Overview
Qualifications
PhD Biological Sciences, University of Exeter
BSc (hons) Microbiology, Imperial College London
Research group links
Research
Research projects
Characterisation of stress resistance mechanisms in the pathogenic yeast Candida glabrata
During infection, pathogenic microorganisms encounter and must overcome a variety of stresses in the host environment. My research focuses on understanding how the pathogenic yeast Candida glabrata survives oxidative and osmotic stresses, both singly and in combination. The generation and sequencing of stress-resistant C. glabrata mutants will facilitate our understanding of such mechanisms through the identification of novel mutations conferring stress resistant phenotypes.
PhD research
Candida glabrata is a significant and increasingly common pathogen of humans yet its mechanism of virulence remains unclear. Comparative genomic studies revealed that C. glabrata is more closely related to the non-pathogenic yeast Saccharomyces cerevisiae and that both these genomes are distinct from C. albicans. In order to explore C. glabrata virulence attributes, C. glabrata ORFs with no orthologue in S. cerevisiae were studied since these ORFs may have accompanied the adaptation of C. glabrata to the human host.
Reciprocal best hit searches identified C. glabrata ORFs with no S. cerevisiae orthologue. A barcoded deletion library targeting 65 C. glabrata-specific ORFswas constructed. To functionally characterise the deletion library, mutants were tested for fitness and phenotypically screened to identify gene products required for growth in response to biologically relevant stresses. As such, novel phenotypes associated with the deletion of previously uncharacterised ORFs were uncovered. Mutants were also tested for infection-related properties including biofilm formation, antifungal agent susceptibility for virulence in a Drosophila melanogaster infection model, resulting in the identification of two ORFswhich were required for virulence.
An adapted genome-wide synthetic genetic interaction approach was used to create genetic interaction networks for C. glabrata ORFs over-expressed in S. cerevisiae. Genetic interaction analysis of a C. glabrata HMG-box chromatin remodeler revealed a putative role for this ORF in DNA damage repair.
Publications
Key publications | Publications by category | Publications by year
Publications by category
Journal articles
Holland LM, Schröder MS, Turner SA, Taff H, Andes D, Grózer Z, Gácser A, Ames L, Haynes K, Higgins DG, et al (2014). Comparative phenotypic analysis of the major fungal pathogens Candida parapsilosis and Candida albicans.
PLoS Pathog,
10(9).
Abstract:
Comparative phenotypic analysis of the major fungal pathogens Candida parapsilosis and Candida albicans.
Candida parapsilosis and Candida albicans are human fungal pathogens that belong to the CTG clade in the Saccharomycotina. In contrast to C. albicans, relatively little is known about the virulence properties of C. parapsilosis, a pathogen particularly associated with infections of premature neonates. We describe here the construction of C. parapsilosis strains carrying double allele deletions of 100 transcription factors, protein kinases and species-specific genes. Two independent deletions were constructed for each target gene. Growth in >40 conditions was tested, including carbon source, temperature, and the presence of antifungal drugs. The phenotypes were compared to C. albicans strains with deletions of orthologous transcription factors. We found that many phenotypes are shared between the two species, such as the role of Upc2 as a regulator of azole resistance, and of CAP1 in the oxidative stress response. Others are unique to one species. For example, Cph2 plays a role in the hypoxic response in C. parapsilosis but not in C. albicans. We found extensive divergence between the biofilm regulators of the two species. We identified seven transcription factors and one protein kinase that are required for biofilm development in C. parapsilosis. Only three (Efg1, Bcr1 and Ace2) have similar effects on C. albicans biofilms, whereas Cph2, Czf1, Gzf3 and Ume6 have major roles in C. parapsilosis only. Two transcription factors (Brg1 and Tec1) with well-characterized roles in biofilm formation in C. albicans do not have the same function in C. parapsilosis. We also compared the transcription profile of C. parapsilosis and C. albicans biofilms. Our analysis suggests the processes shared between the two species are predominantly metabolic, and that Cph2 and Bcr1 are major biofilm regulators in C. parapsilosis.
Abstract.
Author URL.
Kasper L, Seider K, Gerwien F, Allert S, Brunke S, Schwarzmüller T, Ames L, Zubiria-Barrera C, Mansour MK, Becken U, et al (2014). Identification of Candida glabrata genes involved in pH modulation and modification of the phagosomal environment in macrophages.
