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During pulmonary mucormycosis, inhaled sporangiospores adhere to, germinate, and invade airway epithelial cells to establish infection. We provide evidence that HIF1α plays dual roles in airway epithelial cells during Mucorales infection. We observed an increase in HIF1α protein accumulation and increased expression of many known HIF1α-responsive genes during in vitro infection, indicating that HIF1α signaling is activated by Mucorales infection. Inhibition of HIF1α signaling led to a substantial decrease in the ability of R. delemar to invade cultured airway epithelial cells. Transcriptome analysis revealed that R. delemar infection induces the expression of many pro-inflammatory genes whose expression was significantly reduced by HIF1α inhibition. Importantly, pharmacological inhibition of HIF1α increased survival in a mouse model of pulmonary mucormycosis without reducing fungal burden. These results suggest that HIF1α plays two opposing roles during mucormycosis: one that facilitates the ability of Mucorales to invade the host cells and one that facilitates the ability of the host to mount an innate immune response.
Assuntos
Células Epiteliais , Subunidade alfa do Fator 1 Induzível por Hipóxia , Mucorales , Mucormicose , Animais , Feminino , Humanos , Camundongos , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Perfilação da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Pulmão/microbiologia , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Camundongos Endogâmicos C57BL , Mucorales/metabolismo , Mucorales/genética , Mucormicose/microbiologia , Mucormicose/metabolismo , Mucormicose/imunologia , Transdução de SinaisRESUMO
Candida auris is an emerging nosocomial fungal pathogen associated with life-threatening invasive disease due to its persistent colonization, high level of transmissibility and multi-drug resistance. Aggregative and non-aggregative growth phenotypes for C. auris strains with different biofilm forming abilities, drug susceptibilities and virulence characteristics have been described. Using comprehensive transcriptional analysis we identified key cell surface adhesins that were highly upregulated in the aggregative phenotype during in vitro and in vivo grown biofilms using a mouse model of catheter infection. Phenotypic and functional evaluations of generated null mutants demonstrated crucial roles for the adhesins Als5 and Scf1 in mediating cell-cell adherence, coaggregation and biofilm formation. While individual mutants were largely non-aggregative, in combination cells were able to co-adhere and aggregate, as directly demonstrated by measuring cell adhesion forces using single-cell atomic force spectroscopy. This co-adherence indicates their role as complementary adhesins, which despite their limited similarity, may function redundantly to promote cell-cell interaction and biofilm formation. Functional diversity of cell wall proteins may be a form of regulation that provides the aggregative phenotype of C. auris with flexibility and rapid adaptation to the environment, potentially impacting persistence and virulence.
RESUMO
Candida auris is an emerging nosocomial fungal pathogen associated with life-threatening invasive disease due to its persistent colonization, high level of transmissibility and multi-drug resistance. Aggregative and non-aggregative growth phenotypes for C. auris strains with different biofilm forming abilities, drug susceptibilities and virulence characteristics have been described. Using comprehensive transcriptional analysis we identified key cell surface adhesins that were highly upregulated in the aggregative phenotype during in vitro and in vivo grown biofilms using a mouse model of catheter infection. Phenotypic and functional evaluations of generated null mutants demonstrated crucial roles for the adhesins Als5 and Scf1 in mediating cell-cell adherence, coaggregation and biofilm formation. While individual mutants were largely non-aggregative, in combination cells were able to co-adhere and aggregate, as directly demonstrated by measuring cell adhesion forces using single-cell atomic force spectroscopy. This co-adherence indicates their role as complementary adhesins, which despite their limited similarity, may function redundantly to promote cell-cell interaction and biofilm formation. Functional diversity of cell wall proteins may be a form of regulation that provides the aggregative phenotype of C. auris with flexibility and rapid adaptation to the environment, potentially impacting persistence and virulence.
