delivery. ConclusionsOur findings question the prevailing view of LS as a strictly non-infective parasite, demonstrating its capacity to replicate within mammalian macrophages and persist during mono-or LD co-infection. The identification of a stable LD-LS-virus interaction highlights a previously underappreciated "triple-pathogen" biology with potential implications for VL and PKDL pathogenesis.

In yeast and humans, the conserved DENN-domain (Differentially Expressed in Normal and Neoplastic tissue) protein Avl9 is thought to play roles in membrane traffic and secretion, but its precise function remains poorly defined. Since DENN-containing proteins are associated with Rab GTPase function, we sought to understand Avl9 function in the context of Rab regulation. Here, we show that Avl9 localizes to peripheral punctae that are consistent with secretory vesicles. Moreover, we demonstrate genetic interactions and co-localization between Avl9 and numerous Rabs in the secretory and endosomal pathways, suggesting a potential function at the interface of secretion and recycling. Consistent with this role, avl9{Delta} results in defective recycling of the endosomal cargo Snc1 but does not alter plasma membrane delivery of an endocytosis-defective Snc1EN- mutant, suggesting that Avl9 is not directly involved in secretory traffic from the TGN to the plasma membrane. The avl9{Delta} recycling defect is exacerbated by the additional loss of RCY1 or SNX4, but not VPS35. Each of these three genes contributes to a distinct endosomal recycling pathway, indicating that Avl9 acts in conjunction with multiple recycling pathways. Summary StatementIn this study, Rioux et al. describe a role for the DENN domain protein Avl9, previously thought to regulate secretion, as a novel factor involved in recycling of cargos from endosomal compartments.

Entamoeba histolytica is a parasitic amoeba and the cause amoebiasis, a common but understudied human diarrheal disease. E. histolytica trophozoites ("amoebae") kill human cells through a process of cell-nibbling called trogocytosis (trogo-: nibble) that contributes to tissue damage. Amoebae can also perform phagocytosis, in which entire human cells are ingested. Based on studies in which human cells were artificially stiffened, it was suggested that amoebae perform phagocytosis on stiffer cells, and trogocytosis on less stiff cells. A handful of recent studies of macrophages that used artificial targets or artificially stiffened target cells also suggested a similar relationship between target stiffness and trogocytosis/phagocytosis efficiencies. To better evaluate the impact of target cell stiffness on amoebic ingestion, instead of using artificial targets or artificial cell stiffening, we created human cell mutants in which individual Rho-pathway genes were knocked down. Strikingly, amoebae performed quantitatively reduced levels of trogocytosis on all knockdown mutants, regardless of cytoskeletal F-actin organization. In contrast, amoebic phagocytosis efficiency was inversely correlated with human cell cortical F-actin density. Thus, human cell F-actin organization differentially influences amoebic trogocytosis and phagocytosis. This is more complex than the conclusions of studies that used artificial targets or artificially stiffened cells. Our results emphasize that the dynamic nature of the cytoskeleton in living cells impacts trogocytosis. In addition to shedding light on the burgeoning field of eukaryotic trogocytosis, this work extends knowledge of amoebic ingestion processes that contribute to disease.

Mycobacteria have a hydrophobic cell envelope that makes uniform growth in liquid culture challenging. Non-ionic detergents including Tween-80 and tyloxapol are commonly added to media when culturing mycobacterial species in the laboratory. Tyloxapol was reported to exhibit anti-tuberculous activity during animal infection with M. tuberculosis in the 1950s. In the 1980s, microscopy studies suggested that tyloxapol impacted the inter-action between M. tuberculosis and the phagosomal membrane, preventing mycobacterial access to the cytoplasm. It is now known that the ESX-1 Type VII secretion system mediates the interaction between pathogenic mycobacteria and the phagosomal membrane. Mycobacterium marinum is a pathogenic mycobacterial species that has been widely used to understand the molecular mechanisms and host responses to the ESX-1 system. The hemolytic activity of M. marinum allows the study of ESX-1 lytic activity outside of the context of a host cell. We found that tyloxapol inhibits the hemolytic activity of M. marinum in a concentration dependent manner. The addition of 100-fold less tyloxapol than commonly used for mycobacterial growth differentially inhibits the production and secretion of ESX-1 substrates required for lytic activity. Our findings directly impact how the field interprets data from studies where M. marinum, and potentially other mycobacterial species were grown in tyloxapol. Our findings may explain the original ob-servations linking tyloxapol to anti-tuberculosis activity. Author SummaryTuberculosis, which is caused by Mycobacterium tuberculosis, is one of the worlds deadliest diseases. We lack a clear understanding of how M. tuberculosis and related mycobacterial species cause disease. In the 1950s, it was reported that treating M. tuberculosis infected animals with tyloxapol improved the survival and in some cases protected the animals from death. Tyloxapol is a detergent that is commonly added to mycobacterial cultures to promote dispersed growth in the laboratory. Later studies suggested that tyloxapol altered the interaction between M. tuberculosis and the phagosomal membrane during macrophage infection. The ability to escape the phagosome is essential for mycobacteria to cause disease, and is mediated by a Type VII protein secretion system, ESX-1. Using M. marinum, a well-established model for understanding the molecular mechanisms of ESX-1 secretion, we show that tyloxapol used at more than 100-fold less than what is commonly used to grow mycobacteria in the lab, inhibits ESX-1 secretion. Our findings have widespread implications on how we interpret our findings as a field, and may explain why tyloxapol impacted M. tuberculosis infection of both animals and macrophages. Our study also indicates that tyloxapol can be used as a tool to understand the molecular mechanisms of ESX-1 protein secretion.

