Ubiquitination is a posttranslational modification in eukaryotes that plays a significant role in the infection of intracellular microbial pathogens, such as Legionella pneumophila. While the Legionella-containing vacuole (LCV) is coated with ubiquitin (Ub), it avoids recognition by autophagy adaptors. Here, we report that the Sdc and Sde families of effectors work together to build ubiquitinated species around the LCV. The Sdc effectors catalyze canonical polyubiquitination directly on host targets or on phosphoribosyl-Ub conjugated to host targets by Sde. Remarkably, Ub moieties within poly-Ub chains are either modified with a phosphoribosyl group by PDE domain-containing effectors or covalently attached to other host substrates via Sde-mediated phosphoribosyl-ubiquitination. Furthermore, these modifications prevent the recognition by Ub adaptors and therefore exclude host autophagy adaptors from the LCV. In this work, we shed light on the nature of the poly-ubiquitinated species present at the surface of the LCV and provide a molecular mechanism for the avoidance of autophagy adaptors by the Ub-decorated LCV.

The Legionella pneumophila Sde family of translocated proteins promotes host tubular endoplasmic reticulum (ER) rearrangements that are tightly linked to phosphoribosyl-ubiquitin (pR-Ub) modification of Reticulon 4 (Rtn4). Sde proteins have two additional activities of unclear relevance to the infection process: K63 linkage-specific deubiquitination and phosphoribosyl modification of polyubiquitin (pR-Ub). We show here that the deubiquitination activity (DUB) stimulates ER rearrangements while pR-Ub protects the replication vacuole from cytosolic surveillance by autophagy. Loss of DUB activity is tightly linked to lowered pR-Ub modification of Rtn4, consistent with the DUB activity fueling the production of pR-Ub-Rtn4. In parallel, phosphoribosyl modification of polyUb, in a region of the protein known as the isoleucine patch, prevents binding by the autophagy adapter p62. An inability of Sde mutants to modify polyUb results in immediate p62 association, a critical precursor to autophagic attack. The ability of Sde WT to block p62 association decays quickly after bacterial infection, as predicted by the presence of previously characterized L. pneumophila effectors that inactivate Sde and remove polyUb. In sum, these results show that the accessory Sde activities act to stimulate ER rearrangements and protect from host innate immune sensing in a temporal fashion.

An 83-year-old man with a history of monoclonal gammopathy of unknown significance, macrocytic anemia, and cytopenias presented with hemorrhagic bullae on his left hand, alongside intermittent fevers and joint pain. Laboratory findings indicated anemia, elevated mean corpuscular volume, thrombocytopenia, leukopenia, and a high C-reactive protein level. A biopsy of the bullae showed neutrophilic dermatosis, and computed tomography scans of the thorax revealed lung opacities and mediastinal lymphadenopathy, suggesting neutrophilic alveolitis. Bone marrow examination found hypercellularity with myeloid and histiocytic hyperplasia, vacuolated precursors, and 3% blasts of an immature myelomonocytic lineage. Genetic testing uncovered a UBA1 mutation at an 81% allele frequency, confirming a diagnosis of VEXAS syndrome. Treatment commenced with prednisone, initiated at 60 mg daily and tapered to 10 mg, with tocilizumab considered for future symptom management. This regimen has successfully maintained remission, as observed in follow-up appointments. This case highlights the diagnostic complexity and effective management of VEXAS syndrome, underscoring the importance of genetic testing in guiding treatment decisions.
VEXAS syndrome, initially identified in 2020, is a rare disease characterized by overlapping hematologic and rheumatologic conditions, primarily affecting middle-aged to older men.Our case presents a unique instance of VEXAS syndrome, featuring inflammatory symptoms alongside pancytopenia and a history of monoclonal gammopathy of unknown significance, highlighting the diverse clinical spectrum of this condition.The diagnosis was confirmed through bone marrow biopsy revealing vacuoles in myeloid precursors and a UBA1 mutation, reaffirming the diagnostic criteria outlined for VEXAS syndrome.Management challenges persist, with current treatment options limited to glucocorticoids for symptomatic relief, underscoring the need for further research into targeted therapies such as ruxolitinib to improve patient outcomes.