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Triazole antifungals function as ergosterol biosynthesis inhibitors and are frontline therapy for invasive fungal infections, such as invasive aspergillosis. The primary mechanism of action of triazoles is through the specific inhibition of a cytochrome P450 14-α-sterol demethylase enzyme, Cyp51A/B, resulting in depletion of cellular ergosterol. Here, we uncover a clinically relevant secondary mechanism of action for triazoles within the ergosterol biosynthesis pathway. We provide evidence that triazole-mediated inhibition of Cyp51A/B activity generates sterol intermediate perturbations that are likely decoded by the sterol sensing functions of HMG-CoA reductase and Insulin-Induced Gene orthologs as increased pathway activity. This, in turn, results in negative feedback regulation of HMG-CoA reductase, the rate-limiting step of sterol biosynthesis. We also provide evidence that HMG-CoA reductase sterol sensing domain mutations previously identified as generating resistance in clinical isolates of Aspergillus fumigatus partially disrupt this triazole-induced feedback. Therefore, our data point to a secondary mechanism of action for the triazoles: induction of HMG-CoA reductase negative feedback for downregulation of ergosterol biosynthesis pathway activity. Abrogation of this feedback through acquired mutations in the HMG-CoA reductase sterol sensing domain diminishes triazole antifungal activity against fungal pathogens and underpins HMG-CoA reductase-mediated resistance.
Assuntos
Antifúngicos , Aspergillus fumigatus , Ergosterol , Proteínas Fúngicas , Hidroximetilglutaril-CoA Redutases , Triazóis , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/metabolismo , Aspergillus fumigatus/genética , Antifúngicos/farmacologia , Triazóis/farmacologia , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Ergosterol/metabolismo , Ergosterol/biossíntese , Hidroximetilglutaril-CoA Redutases/metabolismo , Hidroximetilglutaril-CoA Redutases/genética , Aspergilose/tratamento farmacológico , Aspergilose/microbiologia , Farmacorresistência Fúngica/genética , Farmacorresistência Fúngica/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Testes de Sensibilidade Microbiana , Esterol 14-Desmetilase/metabolismo , Esterol 14-Desmetilase/genética , Humanos , MutaçãoRESUMO
IMPORTANCE: In October 2022, Mucorales fungi were listed in the "High Priority Group" on the first-ever list of fungal priority pathogens by the World Health Organization. As the causative agent of mucormycosis, Mucorales have become of great clinical and public health importance with growing mucormycosis numbers, notably with the exponential rise of COVID-19-associated mucormycosis cases. Despite the dire need, there are limited therapeutic options to treat mucormycosis. Our research fills in critical gaps of knowledge about how Mucorales fungi evade the host immune system. Specifically, we offer evidence that Mucorales block nitric oxide production, which is a key mediator and signaling molecule of the mammalian innate immune response to microbial pathogens. Our work offers new insight into immune evasion mechanisms by Mucorales fungi.
Assuntos
Mucorales , Mucormicose , Animais , Humanos , Mucormicose/microbiologia , Óxido Nítrico , Macrófagos , Imunidade Inata , MamíferosRESUMO
RNA modifications, such as methylation, can be detected with Oxford Nanopore Technologies direct RNA sequencing. One commonly used tool for detecting 5-methylcytosine (m5C) modifications is Tombo, which uses an "Alternative Model" to detect putative modifications from a single sample. We examined direct RNA sequencing data from diverse taxa including viruses, bacteria, fungi, and animals. The algorithm consistently identified a m5C at the central position of a GCU motif. However, it also identified a m5C in the same motif in fully unmodified in vitro transcribed RNA, suggesting that this is a frequent false prediction. In the absence of further validation, several published predictions of m5C in a GCU context should be reconsidered, including those from human coronavirus and human cerebral organoid samples.