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.

Pseudomonas aeruginosa, an opportunistic pathogen, uses a trio of quorum sensing (QS) systems to regulate the production of some virulence factors. Two of these, the las and rhl systems, involve acyl-homoserine lactone signals; the third, called pqs, primarily uses the signal 2-heptyl-3-hydroxy-4(1H)-quinolone ("PQS"). We aimed to identify how interbacterial interactions are regulated between P. aeruginosa and Stenotrophomonas maltophilia, which co-occur in the airways of people with cystic fibrosis. We explored P. aeruginosa and S. maltophilia interactions using a co-culture model. S. maltophilia in co-culture with P. aeruginosa grows for 12 hours and thereafter exhibits a large decline in CFU, demonstrating that P. aeruginosa is killing S. maltophilia. Co-culture of S. maltophilia with P. aeruginosa deficient in las, rhl, or pqs QS resulted in greater S. maltophilia viability than co-culture with the wildtype. This inhibition was not attributable to las and rhl-regulated toxins. Therefore, we interrogated the role of PQS and found that co-culture of S. maltophilia with P. aeruginosa deficient in PQS biosynthesis showed similar CFUs to monoculture. Exogenous PQS did not complement this phenotype, suggesting that another quinolone is the effector. We found that S. maltophilia killing is reduced in competition with a mutant that cannot make the quinolone HQNO. We show that full killing of S. maltophilia by P. aeruginosa requires three components: HQNO, the chaperone PqsE, and intact PQS biosynthesis. Our work identifies quinolone biosynthesis as a driver for interactions between P. aeruginosa and S. maltophilia and, more generally, in modulating interbacterial interactions.

Protein phosphorylation is a central post-translational modification that regulates signaling pathways across all living organisms. Through the antagonistic activities of protein kinases and phosphatases, phosphorylation modulates protein function by inducing conformational changes that affect enzyme activity, protein-protein interactions, stability, and subcellular localization. These molecular events regulate diverse cellular processes, including cell cycle progression, differentiation, gene expression, and metabolism. In unicellular parasites such as Trypanosoma cruzi, Trypanosoma brucei, and Leishmania spp., specialized signaling pathways have evolved to enable adaptation to the fluctuating environments of insect vectors and mammalian hosts. In many eukaryotes, phosphorylation of histone H3 at serine 10 (H3Ser10p) is essential for proper chromosome condensation during mitosis and is catalyzed by Aurora kinase B. Although trypanosomatids possess an Aurora kinase B homolog and a conserved serine residue at position 10 of histone H3, this modification had not been previously detected in these organisms. Here, using a stage-specific approach, we report the first detection of H3Ser10p in T. cruzi and explore its association with cell cycle progression. Western blot analyses using a specific antibody revealed H3Ser10p in exponentially growing epimastigotes, both in total protein extracts and nucleosome-enriched fractions, indicating its incorporation into chromatin. Fluorescence microscopy showed that this histone mark is restricted to the nuclei of dividing cells. Furthermore, H3Ser10p was detected exclusively in replicative stages of the parasite. Analysis of cell cycle-associated structures and flow cytometry demonstrated that H3Ser10 phosphorylation is dynamically regulated, peaking in the G2/M phase. These findings identify H3Ser10p as a novel epigenetic mark in T. cruzi that is tightly regulated during the cell cycle.

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.

