Vacuolar Acidification Research Articles

Foliar spraying magnesium promotes soluble sugar accumulation by inducing the activities of sucrose biosynthesis and transport in citrus fruits

Accumulation of soluble sugars promotes the formation of fruit nutritional quality and flavor while proper application of Magnesium (Mg) can increase the sugar content in citrus fruits. However, the underlying mechanism is still unclear. In the present study by using ‘Huagan No.2′ (Citrus reticulate cv. Huagan No.2) as research material, foliar spraying of MgSO4 with two or three times was performed during the fruit expansion period. Results indicated that foliar spraying of Mg significantly increased the total soluble solids (TSS), as well as the contents of sucrose, fructose and glucose in fruits; moreover, the activities of sucrose metabolizing enzymes including sucrose synthase (SS), sucrose phosphate synthesis (SPS) and acid invertase (AI), as well as the transcript levels of SS2, SPS1–3 and vacuolar acid invertase gene (VINV) were significantly increased in the Mg-treated fruits. In addition, the transcript levels of the sucrose transport-related gene (SUT3) and the vacuolar pyrophosphate proton pump type I genes (CsVPP-1 and CsVPP-2) in the fruits were also significantly increased by Mg treatment. These results were further verified by soaking the fruit-bearing branches in 0.5% MgSO4 solution. In conclusion, foliar spraying Mg with two or three times promotes soluble sugar accumulation in fruits through enhancing the sink strength by inducing the activity of sucrose synthase via inducing SS2 transcript level, and improving sucrose transport ability by inducing transcript levels of SUT3, CsVPP-1, CsVPP-2 and VINV.

The Responses of Sucrose Metabolism and Carbon Translocation in Tomato Seedlings under Different Light Spectra.

Light plays a dominant role in the biosynthesis and accumulation of photosynthetic products. However, the metabolism and translocation of photosynthetic products in plants under different light spectra remain elusive. In this study, tomato (Solanum lycopersicum L.) seedlings were treated with different light spectra delivered by light-emitting diodes (LEDs) with the same photosynthetic photon flux density at 300 μmol m-2 s-1, including monochromatic red (660 nm, R), blue (450 nm, B), sun-like white (W, 380-780 nm), or a combination of R and B lights (R:B = 1:1, RB). Compared with W, the biomass distribution ratio for leaves under R, B, and RB decreased by 5.01-9.53%, while the ratio for stems and roots increased by 3.71-6.92% and 0.14-2.81%, respectively. The photosynthetic carbon distribution expressed as 13C enrichment was higher in stems and roots under RB and R, while B led to more 13C transported from leaves and enriched in stems when compared with W. Meanwhile, RB led to significant increases in the activities of phosphate synthase (SPS), sucrose synthase (SS), vacuolar acid invertase (VI), and neutral invertase (NI). The R was more efficient in increasing the activity of SPS and SS, while B was more effective in promoting the activity of VI and NI. The transcript levels of SPS, SS3, NI6, and VI were upregulated under R, B, and RB. However, the transcript patterns of SPS, SS3, NI6, and VI were not consistent with the changes in their encoded enzymes, especially the transcript patterns of SPS and SS3. Our study suggests that the red- and blue-light-induced long-distance and short-distance transport of photosynthetic products in plants, respectively, might result from different regulation of sucrose-metabolizing enzymes from transcriptional and post-transcriptional levels.

Regulation of a vacuolar proton-pumping P-ATPase MdPH5 by MdMYB73 and its role in malate accumulation and vacuolar acidification

As the main organic acid in fruits, malate is produced in the cytoplasm and is then transported into the vacuole. It accumulates by vacuolar proton pumps, transporters, and channels, affecting the taste and flavor of fruits. Among the three types of proton pumps (V-ATPases, V-PPases, and P-ATPases), the P-ATPases play an important role in the transport of malate into vacuoles. In this study, the transcriptome data, collected at different stages after blooming and during storage, were analyzed and the results demonstrated that the expression of MdPH5, a vacuolar proton-pumping P-ATPase, was associated with both pre- and post-harvest malate contents. Moreover, MdPH5 is localized at the tonoplast and regulates malate accumulation and vacuolar pH. In addition, MdMYB73, an upstream MYB transcription factor of MdPH5, directly binds to its promoter, thereby transcriptionally activating its expression and enhancing its activity. In this way, MdMYB73 can also affect malate accumulation and vacuolar pH. Overall, this study clarifies how MdMYB73 and MdPH5 act to regulate vacuolar malate transport systems, thereby affecting malate accumulation and vacuolar pH.