Assuntos
Algoritmos , RNA , Animais , Humanos , RNA/genética , Metilação , Análise de Sequência de RNARESUMO
Hematophagous ectoparasites, such as ticks, rely on impaired wound healing for skin attachment and blood feeding. Wound healing has been extensively studied through the lens of inflammatory disorders and cancer, but limited attention has been given to arthropod-borne diseases. Here, we used orthogonal approaches combining single-cell RNA sequencing (scRNAseq), flow cytometry, murine genetics, and intravital microscopy to demonstrate how tick extracellular vesicles (EVs) disrupt networks involved in tissue repair. Impairment of EVs through silencing of the SNARE protein vamp33 negatively impacted ectoparasite feeding and survival in three medically relevant tick species, including Ixodes scapularis. Furthermore, I. scapularis EVs affected epidermal γδ T cell frequencies and co-receptor expression, which are essential for keratinocyte function. ScRNAseq analysis of the skin epidermis in wildtype animals exposed to vamp33-deficient ticks revealed a unique cluster of keratinocytes with an overrepresentation of pathways connected to wound healing. This biological circuit was further implicated in arthropod fitness when tick EVs inhibited epithelial proliferation through the disruption of phosphoinositide 3-kinase activity and keratinocyte growth factor levels. Collectively, we uncovered a tick-targeted impairment of tissue repair via the resident γδ T cell-keratinocyte axis, which contributes to ectoparasite feeding.
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RNA modifications, such as méthylation, can be detected with Oxford Nanopore Technologies direct RNA sequencing. One commonly used tool for detecting 5-methylcytosine (m5C) modifications is Tombo, which uses an "Alternative Model" to detect putative modifications from a single sample. We examined direct RNA sequencing data from diverse taxa including virus, bacteria, fungi, and animals. The algorithm consistently identified a 5-methylcytosine at the central position of a GCU motif. However, it also identified a 5-methylcytosine in the same motif in fully unmodified in vitro transcribed RNA, suggesting that this a frequent false prediction. In the absence of further validation, several published predictions of 5-methylcytosine in human coronavirus and human cerebral organoid RNA in a GCU context should be reconsidered.
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Invasive aspergillosis is initiated when Aspergillus fumigatus adheres to and invades the pulmonary epithelial cells that line the airways and alveoli. To gain deeper insight into how pulmonary epithelial cells respond to A. fumigatus invasion, we used transcriptome sequencing (RNA-seq) to determine the transcriptional response of the A549 type II alveolar epithelial cell line to infection with strains CEA10 and Af293, two clinical isolates of A. fumigatus. Upstream regulator analysis of the data indicated that while both strains activated virtually identical host cell signaling pathways after 16 h of infection, only strain CEA10 activated these pathways after 6 h of infection. Many of the pathways that were predicted to be activated by A. fumigatus, including the tumor necrosis factor (TNF), interleukin-1α (IL-1α), IL-1ß, IL-17A, Toll-like receptor 2 (TLR2), and TLR4 pathways, are known to be critical for the host defense against this fungus. We also found that the platelet-derived growth factor BB (PDGF BB) and progesterone receptor (PGR) pathways were activated by A. fumigatus. Using pharmacologic inhibitors, we determined that blocking the PDGF receptor or PGR inhibited the endocytosis of both strains of A. fumigatus in an additive manner. Both the PDGF BB and PGR pathways are also predicted to be activated by infection of A549 cells with other molds, such as Rhizopus delemar and Rhizopus oryzae. Thus, these pathways may represent a common response of pulmonary epithelial cells to mold infection. IMPORTANCE Invasive aspergillosis is a deadly invasive fungal infection that initiates when Aspergillus fumigatus spores are inhaled and come into contact with the epithelial cells that line the airways and alveoli. Understanding this fungus-host interaction is important for the development of novel therapeutics. To gain a deeper understanding of how these airway epithelial cells respond to A. fumigatus during infection, we used RNA-seq to determine the transcriptional response of alveolar epithelial cells to infection with two different clinical isolates of A. fumigatus. Our analysis identified new host response pathways that have not previously been tied to infection with A. fumigatus. Pharmacological inhibition of two of these pathways inhibited the ability of A. fumigatus to invade airway epithelial cells. These two pathways are also predicted to be activated by infection with other filamentous fungi. Thus, these pathways may represent a common response of alveolar epithelial cells to mold infection.