Phlebotomine sand flies are major vectors of Leishmania parasites, yet the mechanisms underlying their blood-feeding behavior remain poorly understood. In Lutzomyia longipalpis, the primary vector of Leishmania infantum in the Americas, feeding occurs via telmophagy, a pool-feeding method which is known by involving dermal laceration, salivation, and the creation of a blood pool. While the biochemical effects of sand fly saliva on host hemostasis, inflammation, and immunity are well studied, the dynamics of mouthpart movements and saliva at the feeding site remain to be systematically explored. Using intravital microscopy, fluorescent saliva labelling and image analysis, we characterized the mechanical actions of mouthparts and the spatial-temporal patterns of salivation during feeding on mammalian skin. Our recordings indicate that the labrum and hypopharynx are the most prominent mouthparts during feeding and exhibit scissor-like movements during probing. At specific moments, these structures close forcefully, generating small blood splashes in multiple directions. Feeding occurred in two distinct phases: an initial probing phase, often distinguished by ineffective blood intake, and a subsequent engorgement phase that was initiated exclusively upon the activation of small dermal "feeder vessels."Acridine Orange labelling showed abundant early salivation that penetrated progressively deeper into the dermis and remained detectable for over an hour, reflecting both the tissue damage and enzymatic effects. The analysis of images demonstrated the sequential salivation events, highlighting an initial high-frequency phase followed by a more gradual pattern during engorgement. These findings provide the first real-time, detailed view of the coordinated interactions between mouthpart mechanics, targeted salivation, and host microvascular responses in Lu. longipalpis. This study redefines sand fly telmophagy as a non-passive and coordinated process integrating mouthpart mechanics, salivation, and modulation of host vasculature. This work advances our understanding of sand fly vector-host interactions and underscores the potential of salivary molecules as targets for transmission-blocking strategies. Author SummaryPhlebotomine sand flies are the main vectors of Leishmania infantum, the parasite responsible for visceral leishmaniasis in the Americas. Although sand flies are traditionally classified as "pool feeders," meaning they lacerate the skin and feed from small pools of blood, the mechanics of how they obtain blood and deliver saliva into host skin have remained poorly understood. In this study, we used image analysis, intravital microscopy and fluorescent labeling of saliva to visualize, in real time, the feeding behavior of Lutzomyia longipalpis on mammalian skin. We show that blood feeding is not a passive process based solely on blood pooling. Instead, it involves coordinated movements of the mouthparts, modulation of host microvessels with the saliva contribution, and the recruitment of small dermal "feeder vessels" that supply blood directly to the insect. Our findings reveal that sand fly feeding is a highly orchestrated interaction between vector and host, integrating mechanical tissue disruption, salivary secretion, and vascular responses. These processes likely create a favorable microenvironment for Leishmania establishment and transmission. By providing a detailed characterization of mouthpart and salivation dynamics, this study advances our understanding of sand fly biology and highlights salivary components and feeding-site events as potential targets for transmission-blocking strategies.

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.

The phylum Apicomplexa contains [~] 6000 known species of unicellular eukaryotic parasites. A unifying feature among the apicomplexans is the apical complex, which varies in complexity in different lineages, but always contains an annulus (a.k.a. the apical polar ring) into which the minus ends of an array of cortical microtubules are embedded. In Toxoplasma gondii, the apical complex also includes the conoid, which contains several signaling and structural proteins critical for parasite motility. The conoid extends and retracts through the apical polar ring in a calcium-dependent manner. Here we report the identification of several new apical polar ring components, including APR9, which is highly conserved among the apicomplexans and their free-living relative Chromera velia. The loss of APR9 alone has only a moderate impact on the parasite lytic cycle. However, the knockout of both APR9 and KinesinA (another apical polar ring component) paralyzes parasite and drastically impairs invasion, egress and the lytic cycle. The double-knockout displays multiple subcellular abnormalities, including the formation of an apical actin concentration, impaired conoid extension, and significantly reduced secretion of a major adhesin (MIC2) upon stimulation with a calcium ionophore. These findings reveal that the apical polar ring plays a critical role in parasite motility and contributes to multiple subcellular processes.