We previously reported autophagy-mediated degradation of nuclei, nucleophagy, in the filamentous fungus Aspergillus oryzae. In this study, we examined whether nuclei are degraded as a whole. We generated A. oryzae mutants deleted for orthologs of Saccharomyces cerevisiae YPT7 and ATG15 which are required, respectively, for autophagosome-vacuole fusion and vacuolar degradation of autophagic bodies. Degradation of histone H2B-EGFP under starvation conditions was greatly decreased in the ΔAoypt7 and ΔAoatg15 mutants. Fluorescence and electron microscopic observations showed that autophagosomes and autophagic bodies surrounding the entire nuclei were accumulated in the cytoplasm of ΔAoypt7 and the vacuole of ΔAoatg15, respectively. These results indicate that nuclei are engulfed in the autophagosomes as a whole and transported/released into the vacuolar lumen where they are degraded.

Plants have developed precise defense mechanisms against cadmium (Cd) stress, with vacuolar compartmentalization of Cd2+ being a crucial process in Cd detoxification. The transport of Cd into vacuoles by these cation / H+ antiporters is powered by the pH gradient created by proton pumps. In this study, the full-length cDNA of a vacuolar H+-pyrophosphatase (V-PPase) gene from Boehmeria nivea (ramie), BnVP1, was isolated using the rapid amplification of cDNA ends (RACE) method. The open reading frame (ORF) of BnVP1 is 2292 bp, encoding a 763 amino acid V-PPase protein with 15 predicted transmembrane domains. Sequence alignment and phylogenetic analysis revealed that BnVP1 belongs to the Type I V-PPase family. Quantitative RT-PCR assays demonstrated that BnVP1 expression was significantly higher in ramie roots than in shoots. Cd treatments markedly induced BnVP1 expression in both roots and leaves of ramie seedlings, with a more pronounced effect in roots. Additionally, BnVP1 expression was significantly upregulated by the plant hormone methyl jasmonate (MeJA). Heterologous expression of BnVP1 in transgenic Arabidopsis significantly enhanced V-PPase activity in the roots. The growth performance, root elongation, and total chlorophyll content of transgenic plants with high tonoplast H+-PPase (V-PPase) activity were superior to those of wild-type plants. Overexpression of BnVP1 reduced membrane lipid peroxidation and ion leakage, and significantly increased Cd accumulation in the roots of transgenic Arabidopsis seedlings. This study provides new genetic resources for the phytoremediation of Cd-contaminated farmland.

Coxiella burnetii is a globally distributed obligate intracellular pathogen. Although often asymptomatic, infections can cause acute Q fever with influenza-like symptoms and/or severe chronic Q fever. Coxiella burnetii develops a unique replicative niche within host cells called the Coxiella-containing vacuole (CCV), facilitated by the Dot/Icm type IV secretion system translocating a cohort of bacterial effector proteins into the host. The role of some effectors has been elucidated; however, the actions of the majority remain enigmatic and the list of true effectors is disputable. This study examined CBU2016, a unique C. burnetii protein previously designated as an effector with a role in infection. We were unable to validate CBU2016 as a translocated effector protein. Employing targeted knock-out and complemented strains, we found that the loss of CBU2016 did not cause a replication defect within Hela, THP-1, J774, or iBMDM cells or in axenic media, nor did it affect the pathogenicity of C. burnetii in the Galleria mellonella infection model. The absence of CBU2016 did, however, result in a consistent decrease in the size of CCVs in HeLa cells. These results suggest that although CBU2016 may not be a Dot/Icm effector, it is still able to influence the host environment during infection.