Assuntos
Aspergilose , Aspergillus fumigatus , Humanos , Becaplermina , Aspergilose/microbiologia , Células Epiteliais/microbiologia , Pulmão/microbiologiaRESUMO
Aspergillus fumigatus is an environmental fungus that can cause life-threatening pulmonary disease. Infections initiate when conidia are inhaled and land deep inside the small airways and alveoli of the lungs, where they interact with epithelial cells. These cells provide a physical barrier and secrete chemokines to attract innate immune cells to the site of infection. Melanin, a key constituent of the conidial cell wall, is required for the establishment of invasive infection due to its ability to inhibit the function of innate immune cells recruited to clear the infection. Here, we provide evidence for an additional mechanism by which A. fumigatus can alter host innate immune responses. In vitro infection of a normal human small airway epithelial cell line (HSAEC1-KT) caused a decrease in extracellular protein levels of CXCL10 and CCL20, two proinflammatory chemokines that are required for the host defense against aspergillosis, despite a dramatic increase in the levels of each mRNA. A. fumigatus depleted recombinant human CXCL10 and CCL20 from medium in the absence of host cells, suggesting that the block in accumulation is downstream of protein translation and secretion. Melanin is both necessary and sufficient for this chemokine-depleting activity because a dihydroxynaphthalene (DHN)-melanin-deficient strain of A. fumigatus is defective in depleting chemokines and purified melanin ghosts retain potent depletion activity. We propose that A. fumigatus, through the action of melanin, depletes important chemokines, thereby dampening the innate immune response to promote infection. IMPORTANCE Aspergillus fumigatus is the major airborne fungal pathogen that affects humans. In order to cause an invasive infection, inhaled spores must avoid killing by innate immune cells that are recruited to the site of infection. Understanding how A. fumigatus achieves immune evasion is important for the development of novel therapeutics. We provide evidence that melanin, a pigment contained in the spore cell wall, can remove certain chemokines from the extracellular space to suppress the host inflammatory response that is responsible for clearing fungal infection.
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Aspergilose , Melaninas , Humanos , Melaninas/genética , Aspergillus fumigatus/genética , Aspergilose/microbiologia , Quimiocinas/metabolismo , Esporos Fúngicos/genéticaRESUMO
Many mutations in genes for ribosomal proteins (r-proteins) and assembly factors cause cell stress and altered cell fate, resulting in congenital diseases collectively called ribosomopathies. Even though all such mutations depress the cell's protein synthesis capacity, they generate many different phenotypes, suggesting that the diseases are not due simply to insufficient protein synthesis capacity. To learn more, we investigated how the global transcriptome in Saccharomyces cerevisiae responds to reduced protein synthesis generated in two different ways: abolishing the assembly of new ribosomes and inhibiting ribosomal function. Our results showed that the mechanism by which protein synthesis is obstructed affects the ribosomal protein transcriptome differentially: ribosomal protein mRNA abundance increases during the abolition of ribosome formation but decreases during the inhibition of ribosome function. Interestingly, the ratio between mRNAs from some, but not all, pairs of paralogous ribosomal protein genes encoding slightly different versions of a given r-protein changed differently during the two types of stress, suggesting that expression of specific ribosomal protein paralogous mRNAs may contribute to the stress response. Unexpectedly, the abundance of transcripts for ribosome assembly factors and translation factors remained relatively unaffected by the stresses. On the other hand, the state of the translation apparatus did affect cell physiology: mRNA levels for some other proteins not directly related to the translation apparatus also changed differentially, though not coordinately with the r-protein genes, in response to the stresses. IMPORTANCE Mutations in genes for ribosomal proteins or assembly factors cause a variety of diseases called ribosomopathies. These diseases are typically ascribed to a reduction in the cell's capacity for protein synthesis. Paradoxically, ribosomal mutations result in a wide variety of disease phenotypes, even though they all reduce protein synthesis. Here, we show that the transcriptome changes differently depending on how the protein synthesis capacity is reduced. Most strikingly, inhibiting ribosome formation and ribosome function had opposite effects on the abundance of mRNA for ribosomal proteins, while genes for ribosome translation and assembly factors showed no systematic responses. Thus, the process by which the protein synthesis capacity is reduced contributes decisively to global mRNA composition. This emphasis on process is a new concept in understanding ribosomopathies and other stress responses.