PLoS One,
9(5).
Abstract:
Identification of Candida glabrata genes involved in pH modulation and modification of the phagosomal environment in macrophages.
Candida glabrata currently ranks as the second most frequent cause of invasive candidiasis. Our previous work has shown that C. glabrata is adapted to intracellular survival in macrophages and replicates within non-acidified late endosomal-stage phagosomes. In contrast, heat killed yeasts are found in acidified matured phagosomes. In the present study, we aimed at elucidating the processes leading to inhibition of phagosome acidification and maturation. We show that phagosomes containing viable C. glabrata cells do not fuse with pre-labeled lysosomes and possess low phagosomal hydrolase activity. Inhibition of acidification occurs independent of macrophage type (human/murine), differentiation (M1-/M2-type) or activation status (vitamin D3 stimulation). We observed no differential activation of macrophage MAPK or NFκB signaling cascades downstream of pattern recognition receptors after internalization of viable compared to heat killed yeasts, but Syk activation decayed faster in macrophages containing viable yeasts. Thus, delivery of viable yeasts to non-matured phagosomes is likely not triggered by initial recognition events via MAPK or NFκB signaling, but Syk activation may be involved. Although V-ATPase is abundant in C. glabrata phagosomes, the influence of this proton pump on intracellular survival is low since blocking V-ATPase activity with bafilomycin A1 has no influence on fungal viability. Active pH modulation is one possible fungal strategy to change phagosome pH. In fact, C. glabrata is able to alkalinize its extracellular environment, when growing on amino acids as the sole carbon source in vitro. By screening a C. glabrata mutant library we identified genes important for environmental alkalinization that were further tested for their impact on phagosome pH. We found that the lack of fungal mannosyltransferases resulted in severely reduced alkalinization in vitro and in the delivery of C. glabrata to acidified phagosomes. Therefore, protein mannosylation may play a key role in alterations of phagosomal properties caused by C. glabrata.
Abstract.
Author URL.
Full text.
Schwarzmüller T, Ma B, Hiller E, Istel F, Tscherner M, Brunke S, Ames L, Firon A, Green B, Cabral V, et al (2014). Systematic phenotyping of a large-scale Candida glabrata deletion collection reveals novel antifungal tolerance genes.
PLoS Pathog,
10(6).
Abstract:
Systematic phenotyping of a large-scale Candida glabrata deletion collection reveals novel antifungal tolerance genes.
The opportunistic fungal pathogen Candida glabrata is a frequent cause of candidiasis, causing infections ranging from superficial to life-threatening disseminated disease. The inherent tolerance of C. glabrata to azole drugs makes this pathogen a serious clinical threat. To identify novel genes implicated in antifungal drug tolerance, we have constructed a large-scale C. glabrata deletion library consisting of 619 unique, individually bar-coded mutant strains, each lacking one specific gene, all together representing almost 12% of the genome. Functional analysis of this library in a series of phenotypic and fitness assays identified numerous genes required for growth of C. glabrata under normal or specific stress conditions, as well as a number of novel genes involved in tolerance to clinically important antifungal drugs such as azoles and echinocandins. We identified 38 deletion strains displaying strongly increased susceptibility to caspofungin, 28 of which encoding proteins that have not previously been linked to echinocandin tolerance. Our results demonstrate the potential of the C. glabrata mutant collection as a valuable resource in functional genomics studies of this important fungal pathogen of humans, and to facilitate the identification of putative novel antifungal drug target and virulence genes.
Abstract.
Author URL.
Kaloriti D, Tillmann A, Cook E, Jacobsen M, you T, Lenardon M, Ames L, Barahona M, Chandrasekaran K, Coghill G, et al (2012). Combinatorial stresses kill pathogenic Candida species.
Med Mycol,
50(7), 699-709.
Abstract:
Combinatorial stresses kill pathogenic Candida species.