Monoterpenoid indole alkaloid (MIA) biosynthesis in Catharanthus roseus is a paragon of the spatiotemporal complexity achievable by plant specialized metabolism. Spanning a range of tissues, four cell types, and five cellular organelles, MIA metabolism is intricately regulated and organized. This high degree of metabolic differentiation requires inter-cellular and organellar transport, which remains understudied. Here, we have characterized a vacuolar importer of secologanin belonging to the multidrug and toxic compound extrusion (MATE) family, named CrMATE1. Phylogenetic analyses of MATEs suggested a role in alkaloid transport for CrMATE1, and in planta silencing in two varieties of C. roseus resulted in a shift in the secoiridoid and MIA profiles. Subcellular localization of CrMATE1 confirmed tonoplast localization. Biochemical characterization was conducted using the Xenopus laevis oocyte expression system to determine substrate range, directionality, and rate. We can confirm that CrMATE1 is a vacuolar importer of secologanin, translocating 1 mM of substrate within 25 min. The transporter displayed strict directionality and specificity for secologanin and did not accept other secoiridoid substrates. The unique substrate-specific activity of CrMATE1 showcases the utility of transporters as gatekeepers of pathway flux, mediating the balance between a defense arsenal and cellular homeostasis.

Fusarium graminearum is an important plant pathogen that causes head blight in cereal crops such as wheat, barley, and rice worldwide. In this study, we identified and functionally characterized FgVAC1, an essential gene in F. graminearum that encodes a Rab5 effector involved in membrane tethering functions. The essentiality of FgVAC1 was confirmed through a conditional promoter replacement strategy using the zearalenone-inducible promoter (PZEAR). Cytological analyses revealed that FgVac1 colocalizes with FgRab51 on early endosomes and regulates the proper transport of the vacuolar hydrolase FgCpy1 to the vacuole. Suppression of FgVAC1 led to inhibited vegetative growth, reduced asexual and sexual reproduction, decreased deoxynivalenol (DON) biosynthesis, and diminished pathogenicity. Our findings highlight the significant role of FgVac1 in vacuolar protein sorting, fungal development, and plant infection in F. graminearum.

Although some budding yeasts have proved tractable and intensely studied models, others are more recalcitrant. Debaryomyces hansenii, an important yeast species in food and biotechnological industries with curious physiological characteristics, has proved difficult to manipulate genetically and remains poorly defined. To remedy this, we have combined live cell fluorescent dyes with high-resolution imaging techniques to define the sub-cellular features of D. hansenii, such as the mitochondria, nuclei, vacuoles and the cell wall. Using these tools, we define biological processes like the cell cycle, organelle inheritance and various membrane trafficking pathways of D. hansenii for the first time. Beyond this, reagents designed to study Saccharomyces cerevisiae proteins were used to access proteomic information about D. hansenii. Finally, we optimised the use of label-free holotomography to image yeast, defining the physical parameters and visualising sub-cellular features like membranes and vacuoles. Not only does this work shed light on D. hansenii but this combinatorial approach serves as a template for how other cell biological systems, which are not amenable to standard genetic procedures, can be studied.

Trypanosoma cruzi uses various mechanisms to cope with osmotic fluctuations during infection, including the remodeling of organelles such as the contractile vacuole complex (CVC). Little is known about the morphological changes of the CVC during pulsation cycles occurring upon osmotic stress. Here, we investigated the structure-function relationship between the CVC and the flagellar pocket domain where fluid discharge takes place-the adhesion plaque-during the CVC pulsation cycle. Using TcrPDEC2 and TcVps34 overexpressing mutants, known to have low and high efficiency for osmotic responses, we described a structural phenotype for the CVC that matches their corresponding physiological responses. Quantitative tomography provided data on the volume of the CVC and spongiome connections. Changes in the adhesion plaque during the pulsation cycle were also quantified and a dense filamentous network was observed. Together, the results suggest that the adhesion plaque mediates fluid discharge from the central vacuole, revealing new aspects of the osmoregulatory system in T. cruzi.