Coordinated host-pathogen transcriptional dynamics revealed using sorted subpopulations and single macrophages infected with Candida albicans.
Ontology highlight
ABSTRACT:
SUBMITTER: Munoz JF
PROVIDER: S-EPMC6453965 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Purpose: The purpose of this study was to simulataneously examine the host and fungal pathogen transcriptional profiles of four distinct infection fates during macrophage and Candida albicans interactions Methods: Membrane stained (Deep Red),primary, bone marrow derived, murine macrophages and Candida albicans expressing GFP and mCherry were exposed to each other over a four hour time course. Samples were collected at 0, 1, 2 and 4 hours and sorted for four infection subpopulations: 1. Macrophages which phagocytosed live C. albicans (GFP+ /mCherry+ /Deep Red +), 2. Macrophages which phagocytosed dead C. albicans (GFP- /mCherry+ /Deep Red +), 3. Uninfected macrophages(GFP- /mCherry- /Deep Red +) and 4. Unengulfed C. albicans (GFP+ /mCherry + /Deep Red -). Unexposed controls were also collected for some time points (i.e. macrophages never exposed to C. albicans and C. albicans never exposed to macrophages). Single macrophages infected with live or dead C. albicans were also sorted. Smart-seq2 was used to create libraries for both infection subpopulation and single, infected cell samples that were sequenced on Illumina’s Miseqand Nextseq. Basic quality assessment of Illumina reads and sample demultiplexing was done with Picard version 1.107 and Trimmomatic. Samples profiling exclusively the mouse transcriptional response were aligned to the mouse transcriptome generated from the v. Dec. 2011 GRCm38/mm10 and a collection of mouse rRNA sequences from the UCSC genome website. Samples profiling exclusively the yeast transcriptional response were aligned to the C. albicans transcriptome strain SC5314 version A21-s02-m09-r10 downloaded from Candida Genome Database. Samples containing both macrophages and C. albicans were aligned to a “composite transcriptome” made by combining the mouse transcriptome and C. albicans transcriptomes described above and alignment was done via BWA (version 0.7.10-r789.) Multi-reads (reads that aligned to both host and pathogen transcripts) were discarded. Then, each host or pathogen sample file were aligned to its corresponding reference using Bowtie2 and RSEM (v.1.2.21). Transcript abundance was estimated using transcripts per million (TPM). For subpopulation samples, TPM was calculated using edgeR, all as implemented in the Trinity package version 2.1.. Genes were considered differentially expressed only if they had a 4-fold change difference (> 4 FC) in TPM values and a false discovery rate below or equal to 0.001 (FDR < 0.001), unless specified otherwise. For single macrophages infected with C. albicans, samples were aligned to the combined transcriptome as described above and RSEM was used to calculate TPM. Results: We were able to simultaneously measure the host and fungal pathogen transcriptional profiles of four distinct infection fates during macrophage and Candida albicans interactions Conclusions: Our study represents an analysis of both distinct infection populations of macrophages and fungus.