Assuntos
Proteínas Ribossômicas , Proteínas de Saccharomyces cerevisiae , Proteínas Ribossômicas/genética , Saccharomyces cerevisiae/genética , RNA Mensageiro/genética , Ribossomos/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
A side effect of antibiotics is outgrowth of the opportunistic fungus Candida albicans in the oropharynx (oropharyngeal candidiasis, OPC). IL-17 signaling is vital for immunity to OPC, but how the microbiome impacts antifungal immunity is not well understood. Mice in standard specific pathogen-free (SPF) conditions are resistant to OPC, whereas we show that germ-free (GF) or antibiotic-treated mice are susceptible. Oral type 17 cells and IL-17-dependent responses were impaired in antibiotic-treated and GF mice. Susceptibility could be rescued in GF mice by mono-colonization with segmented filamentous bacterium (SFB), an intestine-specific constituent of the microbiota. SFB protection was accompanied by restoration of oral IL-17+CD4+ T cells and gene signatures characteristic of IL-17 signaling. Additionally, RNA-Seq revealed induction of genes in the retinoic acid (RA) and RA receptor-α (RARα) pathway. Administration of RA rescued immunity to OPC in microbiome-depleted or GF mice, while RAR inhibition caused susceptibility in immunocompetent animals. Surprisingly, immunity to OPC was independent of serum amyloids. Moreover, RAR inhibition did not alter oral type 17 cytokine levels. Thus, mono-colonization with a component of the intestinal microflora confers protection against OPC by type 17 and RA/RARα, which act in parallel to promote antifungal immunity. In principle, manipulation of the microbiome could be harnessed to maintain antifungal immunity.
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Candidíase Bucal , Microbioma Gastrointestinal , Animais , Antibacterianos , Antifúngicos/farmacologia , Candidíase Bucal/microbiologia , Interleucina-17/metabolismo , Camundongos , Mucosa Bucal/microbiologia , TretinoínaRESUMO
Puel and Casanova and Kisand et al. challenge our conclusions that interferonopathy and not IL-17/IL-22 autoantibodies promote candidiasis in autoimmune polyendocrinopathycandidiasisectodermal dystrophy. We acknowledge that conclusive evidence for causation is difficult to obtain in complex human diseases. However, our studies clearly document interferonopathy driving mucosal candidiasis with intact IL-17/IL-22 responses in Aire-deficient mice, with strong corroborative evidence in patients.
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Imunidade nas Mucosas , Micoses , Humanos , Mucosa , Animais , CamundongosRESUMO
Library preparation for high-throughput sequencing applications is a critical step in producing representative, unbiased sequencing data. The iGenomX Riptide High Throughput Rapid Library Prep Kit purports to provide high-quality sequencing data with lower costs compared to other Illumina library kits. To test these claims, we compared sequence data quality of Riptide libraries to libraries constructed with KAPA Hyper and NEBNext Ultra. Across several single-source genome samples, mapping performance and de novo assembly of Riptide libraries were similar to conventional libraries prepared with the same DNA. Poor performance of some libraries resulted in low sequencing depth. In particular, degraded DNA samples may be challenging to sequence with Riptide. There was little cross-well plate contamination with the overwhelming majority of reads belong to the proper source genomes. The sequencing of metagenome samples using different Riptide primer sets resulted in variable taxonomic assignment of reads. Increased adoption of the Riptide kit will decrease library preparation costs. However, this method might not be suitable for degraded DNA.
