Molecular and phenotypic characterization of a dpp3Δ knocked-out mutant, lacking a pyrophosphate phosphatase, in the opportunistic yeast Candida lusitaniae : a study on the interaction of yeasts with the host

Candida lusitaniae est une levure pathogène opportuniste émergente, responsable d’infections sévères chez les sujets immunodéprimés, et constitue un important modèle d’étude fonctionnelle de gènes par génétique inverse. Cette levure est souvent associée à des résistances aux antifongiques, d’où la nécessité de mieux comprendre l’interaction entre l’hôte et le pathogène, afin d’identifier de nouvelles cibles thérapeutiques potentielles. Les molécules signal sont décrites comme modulatrices de cette interaction. Au cours de ce travail, nous avons créé un mutant dpp3Δ, inactivé pour une pyrophosphate phosphatase, chez C. lusitaniae. La mutation a pour conséquence de modifier le taux secrété de PEA/tyrosol, des alcools aromatiques récemment décrits comme molécules signal chez les espèces Candida. Les levures du mutant dpp3Δ présentent aussi un défaut de pseudo-filamentation et de reproduction sexuée. Comparé à la souche sauvage, le mutant dpp3Δ module différemment la réponse macrophagique, notamment la production de NO et ROS, ainsi que la sécrétion de TNF-α et d’IL-10. Curieusement, la sécrétion d’IL-10 est modulée par les levures même sans contact physique avec les macrophages. Par ailleurs, nous avons observé des effets du traitement des levures par le PEA ou le tyrosol sur la modification des réponses immunes macrophagiques. Enfin, nous avons montré que le gène DPP3 chez C. lusitaniae était un facteur de virulence essentiel dans des modèles murins d’infection, et qu’il augmentait le taux de TNF-α secrété et la colonisation des cerveaux.C. lusitaniae, an emerging Candida species often associated with antifungal resistance, is an important model to study gene function by reverse genetics. Understanding the host-pathogen interaction helps identifying new virulence factors and potentially new antifungal targets. Signaling molecules are often reported as modulators of this interaction. Here we created a dpp3Δ knocked-out mutant, lacking a pyrophosphate phosphatase, in C. lusitaniae. This mutation modified the ratio of secreted PEA/tyrosol, two newly reported signaling molecules in Candida species. Colony morphology of yeast cells was also altered as yeasts had a defect in pseudo-filamentation, and mating capacity was severely reduced. Compared to the wild-type strain, the dpp3Δ knocked-out mutant differently affected NO and ROS production by macrophages as well as TNF-α and IL-10 secretion. Interestingly IL-10 secretion was found to be modulated by C. lusitaniae without the need of a physical contact with the phagocytes. Moreover we elucidated the effects of PEA and tyrosol on yeast cells leading to modulations in macrophage immune responses. At last, the DPP3 gene was found to be an essential pathogenicity factor in mice models, leading to an increase of TNF-α secretion and brain colonization

Molecular and phenotypic characterization of a dpp3Δ knocked-out mutant, lacking a pyrophosphate phosphatase, in the opportunistic yeast Candida lusitaniae : a study on the interaction of yeasts with the host