2018-03-12 | GSE111731 | GEO
Project description:Revealing parallel host-pathogen transcriptional dynamics using sorted subpopulations and single, Candida albicans infected macrophages
| PRJNA437988 | ENA
Project description:The fungal pathogen Candida albicans causes macrophage death and escapes, but the molecular mechanisms remained unknown. Here we used live-cell imaging to monitor the interaction of C. albicans with macrophages and show that C. albicans kills macrophages in two temporally and mechanistically distinct phases. Early upon phagocytosis, C. albicans triggers pyroptosis, a proinflammatory macrophage death. Pyroptosis is controlled by the developmental yeast-to-hypha transition of Candida. When pyroptosis is inactivated, wild-type C. albicans hyphae cause significantly less macrophage killing for up to 8 h postphagocytosis. After the first 8 h, a second macrophage-killing phase is initiated. This second phase depends on robust hyphal formation but is mechanistically distinct from pyroptosis. The transcriptional regulator Mediator is necessary for morphogenesis of C. albicans in macrophages and the establishment of the wild-type surface architecture of hyphae that together mediate activation of macrophage cell death. Our data suggest that the defects of the Mediator mutants in causing macrophage death are caused, at least in part, by reduced activation of pyroptosis. A Mediator mutant that forms hyphae of apparently wild-type morphology but is defective in triggering early macrophage death shows a breakdown of cell surface architecture and reduced exposed 1,3 ?-glucan in hyphae. Our report shows how Candida uses host and pathogen pathways for macrophage killing. The current model of mechanical piercing of macrophages by C. albicans hyphae should be revised to include activation of pyroptosis by hyphae as an important mechanism mediating macrophage cell death upon C. albicans infection. IMPORTANCE Upon phagocytosis by macrophages, Candida albicans can transition to the hyphal form, which causes macrophage death and enables fungal escape. The current model is that the highly polarized growth of hyphae results in macrophage piercing. This model is challenged by recent reports of C. albicans mutants that form hyphae of wild-type morphology but are defective in killing macrophages. We show that C. albicans causes macrophage cell death by at least two mechanisms. Phase 1 killing (first 6 to 8 h) depends on the activation of the pyroptotic programmed host cell death by fungal hyphae. Phase 2 (up to 24 h) is rapid and depends on robust hyphal formation but is independent of pyroptosis. Our data provide a new model for how the interplay between fungal morphogenesis and activation of a host cell death pathway mediates macrophage killing by C. albicans hyphae.
| S-EPMC3977349 | biostudies-literature
Project description:Glycolysis is a metabolic pathway that is central to the assimilation of carbon for either respiration or fermentation and therefore is critical for the growth of all organisms. Consequently, glycolytic transcriptional regulation is important for the metabolic flexibility of pathogens in their attempts to colonize diverse niches. We investigated the transcriptional control of carbohydrate metabolism in the human fungal pathogen Candida albicans and identified two factors, Tye7p and Gal4p, as key regulators of glycolysis. When respiration was inhibited or oxygen was limited, a gal4tye7 C. albicans strain showed a severe growth defect when cultured on glucose, fructose or mannose as carbon sources. The gal4tye7 strain displayed attenuated virulence in both Galleria and mouse models as well, supporting the connection between pathogenicity and metabolism. Chromatin immunoprecipitation coupled with microarray analysis (ChIP-CHIP) and transcription profiling revealed that Tye7p bound the promoter sequences of the glycolytic genes and activated their expression during growth on either fermentable or non-fermentable carbon sources. Gal4p also bound the glycolytic promoter sequences and activated the genes although to a lesser extent than Tye7p. Intriguingly, binding and activation by Gal4p was carbon source-dependent and much stronger during growth on media containing fermentable sugars than on glycerol. Furthermore, Tye7p and Gal4p were responsible for the complete induction of the glycolytic genes under hypoxic growth conditions. Tye7p and Gal4p also regulated unique sets of carbohydrate metabolic genes; Tye7p bound and activated genes involved in trehalose, glycogen, and glycerol metabolism, while Gal4p regulated the pyruvate dehydrogenase complex. This suggests that Tye7p represents the key transcriptional regulator of carbohydrate metabolism in C. albicans and Gal4p provides a carbon source-dependent fine-tuning of gene expression while regulating the metabolic flux between respiration and fermentation pathways.
