Project description:Candida albicans is the major invasive fungal pathogen of humans, causing diseases ranging from superficial mucosal infections to disseminated, systemic infections which are often life threatening. Hematogenously disseminated candidiasis (HDC) has a 47% mortality rate despite current antifungal therapy. An increase in prevalence, as well as an increasing resistance to most of the clinically important antifungal therapies, provides a strong impetus to understand the molecular mechanisms of pathogenesis and the acquisition of drug resistance. This information holds promise to identify novel therapeutic targets. A complete and accurate characterization of how the transcriptome of C. albicans responds to its interaction with cells from the host is an absolute necessity to accomplish this goal. RNA-seq (deep-sequencing of cDNA) provides an unbiased method to define comprehensively and systematically the transcriptome of an organism. We propose a comprehensive characterization of the C. albicans transcriptome in two different human tissue culture models, using RNA-seq. Such a characterization will shed unprecedented light on how fungal pathogens sense and respond to the host environment and how the host tissue responds to fungal invasion. We performed a comprehensive characterization of the C. albicans transcriptome in two different murine models of candidiasis (HDC and oropharyngeal candidiasis (OPC)), and two different human tissue culture models, using RNA-seq. Because the two murine models accurately recapitulate the pathology that is observed in clinical cases of candidiasis, we are confident that gene expression levels will provide an accurate representation of what occurs during the course of an infection in humans. Such a characterization of the in vivo transcriptome will shed unprecedented light on how fungal pathogens sense and respond to the host environment and how the host tissue responds to fungal invasion. PRJNA211732 C. albicans strain SC5314 was used to infect mice in a murine model of systemic candidiasis as well as a murine model of oropharyngeal candidiasis. For the disseminated model, kidneys were harvested at 6 hours, 12 hours, 24 hours and 48 hours after infection. For the oropharyngeal model, tongues were harvest at 1 day, 2 days, 3 days, and 5 days after infection. Total RNA was extracted from all harvested tissues and subject to RNA-seq. We also tested 3 C. albicans mutants in the disseminated model. For the infections with the mutant strains, only 1 timepoint (24 hours post-infection) was analyzed.

Project description:Candida albicans is the major invasive fungal pathogen of humans, causing diseases ranging from superficial mucosal infections to disseminated, systemic infections which are often life threatening. Hematogenously disseminated candidiasis (HDC) has a 47% mortality rate despite current antifungal therapy. An increase in prevalence, as well as an increasing resistance to most of the clinically important antifungal therapies, provides a strong impetus to understand the molecular mechanisms of pathogenesis and the acquisition of drug resistance. This information holds promise to identify novel therapeutic targets. A complete and accurate characterization of how the transcriptome of C. albicans responds to its interaction with cells from the host is an absolute necessity to accomplish this goal. RNA-seq (deep-sequencing of cDNA) provides an unbiased method to define comprehensively and systematically the transcriptome of an organism. We propose a comprehensive characterization of the C. albicans transcriptome in two different human tissue culture models, using RNA-seq. Such a characterization will shed unprecedented light on how fungal pathogens sense and respond to the host environment and how the host tissue responds to fungal invasion. We performed a comprehensive characterization of the C. albicans transcriptome in two different murine models of candidiasis (HDC and oropharyngeal candidiasis (OPC)), and two different human tissue culture models, using RNA-seq. Because the two murine models accurately recapitulate the pathology that is observed in clinical cases of candidiasis, we are confident that gene expression levels will provide an accurate representation of what occurs during the course of an infection in humans. Such a characterization of the in vivo transcriptome will shed unprecedented light on how fungal pathogens sense and respond to the host environment and how the host tissue responds to fungal invasion. PRJNA86065 Two different strains of C. albicans (SC5314 or WO-1) were used to infect two different types of human tissue culture monolayers (HUVECs or OKF6-TERT2). RNA from the infection mixtures were harvested at 90 minutes, 5 hours and 8 hours post infection. Experiments were performed in biological duplicate.

Project description:Polymicrobial infections are intrinsically less susceptible to antimicrobial therapy, are more invasive and can induce enhanced inflammatory responses leading to negative clinical outcomes. The importance of studying the interaction between microbes themselves and their interactions with their host in combination has only been realised in recent years. However, there is a lack of simple, effective and low cost in vivo models to study these interactions with the host immune response. This study details the responses of Galleria mellonella larvae to disseminated combination infection by C. albicans and S. aureus and demonstrates the efficacy of antimicrobial chemotherapy on treating co-infected larvae. Doses of C. albicans (1 × 105 larva-1) and S. aureus (1 × 104 larva-1) which were non-lethal in mono-infection, when combined significantly lowered larval survival at (70 ± 3.33% [p < 0.01]), 48 (10 ± 3.33% [p < 0.0001]) and 72h (5 ± 6.66% [p < 0.0001]) h relative to larvae receiving S. aureus 2 × 104 larva-1 mono-infection. Co-infected larvae with S. aureus 2 × 104 larva-1 resulted in an increase in the density of circulating hemocytes relative to S. aureus mono-infected larvae. Co-infected larvae displayed a significantly higher overall S. aureus c.f.u larva-1 compared to larvae only infected with S. aureus. Co-infection resulted in dissemination throughout the host and the appearance of large nodules. Proteomic analysis of co-infected larval hemolymph revealed specific immune responses to bacterial and fungal infection by the increased abundance of a range of antimicrobial peptides, peptidoglycan, lipopolysaccharide and β-glucan recognition proteins and proteins associated with tissue invasion and nodule formation. The in vitro and in vivo efficacy of empirical antimicrobial drugs (amphotericin B & meropenem) was examined against C. albicans and S. aureus was also examined. meropenem was more effective than amphotericin B but a combination was most effective at improving survival of co-infected larvae. The effect of antimicrobial therapy on symptoms associated with co-infection was also assessed using Cryo-imaging. The processes of polymicrobial co-infection in G. mellonella are compared to those in mice models and larvae provide an easy to use and ethically acceptable in vivo system to assess polymicrobial interactions with their host.

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Characterization of transcriptome dynamics in response to contact with host cells

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