Platelet-derived microvesicles induce intracellular calcium mobilization in human platelets.

Platelet-derived microvesicles (PMVs) represent a significant proportion of microvesicles in circulation and have been linked to various pathophysiological complications. Recent research suggests that PMVs carry significant amounts of cargo that can affect cellular functions by influencing calcium oscillations in target cells. As calcium is involved in multiple cellular processes, including hemostasis and thrombosis, this study aimed to investigate the impact of PMVs on platelet calcium mobilization. The study found that PMVs increase platelet intracellular calcium levels via both intracellular storage and extracellular space in a dose-dependent manner. The study highlighted the critical role of the dense tubular system, acidic vacuoles, mitochondrial stores, and store-operated calcium entry (SOCE) in PMV-mediated calcium release in human platelets. Moreover, the study revealed that PMV-induced calcium rise in platelets does not occur via sarcoendoplasmic reticulum calcium ATPase, and extracellular calcium addition further increases the calcium level in platelets, demonstrating the involvement of SOCE. These findings provide insights into the platelet stimulation signaling mechanisms and contributes to our understanding of platelet and cell behavior when exposed to PMV-rich environments.

The phenuivirus Toscana virus makes an atypical use of vacuolar acidity to enter host cells.

Toscana virus is a major cause of arboviral disease in humans in the Mediterranean basin during summer. However, early virus-host cell interactions and entry mechanisms remain poorly characterized. Investigating iPSC-derived human neurons and cell lines, we found that virus binding to the cell surface was specific, and 50% of bound virions were endocytosed within 10 min. Virions entered Rab5a+ early endosomes and, subsequently, Rab7a+ and LAMP-1+ late endosomal compartments. Penetration required intact late endosomes and occurred within 30 min following internalization. Virus entry relied on vacuolar acidification, with an optimal pH for viral membrane fusion at pH 5.5. The pH threshold increased to 5.8 with longer pre-exposure of virions to the slightly acidic pH in early endosomes. Strikingly, the particles remained infectious after entering late endosomes with a pH below the fusion threshold. Overall, our study establishes Toscana virus as a late-penetrating virus and reveals an atypical use of vacuolar acidity by this virus to enter host cells.

Topology and Function of the S. cerevisiae Autophagy Protein Atg15.

The putative phospholipase Atg15 is required for the intravacuolar lysis of autophagic bodies and MVB vesicles. Intracellular membrane lysis is a highly sophisticated mechanism that is not fully understood. The amino-terminal transmembrane domain of Atg15 contains the sorting signal for entry into the MVB pathway. By replacing this domain, we generated chimeras located in the cytosol, the vacuole membrane, and the lumen. The variants at the vacuole membrane and in the lumen were highly active. Together with the absence of Atg15 from the phagophore and autophagic bodies, this suggests that, within the vacuole, Atg15 can lyse vesicles where it is not embedded. In-depth topological analyses showed that Atg15 is a single membrane-spanning protein with the amino-terminus in the cytosol and the rest, including the active site motif, in the ER lumen. Remarkably, only membrane-embedded Atg15 variants affected growth when overexpressed. The growth defects depended on its active site serine 332, showing that it was linked to the enzymatic activity of Atg15. Interestingly, the growth defects were independent of vacuolar proteinase A and vacuolar acidification.