Root-knot nematodes (RKNs) are obligate plant parasites that establish biotrophic interactions with a wide range of host plants, causing major crop losses worldwide. During infection, RKNs secrete effector proteins from their esophageal glands into root tissues via a stylet. These effectors target specific subcellular compartments and manipulate host cell functions to induce the formation of hypertrophied and multinucleated giant cells, which serve as feeding sites essential for nematode development. Here we describe a new RKN-specific effector, EFFECTOR17 (EFF17), conserved in the five most damaging RKN species: Meloidogyne incognita, M. enterolobii, M. arenaria, M. javanica, and M. hapla. In situ hybridization showed that EFF17 genes are specifically expressed in the esophageal glands of various RKN species. Silencing EFF17 in M. incognita reduced its reproduction on Nicotiana benthamiana, demonstrating a key role in parasitism. Yeast two-hybrid and split luciferase assays revealed that tomato and Arabidopsis KINESIN LIGHT CHAIN RELATED (KLCR)/CELLULOSE-MICROUBULE UNCOUPLING (CMU) proteins are host targets of MiEFF17. Arabidopsis klcr mutants developed significantly fewer galls and egg masses. Our findings propose that EFF17 manipulates KLCR function to promote parasitism.

HIV-1 (Human Immunodeficiency Virus type 1) infects macrophages, which resist to the cytopathic effects of the virus and are considered as viral reservoirs. However, the cellular factors involved in viral production by human macrophages have not been fully identified. In this study, we focused on the amino acid transporter SNAT7 (small neutral amino-acid transporter 7), member of the SLC38 solute carrier family, which is the main lysosomal transporter of glutamine from the lysosome to the cytoplasm. Its expression was increased by HIV-1 infection. We revealed that the absence of SNAT7 inhibited viral production not only at the level of protein synthesis, but also early at the level of reverse transcription, without affecting global RNA or protein synthesis in the cells. The reduction in HIV expression upon SNAT7 depletion correlated with a reduction in the levels of an inactive form of the SAMHD1 (SAM domain- and HD domain-containing protein) restriction factor and was rescued following SAMHD1 degradation. Lastly, supplementation of the extracellular medium with glutamine in the absence of SNAT7 partially restored viral production. Together, our data reveal that glutamine extracted from lysosomes is involved in the early stages of the HIV-1 cycle and that the SNAT7 glutamine transporter acts as a dependency factor for HIV-1 in human macrophages.

The genomes of malaria parasites (Plasmodium spp.) encode many gene families, which are intimately associated with host interactions and disease in these important pathogens. The largest malaria gene family is the Plasmodium interspersed repeat (pir) genes, present in rodent, primate and most human malaria parasites, which are suggested to have originated from one highly conserved gene, which we call pirC1. The precise function(s) of pir is unknown but to determine their potentially multifarious roles we must understand the evolutionary dynamics of pir repertoire to discriminate among the many genes. Here we estimate the global phylogeny for pir genes in 14 Plasmodium species and one Hepatocystis species. We reveal that pirC1 is not the common ancestor but is one of several orthologous genes conserved in multiple species amidst the rapid turnover of species-specific paralogs. We show that the PIRC1 protein is nonetheless essential for blood stage growth of P. berghei, P. chabaudi and P. knowlesi, as parasites lacking the pirC1 gene could not be generated or had severely reduced growth rates. As this effect was observed both in vivo and in vitro, the role of pirC1 is not related to host immune interaction. Rather, P. berghei and P. knowlesi PIRC1 are secreted from the parasite, pointing to a role in parasite interaction with the host cell or nutrient uptake by blood stages. The phylodynamics of pir genes indicate that old orthologs, like pirC1, and younger within-species paralogs could have fundamentally different roles, and emphasize the need to distinguish between them in future. This study is the first to provide evidence for the existence of an essential pir gene and provides a robust rationale for further experimental approaches to pir gene functions. SIGNFICANCEThe genomes of malaria parasites (Plasmodium) contain many different gene families, of which the pir family is the largest, with more than 1000 members in some species. The PIR proteins are likely important for parasite fitness but their precise functions remain unknown - roles in adherence of infected red blood cells to blood vessels, virulence and immune evasion of have been suggested. How, and why, this highly diverse gene family evolved is a significant question both for understanding malaria physiology and pathogenesis. Here we present a comprehensive pir phylogeny, identifying the origins of gene diversity during Plasmodium evolution and a select group of highly conserved genes. We show that one conserved pir gene (pirC1) encodes a protein that is essential for optimal growth of multiple malaria parasites during the asexual blood stage, both in the host and in vitro. This indicates that pirC1 function relates to interaction with the host cell or nutrient acquisition, and not to immune evasion or sequestration, (although this might still be the function of other pir genes). This study provides a robust rationale for the hitherto baffling diversity of pir genes, and shows why it is important to distinguish old orthologs from young paralogs in future studies on pir gene function.

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.