Pathogenic microbes exist in dynamic niches and have evolved robust adaptive responses to promote survival in their hosts. The major fungal pathogens of humans, Candida albicans and Candida glabrata, are exposed to a range of environmental stresses in their hosts including osmotic, oxidative and nitrosative stresses. Significant efforts have been devoted to the characterization of the adaptive responses to each of these stresses. In the wild, cells are frequently exposed simultaneously to combinations of these stresses and yet the effects of such combinatorial stresses have not been explored. We have developed a common experimental platform to facilitate the comparison of combinatorial stress responses in C. glabrata and C. albicans. This platform is based on the growth of cells in buffered rich medium at 30°C, and was used to define relatively low, medium and high doses of osmotic (NaCl), oxidative (H(2)O(2)) and nitrosative stresses (e.g. dipropylenetriamine (DPTA)-NONOate). The effects of combinatorial stresses were compared with the corresponding individual stresses under these growth conditions. We show for the first time that certain combinations of combinatorial stress are especially potent in terms of their ability to kill C. albicans and C. glabrata and/or inhibit their growth. This was the case for combinations of osmotic plus oxidative stress and for oxidative plus nitrosative stress. We predict that combinatorial stresses may be highly significant in host defences against these pathogenic yeasts.
Abstract.
Author URL.
Publications by year
2014
Holland LM, Schröder MS, Turner SA, Taff H, Andes D, Grózer Z, Gácser A, Ames L, Haynes K, Higgins DG, et al (2014). Comparative phenotypic analysis of the major fungal pathogens Candida parapsilosis and Candida albicans.
PLoS Pathog,
10(9).
Abstract:
Comparative phenotypic analysis of the major fungal pathogens Candida parapsilosis and Candida albicans.
Candida parapsilosis and Candida albicans are human fungal pathogens that belong to the CTG clade in the Saccharomycotina. In contrast to C. albicans, relatively little is known about the virulence properties of C. parapsilosis, a pathogen particularly associated with infections of premature neonates. We describe here the construction of C. parapsilosis strains carrying double allele deletions of 100 transcription factors, protein kinases and species-specific genes. Two independent deletions were constructed for each target gene. Growth in >40 conditions was tested, including carbon source, temperature, and the presence of antifungal drugs. The phenotypes were compared to C. albicans strains with deletions of orthologous transcription factors. We found that many phenotypes are shared between the two species, such as the role of Upc2 as a regulator of azole resistance, and of CAP1 in the oxidative stress response. Others are unique to one species. For example, Cph2 plays a role in the hypoxic response in C. parapsilosis but not in C. albicans. We found extensive divergence between the biofilm regulators of the two species. We identified seven transcription factors and one protein kinase that are required for biofilm development in C. parapsilosis. Only three (Efg1, Bcr1 and Ace2) have similar effects on C. albicans biofilms, whereas Cph2, Czf1, Gzf3 and Ume6 have major roles in C. parapsilosis only. Two transcription factors (Brg1 and Tec1) with well-characterized roles in biofilm formation in C. albicans do not have the same function in C. parapsilosis. We also compared the transcription profile of C. parapsilosis and C. albicans biofilms. Our analysis suggests the processes shared between the two species are predominantly metabolic, and that Cph2 and Bcr1 are major biofilm regulators in C. parapsilosis.
Abstract.
Author URL.
Kasper L, Seider K, Gerwien F, Allert S, Brunke S, Schwarzmüller T, Ames L, Zubiria-Barrera C, Mansour MK, Becken U, et al (2014). Identification of Candida glabrata genes involved in pH modulation and modification of the phagosomal environment in macrophages.
PLoS One,
9(5).
Abstract:
Identification of Candida glabrata genes involved in pH modulation and modification of the phagosomal environment in macrophages.