Molecular mechanism underlying regulation of Arabidopsis CLCa transporter by nucleotides and phospholipids

Chloride channels (CLCs) transport anion across membrane to regulate ion homeostasis and acidification of intracellular organelles, and are divided into anion channels and anion/proton antiporters. Arabidopsis thaliana CLCa (AtCLCa) transporter localizes to the tonoplast which imports NO3− and to a less extent Cl− from cytoplasm. The activity of AtCLCa and many other CLCs is regulated by nucleotides and phospholipids, however, the molecular mechanism remains unclear. Here we determine the cryo-EM structures of AtCLCa bound with NO3− and Cl−, respectively. Both structures are captured in ATP and PI(4,5)P2 bound conformation. Structural and electrophysiological analyses reveal a previously unidentified N-terminal β-hairpin that is stabilized by ATP binding to block the anion transport pathway, thereby inhibiting the AtCLCa activity. While AMP loses the inhibition capacity due to lack of the β/γ- phosphates required for β-hairpin stabilization. This well explains how AtCLCa senses the ATP/AMP status to regulate the physiological nitrogen-carbon balance. Our data further show that PI(4,5)P2 or PI(3,5)P2 binds to the AtCLCa dimer interface and occupies the proton-exit pathway, which may help to understand the inhibition of AtCLCa by phospholipids to facilitate guard cell vacuole acidification and stomatal closure. In a word, our work suggests the regulatory mechanism of AtCLCa by nucleotides and phospholipids under certain physiological scenarios and provides new insights for future study of CLCs.

Photodynamic Activity of Acridine Orange in Keratinocytes under Blue Light Irradiation

Acridine orange (AO) is a metachromatic fluorescent dye that stains various cellular compartments, specifically accumulating in acidic vacuoles (AVOs). AO is frequently used for cell and tissue staining (in vivo and in vitro), mainly because it marks different cellular compartments with different colors. However, AO also forms triplet excited states and its role as a photosensitizer is not yet completely understood. Human immortalized keratinocytes (HaCaT) were incubated for either 10 or 60 min with various concentrations (nanomolar range) of AO that were significantly lower than those typically used in staining protocols (micromolar). After incubation, the cells were irradiated with a 490 nm LED. As expected, cell viability (measured by MTT, NRU and crystal violet staining) decreased with the increase in AO concentration. Interestingly, at the same AO concentration, altering the incubation time with HaCaT substantially decreased the 50% lethal dose (LD50) from 300 to 150 nM. The photoinduced cell death correlated primarily with lysosomal disfunction, and the correlation was stronger for the 60 min AO incubation results. Furthermore, the longer incubation time favored monomers of AO and a distribution of the dye to intracellular sites other than lysosomes. Studies with mimetic systems indicated that monomers, which have higher yields of fluorescence emission and singlet oxygen generation, are favored in acidic environments, consistent with the more intense emission from cells submitted to the longer AO incubation period. Our results indicate that AO is an efficient PDT photosensitizer, with a photodynamic efficiency that is enhanced in acidic environments when multiple intracellular locations are targeted. Consequently, when using AO as a probe for live cell tracking and tissue staining, care must be taken to avoid excessive exposure to light to avoid undesirable photosensitized oxidation reactions in the tissue or cell under investigation.

Host Lipid Transport Protein ORP1 Is Necessary for Coxiella burnetii Growth and Vacuole Expansion in Macrophages.

Coxiella burnetii is an intracellular bacterium that causes the human disease Q fever. C. burnetii forms a large, acidic Coxiella-containing vacuole (CCV) and uses a type 4B secretion system to secrete effector proteins into the host cell cytoplasm. While the CCV membrane is rich in sterols, cholesterol accumulation in the CCV is bacteriolytic, suggesting that C. burnetii regulation of lipid transport and metabolism is critical for successful infection. The mammalian lipid transport protein ORP1L (oxysterol binding protein-like protein 1 Long) localizes to the CCV membrane and mediates CCV-endoplasmic reticulum (ER) membrane contact sites. ORP1L functions in lipid sensing and transport, including cholesterol efflux from late endosomes and lysosomes (LELs), and the ER. Its sister isoform, ORP1S (oxysterol binding protein-like protein 1 Short) also binds cholesterol but has cytoplasmic and nuclear localization. In ORP1-null cells, we found that CCVs were smaller than in wild-type cells, highlighting the importance of ORP1 in CCV development. This effect was consistent between HeLa cells and murine alveolar macrophages (MH-S cells). CCVs in ORP1-null cells had higher cholesterol content than CCVs in wild-type cells at 4 days of infection, suggesting ORP1 functions in cholesterol efflux from the CCV. While the absence of ORP1 led to a C. burnetii growth defect in MH-S cells, there was no growth defect in HeLa cells. Together, our data demonstrated that C. burnetii uses the host sterol transport protein ORP1 to promote CCV development, potentially by using ORP1 to facilitate cholesterol efflux from the CCV to diminish the bacteriolytic effects of cholesterol. IMPORTANCE Coxiella burnetii is an emerging zoonotic pathogen and bioterrorism threat. No licensed vaccine exists in the United States, and the chronic form of the disease is difficult to treat and potentially lethal. Postinfectious sequelae of C. burnetii infection, including debilitating fatigue, place a significant burden on individuals and communities recovering from an outbreak. C. burnetii must manipulate host cell processes in order to promote infection. Our results establish a link between host cell lipid transport processes and C. burnetii's avoidance of cholesterol toxicity during infection of alveolar macrophages. Elucidating the mechanisms behind bacterial manipulation of the host will yield insight for new strategies to combat this intracellular pathogen.