Assuntos
Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/economia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise Custo-Benefício , DNA/genética , Metagenoma/genética , Análise de Sequência de DNA/economia , Análise de Sequência de DNA/métodosRESUMO
Microbial penetration of the blood-brain barrier, a prerequisite for the development of central nervous system (CNS) infection, involves microbial invasion, intracellular traversal, and exocytosis. Microbial invasion of the blood-brain barrier has been investigated, but the molecular basis for microbial traversal and exit from the blood-brain barrier remains unknown. We performed transcriptome analysis of human brain microvascular endothelial cells (HBMEC) infected with Escherichia coli and Cryptococcus neoformans, representative bacterial and fungal pathogens common in CNS infections. Among the targets upregulated in response to E. coli and C. neoformans infection, PDLIM2 was knocked down by small hairpin RNA (shRNA) in HBMEC for further investigation. We demonstrated that Pdlim2 specifically regulated microbial traversal and exit from HBMEC by assessing microbial invasion, transcytosis, intracellular multiplication, and egression. Additionally, the defective exocytosis of internalized E. coli cells from the PDLIM2 shRNA knockdown cells was restored by treatment with a calcium ionophore (ionomycin). Moreover, we performed proximity-dependent biotin labeling with the biotin ligase BioID2 and identified 210 potential Pdlim2 interactors. Among the nine Pdlim2 interactors enriched in response to both E. coli and C. neoformans infection, we selected MPRIP and showed that HBMEC with knockdown of MPRIP mimicked the phenotype of PDLIM2 knockdown cells. These results suggest that the CNS-infecting microbes hijack Pdlim2 and Mprip for intracellular traversal and exocytosis in the blood-brain barrier.
Assuntos
Barreira Hematoencefálica/imunologia , Infecções do Sistema Nervoso Central/imunologia , Criptococose/imunologia , Cryptococcus neoformans/imunologia , Infecções por Escherichia coli/imunologia , Escherichia coli/imunologia , Exocitose/imunologia , Proteínas com Domínio LIM/metabolismo , Proteínas dos Microfilamentos/metabolismo , Transporte Biológico/imunologia , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/microbiologia , Células Cultivadas , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/microbiologia , Infecções do Sistema Nervoso Central/metabolismo , Infecções do Sistema Nervoso Central/microbiologia , Criptococose/metabolismo , Criptococose/microbiologia , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Células Endoteliais/microbiologia , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Humanos , Proteínas com Domínio LIM/imunologia , Proteínas dos Microfilamentos/imunologia , Fosforilação/imunologiaRESUMO
Oral mucositis (OM) is a treatment-limiting adverse side effect of radiation and chemotherapy. Approximately 80% of patients undergoing radiotherapy (RT) for head and neck cancers (HNC) develop OM, representing a major unmet medical condition. Our understanding of the immunopathogenesis of OM is limited, due in part to the surprising paucity of information regarding healing mechanisms in the oral mucosa. RNAseq of oral tissue in a murine model that closely mimics human OM, showed elevated expression of IL-17 and related immune pathways in response to head and neck irradiation (HNI). Strikingly, mice lacking the IL-17 receptor (IL-17RA) exhibited markedly more severe OM. Restoration of the oral mucosa was compromised in Il17ra-/- mice and components associated with healing, including matrix metalloproteinase 3, 10 and IL-24 were diminished. IL-17 is typically associated with recruitment of neutrophils to mucosal sites following oral infections. Unexpectedly, in OM the absence of IL-17RA resulted in excessive neutrophil recruitment and immunopathology. Instead, neutrophil activation was IL-1R-driven in Il17ra-/- mice. Blockade of IL-1R and depletion of neutrophils lessened the severity of damage in these mice. Overall, we show IL-17 is protective in OM through multiple mechanisms including restoration of the damaged epithelia and control of the neutrophil response. We also present a clinically relevant murine model of human OM to improve mechanistic understanding and develop rational translational therapeutics.