| S-EPMC2749448 | biostudies-literature
Project description:BackgroundPeptidorhamnomannan is a glycoconjugate that consists of a peptide chain substituted by O- and N-linked glycans, present on the cell surface of Lomentospora prolificans, a saprophytic fungus which is widely distributed in regions with temperate climates. O-linked oligosaccharides from peptidorhamnomannan isolated from Lomentospora prolificans conidia are recognized by macrophages mediating macrophage - conidia interaction. In this work, peptidorhamnomannan was isolated from L. prolificans mycelium cell wall and its role in macrophage - Candida albicans interaction was evaluated.ResultsPurified peptidorhamnomannan inhibits the reactivity of rabbit immune sera to mycelial and conidia forms of L. prolificans, indicating that this glycoconjugate is exposed on the fungal surface and can mediate interaction with host immune cells. We demonstrated that peptidorhamnomannan leads to TNF-α production in J774 macrophages for 1, 2 and 3 h of incubation, suggesting that this glycoconjugate may have a beneficial role in the response to fungal infections. In order to confirm this possibility, the effect of peptidorhamnomannan on the macrophage - C. albicans interaction was evaluated. Macrophages treated with peptidorhamnomannan led to a lower fungal survival, suggesting that peptidorhamnomannan induces an increased fungicidal activity in macrophages. Furthermore, TNF-α levels were measured in supernatants after macrophage - C. albicans interaction for 1, 2 and 3 h. Peptidorhamnomannan treatment led to a higher TNF-α production at the beginning of the interaction. However, the release of TNF-α was not maintained after 1 h of incubation. Besides, peptidorhamnomannan did not show any inhibitory or fungicidal effect in C. albicans when used at 100 μg/ml but it was able to kill C. albicans at a concentration of 400 μg/ml.ConclusionWe suggest that peptidorhamnomannan acts as a molecular pattern on the invading pathogen, promotes TNF-α production and, thus, increases macrophage fungicidal activity against Candida albicans.
| S-EPMC7412847 | biostudies-literature
Project description:The ability of Candida albicans, an important human fungal pathogen, to develop filamentous forms is a crucial determinant for host invasion and virulence. While hypoxia is one of the predominant host cues that promote C. albicans filamentous growth, the regulatory circuits that link oxygen availability to filamentation remain poorly characterized. We have undertaken a genetic screen and identified the two transcription factors Ahr1 and Tye7 as central regulators of the hypoxic filamentation. Both ahr1 and tye7 mutants exhibited a hyperfilamentous phenotype specifically under an oxygen-depleted environment suggesting that these transcription factors act as negative regulators of hypoxic filamentation. By combining microarray and ChIP-chip analyses, we have characterized the set of genes that are directly modulated by Ahr1 and Tye7. We found that both Ahr1 and Tye7 modulate a distinct set of genes and biological processes. Our genetic epistasis analysis supports our genomic finding and suggests that Ahr1 and Tye7 act independently to modulate hyphal growth in response to hypoxia. Furthermore, our genetic interaction experiments uncovered that Ahr1 and Tye7 repress the hypoxic filamentation via the Efg1 and Ras1/Cyr1 pathways, respectively. This study yielded a new and an unprecedented insight into the oxygen-sensitive regulatory circuit that control morphogenesis in a fungal pathogen.
| S-EPMC8807691 | biostudies-literature
Project description:Neutrophils are the most abundant white blood cell in the body and are key participants in the defense against fungal infections. Fungal infections occur often in patients with cirrhosis and are associated with increased 30-day and 90-day mortality. Previous studies have shown that specific neutrophil functions are abnormal in patients with cirrhosis, although the extent of neutrophil dysfunction is not well understood. We tested the ability of neutrophils from 21 hospitalized patients with cirrhosis and 23 healthy control patients to kill Candida albicans, a common fungal pathogen in patients with cirrhosis. Using an assay, we also measured the ability of neutrophils to coordinate multicellular, synchronized control of C. albicans hyphae through a process known as swarming. We found that neutrophils from patients with cirrhosis have significantly decreased fungicidal capacity compared with healthy control neutrophils (53% vs. 74%, P < 0.0001) and diminished ability to control hyphal growth normalized as a ratio to healthy control (0.22 vs. 0.65, P < 0.0001). Moreover, serum from patients with cirrhosis decreases the ability of healthy control neutrophils to kill C. albicans (from 60% to 41%, P < 0.003). Circulating concentration of the inflammatory cytokines tumor necrosis factor α, interleukin-6, and interleukin-8 were found to be significantly elevated in patients with cirrhosis compared to healthy controls. Following pretreatment with granulocyte-colony stimulating factor and granulocyte-macrophage colony-stimulating factor, neutrophil function was restored to almost that of healthy controls. Conclusion: Our data establish profound neutrophil dysfunction against, and altered swarming to, C. albicans in patients with cirrhosis. This dysfunction can be partially reversed with cytokine augmentation ex vivo.