Codon-optimized TDP-43 mediates neurodegeneration in a Drosophila model of ALS/FTLD.

Transactive response DNA binding protein-43 (TDP-43) is known to mediate neurodegeneration associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). The exact mechanism by which TDP-43 exerts toxicity in the brains, spinal cord, and lower motor neurons of affected patients remains unclear. In a novel Drosophila melanogaster model, we report gain-of-function phenotypes due to misexpression of insect codon-optimized version of human wild-type TDP-43 (CO-TDP-43) using both the binary GAL4/UAS system and direct promoter fusion constructs. The CO-TDP-43 model showed robust tissue specific phenotypes in the adult eye, wing, and bristles in the notum. Compared to non-codon optimized transgenic flies, the CO-TDP-43 flies produced increased amount of high molecular weight protein, exhibited pathogenic phenotypes, and showed cytoplasmic aggregation with both nuclear and cytoplasmic expression of TDP-43. Further characterization of the adult retina showed a disruption in the morphology and function of the photoreceptor neurons with the presence of acidic vacuoles that are characteristic of autophagy. Based on our observations, we propose that TDP-43 has the propensity to form toxic protein aggregates via a gain-of-function mechanism, and such toxic overload leads to activation of protein degradation pathways such as autophagy. The novel codon optimized TDP-43 model is an excellent resource that could be used in genetic screens to identify and better understand the exact disease mechanism of TDP-43 proteinopathies and find potential therapeutic targets.

Temozolomide, Simvastatin and Acetylshikonin Combination Induces Mitochondrial-Dependent Apoptosis in GBM Cells, Which Is Regulated by Autophagy

Glioblastoma multiforme (GBM) is one of the deadliest cancers. Temozolomide (TMZ) is the most common chemotherapy used for GBM patients. Recently, combination chemotherapy strategies have had more effective antitumor effects and focus on slowing down the development of chemotherapy resistance. A combination of TMZ and cholesterol-lowering medications (statins) is currently under investigation in in vivo and clinical trials. In our current investigation, we have used a triple-combination therapy of TMZ, Simvastatin (Simva), and acetylshikonin, and investigated its apoptotic mechanism in GBM cell lines (U87 and U251). We used viability, apoptosis, reactive oxygen species, mitochondrial membrane potential (MMP), caspase-3/-7, acridine orange (AO) and immunoblotting autophagy assays. Our results showed that a TMZ/Simva/ASH combination therapy induced significantly more apoptosis compared to TMZ, Simva, ASH, and TMZ/Simva treatments in GBM cells. Apoptosis via TMZ/Simva/ASH treatment induced mitochondrial damage (increase of ROS, decrease of MMP) and caspase-3/7 activation in both GBM cell lines. Compared to all single treatments and the TMZ/Simva treatment, TMZ/Simva/ASH significantly increased positive acidic vacuole organelles. We further confirmed that the increase of AVOs during the TMZ/Simva/ASH treatment was due to the partial inhibition of autophagy flux (accumulation of LC3β-II and a decrease in p62 degradation) in GBM cells. Our investigation also showed that TMZ/Simva/ASH-induced cell death was depended on autophagy flux, as further inhibition of autophagy flux increased TMZ/Simva/ASH-induced cell death in GBM cells. Finally, our results showed that TMZ/Simva/ASH treatment potentially depends on an increase of Bax expression in GBM cells. Our current investigation might open new avenues for a more effective treatment of GBM, but further investigations are required for a better identification of the mechanisms.