Candida lusitaniae est une levure pathogène opportuniste émergente, responsable d’infections sévères chez les sujets immunodéprimés, et constitue un important modèle d’étude fonctionnelle de gènes par génétique inverse. Cette levure est souvent associée à des résistances aux antifongiques, d’où la nécessité de mieux comprendre l’interaction entre l’hôte et le pathogène, afin d’identifier de nouvelles cibles thérapeutiques potentielles. Les molécules signal sont décrites comme modulatrices de cette interaction. Au cours de ce travail, nous avons créé un mutant dpp3Δ, inactivé pour une pyrophosphate phosphatase, chez C. lusitaniae. La mutation a pour conséquence de modifier le taux secrété de PEA/tyrosol, des alcools aromatiques récemment décrits comme molécules signal chez les espèces Candida. Les levures du mutant dpp3Δ présentent aussi un défaut de pseudo-filamentation et de reproduction sexuée. Comparé à la souche sauvage, le mutant dpp3Δ module différemment la réponse macrophagique, notamment la production de NO et ROS, ainsi que la sécrétion de TNF-α et d’IL-10. Curieusement, la sécrétion d’IL-10 est modulée par les levures même sans contact physique avec les macrophages. Par ailleurs, nous avons observé des effets du traitement des levures par le PEA ou le tyrosol sur la modification des réponses immunes macrophagiques. Enfin, nous avons montré que le gène DPP3 chez C. lusitaniae était un facteur de virulence essentiel dans des modèles murins d’infection, et qu’il augmentait le taux de TNF-α secrété et la colonisation des cerveaux.C. lusitaniae, an emerging Candida species often associated with antifungal resistance, is an important model to study gene function by reverse genetics. Understanding the host-pathogen interaction helps identifying new virulence factors and potentially new antifungal targets. Signaling molecules are often reported as modulators of this interaction. Here we created a dpp3Δ knocked-out mutant, lacking a pyrophosphate phosphatase, in C. lusitaniae. This mutation modified the ratio of secreted PEA/tyrosol, two newly reported signaling molecules in Candida species. Colony morphology of yeast cells was also altered as yeasts had a defect in pseudo-filamentation, and mating capacity was severely reduced. Compared to the wild-type strain, the dpp3Δ knocked-out mutant differently affected NO and ROS production by macrophages as well as TNF-α and IL-10 secretion. Interestingly IL-10 secretion was found to be modulated by C. lusitaniae without the need of a physical contact with the phagocytes. Moreover we elucidated the effects of PEA and tyrosol on yeast cells leading to modulations in macrophage immune responses. At last, the DPP3 gene was found to be an essential pathogenicity factor in mice models, leading to an increase of TNF-α secretion and brain colonization

Host-pathogen interaction and signaling molecule secretion are modified in the dpp3 knockout mutant of Candida lusitaniae.

International audienceCandida lusitaniae is an emerging opportunistic yeast and an attractive model to discover new virulence factors in Candida species by reverse genetics. Our goal was to create a dpp3Δ knockout mutant and to characterize the effects of this gene inactivation on yeast in vitro and in vivo interaction with the host. The secretion of two signaling molecules in Candida species, phenethyl alcohol (PEA) and tyrosol, but not of farnesol was surprisingly altered in the dpp3Δ knockout mutant. NO and reactive oxygen species (ROS) production as well as tumor necrosis factor alpha (TNF-α) and interleukin 10 (IL-10) secretion were also modified in macrophages infected with this mutant. Interestingly, we found that the wild-type (WT) strain induced an increase in IL-10 secretion by zymosan-activated macrophages without the need for physical contact, whereas the dpp3Δ knockout mutant lost this ability. We further showed a striking role of PEA and tyrosol in this modulation. Last, the DPP3 gene was found to be an essential contributor to virulence in mice models, leading to an increase in TNF-α secretion and brain colonization. Although reinsertion of a WT DPP3 copy in the dpp3Δ knockout mutant was not sufficient to restore the WT phenotypes in vitro, it allowed a restoration of those observed in vivo. These data support the hypothesis that some of the phenotypes observed following DPP3 gene inactivation may be directly dependent on DPP3, while others may be the indirect consequence of another genetic modification that systematically arises when the DPP3 gene is inactivated