| S-EPMC7917271 | biostudies-literature
Project description:The release of fungal cells following macrophage phagocytosis, called non-lytic expulsion, is reported for several fungal pathogens. On one hand, non-lytic expulsion may benefit the fungus in escaping the microbicidal environment of the phagosome. On the other hand, the macrophage could profit in terms of avoiding its own lysis and being able to undergo proliferation. To analyse the causes of non-lytic expulsion and the relevance of macrophage proliferation in the macrophage-Candida albicans interaction, we employ Evolutionary Game Theory and dynamic optimization in a sequential manner. We establish a game-theoretical model describing the different strategies of the two players after phagocytosis. Depending on the parameter values, we find four different Nash equilibria and determine the influence of the systems state of the host upon the game. As our Nash equilibria are a direct consequence of the model parameterization, we can depict several biological scenarios. A parameter region, where the host response is robust against the fungal infection, is determined. We further apply dynamic optimization to analyse whether macrophage mitosis is relevant in the host-pathogen interaction of macrophages and C. albicans For this, we study the population dynamics of the macrophage-C. albicans interactions and the corresponding optimal controls for the macrophages, indicating the best macrophage strategy of switching from proliferation to attacking fungal cells.
| S-EPMC5550964 | biostudies-literature
Project description:There is currently a small number of classes of antifungal drugs, and these drugs are known to target a very limited set of cellular functions. We derived a set of approximately 900 nonessential, transactivator-defective disruption strains from the tetracycline-regulated GRACE collection of strains of the fungal pathogen Candida albicans This strain set was screened against classic antifungal drugs to identify gene inactivations that conferred either enhanced sensitivity or increased resistance to the compounds. We examined two azoles, fluconazole and posaconazole; two echinocandins, caspofungin and anidulafungin; and a polyene, amphotericin B. Overall, the chemogenomic profiles within drug classes were highly similar, but there was little overlap between classes, suggesting that the different drug classes interacted with discrete networks of genes in C. albicans We also tested two pyridine amides, designated GPI-LY7 and GPI-C107; these drugs gave very similar profiles that were distinct from those of the echinocandins, azoles, or polyenes, supporting the idea that they target a distinct cellular function. Intriguingly, in cases where these gene sets can be compared to genetic disruptions conferring drug sensitivity in other fungi, we find very little correspondence in genes. Thus, even though the drug targets are the same in the different species, the specific genetic profiles that can lead to drug sensitivity are distinct. This implies that chemogenomic screens of one organism may be poorly predictive of the profiles found in other organisms and that drug sensitivity and resistance profiles can differ significantly among organisms even when the apparent target of the drug is the same.
| S-EPMC5786791 | biostudies-literature
Project description:The genomic plasticity of Candida albicans, a commensal and common opportunistic fungal pathogen, continues to reveal unexpected surprises. Once thought to be asexual, we now know that the organism can generate genetic diversity through several mechanisms, including mating between cells of the opposite or of the same mating type and by a parasexual reduction in chromosome number that can be accompanied by recombination events (2, 12, 14, 53, 77, 115). In addition, dramatic genome changes can appear quite rapidly in mitotic cells propagated in vitro as well as in vivo. The detection of aneuploidy in other fungal pathogens isolated directly from patients (145) and from environmental samples (71) suggests that variations in chromosome organization and copy number are a common mechanism used by pathogenic fungi to rapidly generate diversity in response to stressful growth conditions, including, but not limited to, antifungal drug exposure. Since cancer cells often become polyploid and/or aneuploid, some of the lessons learned from studies of genome plasticity in C. albicans may provide important insights into how these processes occur in higher-eukaryotic cells exposed to stresses such as anticancer drugs.
| S-EPMC2901674 | biostudies-literature