Bisphenol S exposure promotes cell apoptosis and mitophagy in murine osteocytes by regulating mtROS signaling.

Bisphenol S (BPS), a safer alternative to bisphenol A, is commonly used as a plasticizer to manufacture various food-packaging materials. The accumulated BPS inhibits osteoblastic bone formation and promotes osteoclastogenesis, thereby accelerating remarkable bone destruction, but it is unclear whether BPS affects osteocytes, comprising over 95% of all bone cells. This study aimed to investigate the biological effect of BPS on osteocytes in vitro, as well as the detailed mechanism. Results showed that BPS (200, 400 μmol/L) exposure caused dose-dependently cell death of osteocytes MLO-Y4, and increased cell apoptosis. BPS induced loss of mitochondrial membrane potential (MMP) and mitochondria impairment. Furthermore, BPS upregulated expressions of mitophagy-related proteins including microtubule-associated protein light chain 3 (LC-3) II and PTEN-induced putative kinase (PINK) 1, accompanied by elevation of autophagy flux and the accumulation of acidic vacuoles; whereas p62 level was downregulated after BPS treatment. Additionally, BPS triggered the production of intracellular reactive oxygen species (ROS) and mitochondrial ROS (mtROS), while it decreased expression levels of nuclear factor E2-related factor 2 (Nrf2) and quinone oxidoreductase 1 (NQO1). The specific mtROS scavenger MitoTEMPO reversed cell apoptosis and mitophagy, suggesting that mtROS contributes to BPS exposure-induced apoptosis and mitophagy in MLO-Y4 cells. Our data first provide novel evidence that apoptosis and mitophagy as cellular mechanisms for the toxic effect of BPS on osteocytes, thereby helping our understanding of the potential role of osteocytes in the adverse effect of BPS and its analogs on bone growth, and supporting strategies targeting bone destruction caused by BPS.

Plasmodium berghei Purified Hemozoin Associated with DNA Strongly Inhibits P. berghei Liver-Stage Development in BALB/c Mice after Intravenous Inoculation.

In the acidic lysosome-like digestive vacuole, Plasmodium parasites crystallize heme from hemoglobin into hemozoin, or malaria pigment. Upon release of progeny merozoites, the residual hemozoin is phagocytized by macrophages principally in the liver and spleen where the heme crystals can persist for months to years, as heme oxygenase does not readily degrade the crystal. Previous studies demonstrated hemozoin modulation of monocytes and macrophages. Hemozoin modulates immune function activity of monocytes/macrophages. Here, we used purified/washed hemozoin (W-Hz) isolated from murine Plasmodium berghei infections and intravenously (i.v.) injected it back into naive mice. We characterized the modulating effect of W-Hz on liver-stage replication. Purified washed hemozoin decreases P. berghei liver levels both at 1 week and 1 month after i.v. injection in a dose and time dependent fashion. The injected hemozoin fully protected in nine out of 10 mice given a 50 sporozoite inoculum, and in 10 out of 10 mice against 2,000 sporozoites when they were infected an hour or a day after hemozoin inoculation. DNase treatment at the hemozoin reversed the observed liver load reduction. The liver load reduction was similar in mature B- and T-cell-deficient RAG-1 knockout (KO) mice suggesting an innate immune protection mechanism. This work indicates a role for residual hemozoin in down modulation of Plasmodium liver stages.

Autophagy induced macrophages by α-alumina(α-AL2O3) conjugated cysteine peptidase, enhances the cytotoxic activity of CD8+ T lymphocytes against Leishmania major.