Assuntos
Interleucina-17/metabolismo , Lesões por Radiação/metabolismo , Receptores de Interleucina-17/metabolismo , Estomatite/metabolismo , Língua/metabolismo , Cicatrização , Animais , Proliferação de Células , Sobrevivência Celular , Modelos Animais de Doenças , Interleucina-1/metabolismo , Interleucina-17/genética , Camundongos Knockout , Infiltração de Neutrófilos , Lesões por Radiação/genética , Lesões por Radiação/imunologia , Lesões por Radiação/patologia , Receptores de Interleucina-1/metabolismo , Receptores de Interleucina-17/genética , Transdução de Sinais , Estomatite/genética , Estomatite/imunologia , Estomatite/patologia , Língua/imunologia , Língua/patologia , TranscriptomaRESUMO
The fungal pathogen Candida albicans is surrounded by a cell wall that is the target of caspofungin and other echinocandin antifungals. Candida albicans can grow in several morphological forms, notably budding yeast and hyphae. Yeast and hyphal forms differ in cell wall composition, leading us to hypothesize that there may be distinct genes required for yeast and hyphal responses to caspofungin. Mutants in 27 genes reported previously to be caspofungin hypersensitive under yeast growth conditions were all caspofungin hypersensitive under hyphal growth conditions as well. However, a screen of mutants defective in transcription factor genes revealed that Cup9 is required for normal caspofungin tolerance under hyphal and not yeast growth conditions. In a hyphal-defective efg1Δ/Δ background, Cup9 is still required for normal caspofungin tolerance. This result argues that Cup9 function is related to growth conditions rather than cell morphology. RNA-seq conducted under hyphal growth conditions indicated that 361 genes were up-regulated and 145 genes were down-regulated in response to caspofungin treatment. Both classes of caspofungin-responsive genes were enriched for cell wall-related proteins, as expected for a response to disruption of cell wall integrity and biosynthesis. The cup9Δ/Δ mutant, treated with caspofungin, had reduced RNA levels of 40 caspofungin up-regulated genes, and had increased RNA levels of 8 caspofungin down-regulated genes, an indication that Cup9 has a narrow rather than global role in the cell wall integrity response. Five Cup9-activated surface-protein genes have roles in cell wall integrity, based on mutant analysis published previously (PGA31 and IFF11) or shown here (ORF19.3499, ORF19.851, or PGA28), and therefore may explain the hypersensitivity of the cup9Δ/Δmutant to caspofungin. Our findings define Cup9 as a new determinant of caspofungin susceptibility.
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Candida albicans/metabolismo , Parede Celular/metabolismo , Farmacorresistência Fúngica , Proteínas Fúngicas/metabolismo , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/metabolismo , Antifúngicos/toxicidade , Candida albicans/efeitos dos fármacos , Candida albicans/genética , Caspofungina/toxicidade , Parede Celular/efeitos dos fármacos , Parede Celular/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Proteínas de Homeodomínio/genética , Fatores de Transcrição/genéticaRESUMO
Advances in transcriptome sequencing allow for simultaneous interrogation of differentially expressed genes from multiple species originating from a single RNA sample, termed dual or multi-species transcriptomics. Compared to single-species differential expression analysis, the design of multi-species differential expression experiments must account for the relative abundances of each organism of interest within the sample, often requiring enrichment methods and yielding differences in total read counts across samples. The analysis of multi-species transcriptomics datasets requires modifications to the alignment, quantification, and downstream analysis steps compared to the single-species analysis pipelines. We describe best practices for multi-species transcriptomics and differential gene expression.