Candida glabrata currently ranks as the second most frequent cause of invasive candidiasis. Our previous work has shown that C. glabrata is adapted to intracellular survival in macrophages and replicates within non-acidified late endosomal-stage phagosomes. In contrast, heat killed yeasts are found in acidified matured phagosomes. In the present study, we aimed at elucidating the processes leading to inhibition of phagosome acidification and maturation. We show that phagosomes containing viable C. glabrata cells do not fuse with pre-labeled lysosomes and possess low phagosomal hydrolase activity. Inhibition of acidification occurs independent of macrophage type (human/murine), differentiation (M1-/M2-type) or activation status (vitamin D3 stimulation). We observed no differential activation of macrophage MAPK or NFκB signaling cascades downstream of pattern recognition receptors after internalization of viable compared to heat killed yeasts, but Syk activation decayed faster in macrophages containing viable yeasts. Thus, delivery of viable yeasts to non-matured phagosomes is likely not triggered by initial recognition events via MAPK or NFκB signaling, but Syk activation may be involved. Although V-ATPase is abundant in C. glabrata phagosomes, the influence of this proton pump on intracellular survival is low since blocking V-ATPase activity with bafilomycin A1 has no influence on fungal viability. Active pH modulation is one possible fungal strategy to change phagosome pH. In fact, C. glabrata is able to alkalinize its extracellular environment, when growing on amino acids as the sole carbon source in vitro. By screening a C. glabrata mutant library we identified genes important for environmental alkalinization that were further tested for their impact on phagosome pH. We found that the lack of fungal mannosyltransferases resulted in severely reduced alkalinization in vitro and in the delivery of C. glabrata to acidified phagosomes. Therefore, protein mannosylation may play a key role in alterations of phagosomal properties caused by C. glabrata.
Abstract.
Author URL.
Full text.
Schwarzmüller T, Ma B, Hiller E, Istel F, Tscherner M, Brunke S, Ames L, Firon A, Green B, Cabral V, et al (2014). Systematic phenotyping of a large-scale Candida glabrata deletion collection reveals novel antifungal tolerance genes.
PLoS Pathog,
10(6).
Abstract:
Systematic phenotyping of a large-scale Candida glabrata deletion collection reveals novel antifungal tolerance genes.
The opportunistic fungal pathogen Candida glabrata is a frequent cause of candidiasis, causing infections ranging from superficial to life-threatening disseminated disease. The inherent tolerance of C. glabrata to azole drugs makes this pathogen a serious clinical threat. To identify novel genes implicated in antifungal drug tolerance, we have constructed a large-scale C. glabrata deletion library consisting of 619 unique, individually bar-coded mutant strains, each lacking one specific gene, all together representing almost 12% of the genome. Functional analysis of this library in a series of phenotypic and fitness assays identified numerous genes required for growth of C. glabrata under normal or specific stress conditions, as well as a number of novel genes involved in tolerance to clinically important antifungal drugs such as azoles and echinocandins. We identified 38 deletion strains displaying strongly increased susceptibility to caspofungin, 28 of which encoding proteins that have not previously been linked to echinocandin tolerance. Our results demonstrate the potential of the C. glabrata mutant collection as a valuable resource in functional genomics studies of this important fungal pathogen of humans, and to facilitate the identification of putative novel antifungal drug target and virulence genes.
Abstract.
Author URL.
2012
Kaloriti D, Tillmann A, Cook E, Jacobsen M, you T, Lenardon M, Ames L, Barahona M, Chandrasekaran K, Coghill G, et al (2012). Combinatorial stresses kill pathogenic Candida species.
Med Mycol,
50(7), 699-709.
Abstract:
Combinatorial stresses kill pathogenic Candida species.
Pathogenic microbes exist in dynamic niches and have evolved robust adaptive responses to promote survival in their hosts. The major fungal pathogens of humans, Candida albicans and Candida glabrata, are exposed to a range of environmental stresses in their hosts including osmotic, oxidative and nitrosative stresses. Significant efforts have been devoted to the characterization of the adaptive responses to each of these stresses. In the wild, cells are frequently exposed simultaneously to combinations of these stresses and yet the effects of such combinatorial stresses have not been explored. We have developed a common experimental platform to facilitate the comparison of combinatorial stress responses in C. glabrata and C. albicans. This platform is based on the growth of cells in buffered rich medium at 30°C, and was used to define relatively low, medium and high doses of osmotic (NaCl), oxidative (H(2)O(2)) and nitrosative stresses (e.g. dipropylenetriamine (DPTA)-NONOate). The effects of combinatorial stresses were compared with the corresponding individual stresses under these growth conditions. We show for the first time that certain combinations of combinatorial stress are especially potent in terms of their ability to kill C. albicans and C. glabrata and/or inhibit their growth. This was the case for combinations of osmotic plus oxidative stress and for oxidative plus nitrosative stress. We predict that combinatorial stresses may be highly significant in host defences against these pathogenic yeasts.
Abstract.
Author URL.
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