Induction of a protective immune response against Leishmania major requires the activation of both TH1 and CD8+ T lymphocytes. Because L. major is an intra-phagosomal parasite, its antigens do not have access to MHC-I. The present study aimed to evaluate the effect of cysteine peptidase A (CPA)/cysteine peptidase B (CPB) conjugated to α-AL2O3 on autophagy induction in L. major infected macrophages and subsequent activation of cytotoxic CD8+ T lymphocytes. Recombinant CPA and CPB of L. major were produced in expression vectors and purified. Aldehyde functionalized α-AL2O3 were conjugated to hydrazine-modified CPA/CPB by a chemical bond was confirmed by Fourier-transform infrared spectroscopy (FTIR). The High efficient internalization of α-AL2O3 conjugated CPA/CPB to macrophages was confirmed using a fluorescence microscope and flowcytometry. Induction of the acidic autophagosome and LC3 conversion in macrophages was determined by acridine orange (AO) staining and western blot. Autophagy-activated macrophages were used for CD8+ T cell priming. Cytotoxic activity of the primed CD8+ T cell against L. major infected macrophages was measured using apoptosis assay. α-AL2O3 conjugated CPA/CPB enhances macrophages antigen uptake and increases acidic vacuole formation and LC-3I to LC-3II conversion. Co-culture of autophagy-activated macrophages with CD8+ T cells augmented CD8+ T cells priming and proliferation more than in other study groups. These primed CD8+ T cells induce significant apoptotic death of L. major infected macrophages compared with non-primed CD8+ T cells. α-AL2O3 nanoparticles enhance the cross-presentation of L. major antigens to CD8+ T cells by inducing autophagy. This finding supports the positive role of autophagy and encourages the use of α-AL2O3 in vaccine design.

An alternative pathway to plant cold tolerance in the absence of vacuolar invertase activity.

To cope with cold stress, plants have developed antioxidation strategies combined with osmoprotection by sugars. In potato (Solanum tuberosum) tubers, which are swollen stems, exposure to cold stress induces starch degradation and sucrose synthesis. Vacuolar acid invertase (VInv) activity is a significant part of the cold-induced sweetening (CIS) response, by rapidly cleaving sucrose into hexoses and increasing osmoprotection. To discover alternative plant tissue pathways for coping with cold stress, we produced VInv-knockout lines in two cultivars. Genome editing of VInv in 'Désirée' and 'Brooke' was done using stable and transient expression of CRISPR/Cas9 components, respectively. After storage at 4°C, sugar analysis indicated that the knockout lines showed low levels of CIS and maintained low acid invertase activity in storage. Surprisingly, the tuber parenchyma of vinv lines exhibited significantly reduced lipid peroxidation and reduced H2 O2 levels. Furthermore, whole plants of vinv lines exposed to cold stress without irrigation showed normal vigor, in contrast to WT plants, which wilted. Transcriptome analysis of vinv lines revealed upregulation of an osmoprotectant pathway and ethylene-related genes during cold temperature exposure. Accordingly, higher expression of antioxidant-related genes was detected after exposure to short and long cold storage. Sugar measurements showed an elevation of an alternative pathway in the absence of VInv activity, raising the raffinose pathway with increasing levels of myo-inositol content as a cold tolerance response.

Calciprotein Particles Cause Physiologically Significant Pro-Inflammatory Response in Endothelial Cells and Systemic Circulation

Calciprotein particles (CPPs) represent an inherent mineral buffering system responsible for the scavenging of excessive Ca2+ and PO43- ions in order to prevent extraskeletal calcification, although contributing to the development of endothelial dysfunction during the circulation in the bloodstream. Here, we performed label-free proteomic profiling to identify the functional consequences of CPP internalisation by endothelial cells (ECs) and found molecular signatures of significant disturbances in mitochondrial and lysosomal physiology, including oxidative stress, vacuolar acidification, accelerated proteolysis, Ca2+ cytosolic elevation, and mitochondrial outer membrane permeabilisation. Incubation of intact ECs with conditioned medium from CPP-treated ECs caused their pro-inflammatory activation manifested by vascular cell adhesion molecule 1 (VCAM1) and intercellular adhesion molecule 1 (ICAM1) upregulation and elevated release of interleukin (IL)-6, IL-8, and monocyte chemoattractant protein-1/ C-C motif ligand 2 (MCP-1/CCL2). Among the blood cells, monocytes were exclusively responsible for CPP internalisation. As compared to the co-incubation of donor blood with CPPs in the flow culture system, intravenous administration of CPPs to Wistar rats caused a considerably higher production of chemokines, indicating the major role of monocytes in CPP-triggered inflammation. Upregulation of sICAM-1 and IL-8 also suggested a notable contribution of endothelial dysfunction to systemic inflammatory response after CPP injections. Collectively, our results demonstrate the pathophysiological significance of CPPs and highlight the need for the development of anti-CPP therapies.