J Fungi (Basel)

is an emerging opportunistic pathogenic yeast capable of shifting from yeast to pseudohyphae form, and it is one of the few species with the ability to reproduce sexually. In this study, we showed that a Δ mutant, inactivated for a putative pyrophosphatase, is impaired in cell separation, pseudohyphal growth and mating. The defective phenotypes were not restored after the reconstruction of a wild-type locus, reinforcing the hypothesis of the presence of an additional mutation that we suspected in our previous study. Genetic crosses and genome sequencing identified an additional mutation in , encoding a subunit of the mediator complex that functions as a general transcriptional co-activator in Eukaryotes. We confirmed that inactivation of was responsible for the defective phenotypes by rescuing the Δ mutant with a wild-type copy of and constructing a Δ knockout mutant that mimics the phenotypes of Δ in vitro. Proteomic analyses revealed the biological processes under the control of Med15 and involved in hyphal growth, cell separation and mating. This is the first description of the functions of in the regulation of hyphal growth, cell separation and mating, and the pathways involved in

Pycnosomes: Condensed Endosomal Structures Secreted by Dictyostelium Amoebae.

Dictyostelium discoideum has been used largely as a model organism to study the organization and function of the endocytic pathway. Here we describe dense structures present in D. discoideum endocytic compartments, which we named pycnosomes. Pycnosomes are constitutively secreted in the extracellular medium, from which they can be recovered by differential centrifugation. We identified the most abundant protein present in secreted pycnosomes, that we designated SctA. SctA defines a new family of proteins with four members in D. discoideum, and homologous proteins in other protists and eumetazoa. We developed a monoclonal antibody specific for SctA and used it to further characterize secreted and intracellular pycnosomes. Within cells, immunofluorescence as well as electron microscopy identified pycnosomes as SctA-enriched dense structures in the lumen of endocytic compartments. Pycnosomes are occasionally seen in continuity with intra-endosomal membranes, particularly in U18666A-treated cells where intraluminal budding is highly enhanced. While the exact nature, origin and cellular function of pycnosomes remain to be established, this study provides a first description of these structures as well as a characterization of reagents that can be used for further studies

MitoNEET-dependent formation of intermitochondrial junctions

MitoNEET (mNEET) is a dimeric mitochondrial outer membrane protein implicated in many facets of human pathophysiology, notably diabetes and cancer, but its molecular function remains poorly characterized. In this study, we generated and analyzed mNEET KO cells and found that in these cells the mitochondrial network was disturbed. Analysis of 3D-EM reconstructions and of thin sections revealed that genetic inactivation of mNEET did not affect the size of mitochondria but that the frequency of intermitochondrial junctions was reduced. Loss of mNEET decreased cellular respiration, because of a reduction in the total cellular mitochondrial volume, suggesting that intermitochondrial contacts stabilize individual mitochondria. Reexpression of mNEET in mNEET KO cells restored the WT morphology of the mitochondrial network, and reexpression of a mutant mNEET resistant to oxidative stress increased in addition the resistance of the mitochondrial network to H2O2-induced fragmentation. Finally, overexpression of mNEET increased strongly intermitochondrial contacts and resulted in the clustering of mitochondria. Our results suggest that mNEET plays a specific role in the formation of intermitochondrial junctions and thus participates in the adaptation of cells to physiological changes and to the control of mitochondrial homeostasis

Water productivity of barley crop under laser land leveling technique and minimum tillage

ABSTRACTLaser land leveling is an important method that helps to improve the spatial distribution of irrigation water and fertilizers applications, subsequently reduce water, nutrient, and energy inputs to agriculture and contributes to increasing productivity. Thus, the aim of the study was to improve the productivity of the barley crop grown in sandy lands under conditions of water scarcity and the negative impact of climate change in Egypt by using laser leveling and the minimum tillage method. Two experiments were conducted during the 2020/2021 and 2021/2022 seasons at the Nubariya farm, Buhaira Governorate, Egypt to study the effects of laser leveling and minimum tillage (zero – tillage, 10 cm, 20 cm, and 30 cm) affecting the distribution of soil moisture, water stress, effectiveness of water application, yield characteristics, water productivity, and some quality parameters of the barley crop. The statistical analysis’ findings revealed a considerable influence of both laser soil leveling and minimum depth of plowing on productivity, water productivity and quality properties of barley crop. Laser leveling with a plowing depth of 10 cm gave the most favorable values of the soil moisture content at the root-zone as well as better grain yield and water productivity in addition to improve the quality properties of barley. The grain yield has improved by 12.65% and 10.41%, while water productivity has increased by 12.75% and 10.06% during the seasons 2020/2021 and 2021/2022, respectively. This increase is likely the result of improving soil moisture distribution and increasing irrigation application efficiency, which resulted in less water stress in the root zone and subsequently increased yield, water productivity and quality properties of barley during the two growing seasons. Generally, the application of laser land leveling as eco-friendly practice will help in sustaining barley productivity in Egypt particularly in the sandy soil regions. According to this study, laser-assisted precision field leveling has the potential to improve grain yield and crop establishment, water productivity and barley quality properties in addition to achievement of the highest net income for farmers