Assuntos
Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , RNA-Seq/métodos , Transcriptoma , Animais , Eucariotos/genética , Perfilação da Expressão Gênica/normas , Regulação da Expressão Gênica , Humanos , Especificidade de Órgãos , Células Procarióticas/metabolismo , RNA/genética , RNA-Seq/normas , Curva ROC , Alinhamento de Sequência , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Fluxo de TrabalhoRESUMO
To gain a better understanding of the transcriptional response of Aspergillus fumigatus during invasive pulmonary infection, we used a NanoString nCounter to assess the transcript levels of 467 A. fumigatus genes during growth in the lungs of immunosuppressed mice. These genes included ones known to respond to diverse environmental conditions and those encoding most transcription factors in the A. fumigatus genome. We found that invasive growth in vivo induces a unique transcriptional profile as the organism responds to nutrient limitation and attack by host phagocytes. This in vivo transcriptional response is largely mimicked by in vitro growth in Aspergillus minimal medium that is deficient in nitrogen, iron, and/or zinc. From the transcriptional profiling data, we selected 9 transcription factor genes that were either highly expressed or strongly up-regulated during in vivo growth. Deletion mutants were constructed for each of these genes and assessed for virulence in mice. Two transcription factor genes were found to be required for maximal virulence. One was rlmA, which is required for the organism to achieve maximal fungal burden in the lung. The other was sltA, which regulates of the expression of multiple secondary metabolite gene clusters and mycotoxin genes independently of laeA. Using deletion and overexpression mutants, we determined that the attenuated virulence of the ΔsltA mutant is due in part to decreased expression aspf1, which specifies a ribotoxin, but is not mediated by reduced expression of the fumigaclavine gene cluster or the fumagillin-pseruotin supercluster. Thus, in vivo transcriptional profiling focused on transcription factors genes provides a facile approach to identifying novel virulence regulators.
Assuntos
Aspergillus fumigatus/genética , Regulação Fúngica da Expressão Gênica/genética , Pulmão/virologia , Fatores de Transcrição/metabolismo , Animais , Aspergilose/microbiologia , Aspergillus fumigatus/patogenicidade , Proteínas Fúngicas/metabolismo , Perfilação da Expressão Gênica/métodos , Ferro/metabolismo , Pulmão/metabolismo , Camundongos , Virulência/genéticaRESUMO
Biofilm-associated polymicrobial infections tend to be challenging to treat. Candida albicans and Staphylococcus aureus are leading pathogens due to their ability to form biofilms on medical devices. However, the therapeutic implications of their interactions in a host is largely unexplored. In this study, we used a mouse subcutaneous catheter model for in vivo-grown polymicrobial biofilms to validate our in vitro findings on C. albicans-mediated enhanced S. aureus tolerance to vancomycin in vivo. Comparative assessment of S. aureus recovery from catheters with single- or mixed-species infection demonstrated failure of vancomycin against S. aureus in mice with co-infected catheters. To provide some mechanistic insights, RNA-seq analysis was performed on catheter biofilms to delineate transcriptional modulations during polymicrobial infections. C. albicans induced the activation of the S. aureus biofilm formation network via down-regulation of the lrg operon, repressor of autolysis, and up-regulation of the ica operon and production of polysaccharide intercellular adhesin (PIA), indicating an increase in eDNA production, and extracellular polysaccharide matrix, respectively. Interestingly, virulence factors important for disseminated infections, and superantigen-like proteins were down-regulated during mixed-species infection, whereas capsular polysaccharide genes were up-regulated, signifying a strategy favoring survival, persistence and host immune evasion. In vitro follow-up experiments using DNA enzymatic digestion, lrg operon mutant strains, and confocal scanning microscopy confirmed the role of C. albicans-mediated enhanced eDNA production in mixed-biofilms on S. aureus tolerance to vancomycin. Combined, these findings provide mechanistic insights into the therapeutic implications of interspecies interactions, underscoring the need for novel strategies to overcome limitations of current therapies.