Subunit C of V-ATPase-VmaC Is Required for Hyphal Growth and Conidiation in A. fumigatus by Affecting Vacuolar Calcium Homeostasis and Cell Wall Integration.

Aspergillus fumigatus is a widespread airborne fungal pathogen in humans. However, the functional genes in A. fumigatus that may contribute to its pathogenesis have not yet been fully identified. Vacuolar H+-ATPase is universal in eukaryotic organisms but exhibits specific roles in various species. Here, we identified VmaC as a putative subunit of vacuolar H+-ATPase in A. fumigatus that is widely conserved through evolution. The C-terminal hydrophobic domain of VmaC plays a critical role in its vacuolar localization and growth and conidiation. Deletion or turn-off of VmaC encoding gene-AfvmaC expression is not lethal but leads to a very sick and tiny colony phenotype, which is different from that of yeast with conditional ScvmaC defects. Furthermore, we found that AfvmaC not only participates in maintaining calcium homeostasis and vacuolar acidity but is also involved in cell wall integration pathway regulation, highlighting the importance of the vacuole as a storage organelle associated with many aspects of cellular homeostasis. This study indicates that fungal VmaC is relatively conserved. When compared to that in model yeasts, VmaC in A. fumigatus is required for hyphal growth and conidiation, suggesting that specific motifs in VmaC might be functioned in Aspergilli.

Pseudomonas aeruginosa Can Diversify after Host Cell Invasion to Establish Multiple Intracellular Niches.

Within epithelial cells, Pseudomonas aeruginosa depends on its type III secretion system (T3SS) to escape vacuoles and replicate rapidly in the cytosol. Previously, it was assumed that intracellular subpopulations remaining T3SS-negative (and therefore in vacuoles) were destined for degradation in lysosomes, supported by data showing vacuole acidification. Here, we report in both corneal and bronchial human epithelial cells that vacuole-associated bacteria can persist, sometimes in the same cells as cytosolic bacteria. Using a combination of phase-contrast, confocal, and correlative light-electron microscopy (CLEM), we also found they can demonstrate biofilm-associated markers: cdrA and cyclic-di-GMP (c-di-GMP). Vacuolar-associated bacteria, but not their cytosolic counterparts, tolerated the cell-permeable antibiotic ofloxacin. Surprisingly, use of mutants showed that both persistence in vacuoles and ofloxacin tolerance were independent of the biofilm-associated protein CdrA or exopolysaccharides (Psl, Pel, alginate). A T3SS mutant (ΔexsA) unable to escape vacuoles phenocopied vacuole-associated subpopulations in wild-type PAO1-infected cells, with results revealing that epithelial cell death depended upon bacterial viability. Intravital confocal imaging of infected mouse corneas confirmed that P. aeruginosa formed similar intracellular subpopulations within epithelial cells in vivo. Together, these results show that P. aeruginosa differs from other pathogens by diversifying intracellularly into vacuolar and cytosolic subpopulations that both contribute to pathogenesis. Their different gene expression and behavior (e.g., rapid replication versus slow replication/persistence) suggest cooperation favoring both short- and long-term interests and another potential pathway to treatment failure. How this intracellular diversification relates to previously described "acute versus chronic" virulence gene-expression phenotypes of P. aeruginosa remains to be determined. IMPORTANCE Pseudomonas aeruginosa can cause sight- and life-threatening opportunistic infections, and its evolving antibiotic resistance is a growing concern. Most P. aeruginosa strains can invade host cells, presenting a challenge to therapies that do not penetrate host cell membranes. Previously, we showed that the P. aeruginosa type III secretion system (T3SS) plays a pivotal role in survival within epithelial cells, allowing escape from vacuoles, rapid replication in the cytoplasm, and suppression of host cell death. Here, we report the discovery of a novel T3SS-negative subpopulation of intracellular P. aeruginosa within epithelial cells that persist in vacuoles rather than the cytoplasm and that tolerate a cell-permeable antibiotic (ofloxacin) that is able to kill cytosolic bacteria. Classical biofilm-associated markers, although demonstrated by this subpopulation, are not required for vacuolar persistence or antibiotic tolerance. These findings advance our understanding of how P. aeruginosa hijacks host cells, showing that it diversifies into multiple populations with T3SS-negative members enabling persistence while rapid replication is accomplished by more vulnerable T3SS-positive siblings. Intracellular P. aeruginosa persisting and tolerating antibiotics independently of the T3SS or biofilm-associated factors could present additional challenges to development of more effective therapeutics.