Deletion of the uracil permease gene confers cross-resistance to 5-fluorouracil and azoles in Candida lusitaniae and highlights antagonistic interaction between fluorinated nucleotides and fluconazole.

International audienceWe characterized two additional membrane transporters (Fur4p and Dal4p) of the nucleobase cation symporter 1 (NCS1) family involved in the uptake transport of pyrimidines and related molecules in the opportunistic pathogenic yeast Candida lusitaniae. Simple and multiple null mutants were constructed by gene deletion and genetic crosses. The function of each transporter was characterized by supplementation experiments, and the kinetic parameters of the uptake transport of uracil were measured using radiolabeled substrate. Fur4p specifically transports uracil and 5-fluorouracil. Dal4p is very close to Fur4p and transports allantoin (glyoxyldiureide). Deletion of the FUR4 gene confers resistance to 5-fluorouracil as well as cross-resistance to triazoles and imidazole antifungals when they are used simultaneously with 5-fluorouracil. However, the nucleobase transporters are not involved in azole uptake. Only fluorinated pyrimidines, not pyrimidines themselves, are able to promote cross-resistance to azoles by both the salvage and the de novo pathway of pyrimidine synthesis. A reinterpretation of the data previously obtained led us to show that subinhibitory doses of 5-fluorocytosine, 5-fluorouracil, and 5-fluorouridine also were able to trigger resistance to fluconazole in susceptible wild-type strains of C. lusitaniae and of different Candida species. Our results suggest that intracellular fluorinated nucleotides play a key role in azole resistance, either by preventing azoles from targeting the lanosterol 14-alpha-demethylase or its catalytic site or by acting as a molecular switch for the triggering of efflux transport

LrrKa, a kinase with leucine-rich repeats links folate sensing with Kil2 activity and intracellular killing

Phagocytic cells ingest bacteria by phagocytosis and kill them efficiently inside phagolysosomes. The molecular mechanisms involved in intracellular killing and their regulation are complex and still incompletely understood. Dictyostelium discoideum has been used as a model to discover and to study new gene products involved in intracellular killing of ingested bacteria. In this study, we performed random mutagenesis of Dictyostelium cells, and isolated a mutant defective for growth on bacteria. This mutant is characterized by the genetic inactivation of the lrrkA gene, which encodes a protein with a kinase domain and leucine-rich repeats. LrrkA knockout (KO) cells kill ingested K. pneumoniae bacteria inefficiently. This defect is not additive to the killing defect observed in kil2 KO cells, suggesting that the function of Kil2 is partially controlled by LrrkA. Indeed, lrrkA KO cells exhibit a phenotype similar to that of kil2 KO cells: intraphagosomal proteolysis is inefficient and both intraphagosomal killing and proteolysis are restored upon exogenous supplementation with magnesium ions. Bacterially secreted folate stimulates intracellular killing in Dictyostelium cells, but this stimulation is lost in cells with genetic inactivation of kil2, lrrkA or far1. Together these results indicate that the stimulation of intracellular killing by folate involves Far1 (the cell surface receptor for folate), LrrkA and Kil2. This study is the first identification of a signaling pathway regulating intraphagosomal bacterial killing in Dictyostelium cells