The interdependent transport of yeast vacuole Ca2+ and H+ and the role of phosphatidylinositol 3,5-bisphosphate

Yeast vacuoles are acidified by the v-type H+-ATPase (V-ATPase) that is comprised of the membrane embedded VO complex and the soluble cytoplasmic V1 complex. The assembly of the V1-VO holoenzyme on the vacuole is stabilized in part through interactions between the VO a-subunit ortholog Vph1 and the lipid phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2). PI(3,5)P2 also affects vacuolar Ca2+ release through the channel Yvc1 and uptake through the Ca2+ pump Pmc1. Here, we asked if H+ and Ca2+ transport activities were connected through PI(3,5)P2. We found that overproduction of PI(3,5)P2 by the hyperactive fab1T2250A mutant augmented vacuole acidification, whereas the kinase-inactive fab1EEE mutant attenuated the formation of a H+ gradient. Separately, we tested the effects of excess Ca2+ on vacuole acidification. Adding micromolar Ca2+ blocked vacuole acidification, whereas chelating Ca2+ accelerated acidification. The effect of adding Ca2+ on acidification was eliminated when the Ca2+/H+ antiporter Vcx1 was absent, indicating that the vacuolar H+ gradient can collapse during Ca2+ stress through Vcx1 activity. This, however, was independent of PI(3,5)P2, suggesting that PI(3,5)P2 plays a role in submicromolar Ca2+ flux but not under Ca2+ shock. To see if the link between Ca2+ and H+ transport was bidirectional, we examined Ca2+ transport when vacuole acidification was inhibited. We found that Ca2+ transport was inhibited by halting V-ATPase activity with Bafilomycin or neutralizing vacuolar pH with chloroquine. Together, these data show that Ca2+ transport and V-ATPase efficacy are connected but not necessarily through PI(3,5)P2.

Sucrose Facilitates Rhizome Development of Perennial Rice (Oryza longistaminata)

Compared with annual crops, perennial crops with longer growing seasons and deeper root systems can fix more sunlight energy, and have advantages in reducing soil erosion and saving water, fertilizer and pesticide inputs. Rice is one of the most important food crops in the world. Perennial rice can be of great significance for protecting the ecological environment and coping with the shortage of young farmers due to urbanization. Oryza longistaminata (OL) is a rhizomatous wild rice with an AA genome and has strong biotic and abiotic resistances. The AA genome makes OL easy to cross with cultivated rice, thus making it an ideal donor material for perennial rice breeding. Sucrose plays an important role in the development and growth of plants. In this study, OL seedlings were cultured in medium with different concentrations of sucrose, and it was found that sucrose of appropriate concentrations can promote the sprout of basal axillary buds and the subsequent development of rhizomes. In order to explore the molecular mechanism, comparative transcriptome analysis was carried out with OL cultured under two concentrations of sucrose, 20 g/L and 100 g/L, respectively. The results showed that the boost of sucrose to rhizome elongation may be due to the glucose and fructose, hydrolyzed from the absorbed sucrose by vacuolar acid invertase. In addition, the consequent increased osmotic pressure of the cells would promote water absorption, which is benefit for the cell elongation, eventually causing the rhizome elongation. These results may provide a reference for elucidating the regulatory mechanism of sucrose on the rhizome development of OL.