Drug resistance is a rapidly emerging concern, thus prompting the development of novel therapeutics or combinatorial therapy. Currently, combinatorial therapy targets are based on knowledge of drug ...mode of action and/or resistance mechanisms, constraining the number of target proteins. Unbiased genome-wide screens could reveal novel genetic components within interaction networks as potential targets in combination therapies. Testing this, in the context of antimicrobial resistance, we implemented an unbiased genome-wide screen, performed in Saccharomyces cerevisiae expressing a Candida glabrata PDR1+ gain-of-function allele. Gain-of-function mutations in this gene are the principal mediators of fluconazole resistance in this human fungal pathogen. Eighteen synthetically lethal S. cerevisiae genetic mutants were identified in cells expressing C. glabrata PDR1+. One mutant, lacking the histone acetyltransferase Gcn5, was investigated further. Deletion or drug-mediated inhibition of Gcn5 caused a lethal phenotype in C. glabrata cells expressing PDR1+ alleles. Moreover, deletion or drug-mediated inactivation of Gcn5, inhibited the emergence of fluconazole-resistant C. glabrata isolates in evolution experiments. Thus, taken together, the data generated in this study provides proof of concept that synthetically lethal genetic screens can identify novel candidate proteins that when therapeutically targeted could allow effective treatment of drug-resistant infections.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Fungal pathogenicity has arisen in polyphyletic manner during evolution, yielding fungal pathogens with diverse infection strategies and with differing degrees of evolutionary adaptation to their ...human host. Not surprisingly, these fungal pathogens display differing degrees of resistance to the reactive oxygen and nitrogen species used by human cells to counteract infection. Furthermore, whilst evolutionarily conserved regulators, such as Hog1, are central to such stress responses in many fungal pathogens, species-specific differences in their roles and regulation abound. In contrast, there is a high degree of commonality in the cellular responses to reactive oxygen and nitrogen species evoked in evolutionarily divergent fungal pathogens.
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.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Siderophore biosynthesis by the highly lethal mould Aspergillus fumigatus is essential for virulence, but non-existent in humans, presenting a rare opportunity to strategize therapeutically against ...this pathogen. We have previously demonstrated that A. fumigatus excretes fusarinine C and triacetylfusarinine C to capture extracellular iron, and uses ferricrocin for hyphal iron storage. Here, we delineate pathways of intra- and extracellular siderophore biosynthesis and show that A. fumigatus synthesizes a developmentally regulated fourth siderophore, termed hydroxyferricrocin, employed for conidial iron storage. By inactivation of the nonribosomal peptide synthetase SidC, we demonstrate that the intracellular siderophores are required for germ tube formation, asexual sporulation, resistance to oxidative stress, catalase A activity, and virulence. Restoration of the conidial hydroxyferricrocin content partially rescues the virulence of the apathogenic siderophore null mutant Delta sidA, demonstrating an important role for the conidial siderophore during initiation of infection. Abrogation of extracellular siderophore biosynthesis following inactivation of the acyl transferase SidF or the nonribosomal peptide synthetase SidD leads to complete dependence upon reductive iron assimilation for growth under iron-limiting conditions, partial sensitivity to oxidative stress, and significantly reduced virulence, despite normal germ tube formation. Our findings reveal distinct cellular and disease-related roles for intra- and extracellular siderophores during mammalian Aspergillus infection.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Aspergillus fumigatus is a common mould whose spores are a component of the normal airborne flora. Immune dysfunction permits developmental growth of inhaled spores in the human lung causing ...aspergillosis, a significant threat to human health in the form of allergic, and life-threatening invasive infections. The success of A. fumigatus as a pathogen is unique among close phylogenetic relatives and is poorly characterised at the molecular level. Recent genome sequencing of several Aspergillus species provides an exceptional opportunity to analyse fungal virulence attributes within a genomic and evolutionary context. To identify genes preferentially expressed during adaptation to the mammalian host niche, we generated multiple gene expression profiles from minute samplings of A. fumigatus germlings during initiation of murine infection. They reveal a highly co-ordinated A. fumigatus gene expression programme, governing metabolic and physiological adaptation, which allows the organism to prosper within the mammalian niche. As functions of phylogenetic conservation and genetic locus, 28% and 30%, respectively, of the A. fumigatus subtelomeric and lineage-specific gene repertoires are induced relative to laboratory culture, and physically clustered genes including loci directing pseurotin, gliotoxin and siderophore biosyntheses are a prominent feature. Locationally biased A. fumigatus gene expression is not prompted by in vitro iron limitation, acid, alkaline, anaerobic or oxidative stress. However, subtelomeric gene expression is favoured following ex vivo neutrophil exposure and in comparative analyses of richly and poorly nourished laboratory cultured germlings. We found remarkable concordance between the A. fumigatus host-adaptation transcriptome and those resulting from in vitro iron depletion, alkaline shift, nitrogen starvation and loss of the methyltransferase LaeA. This first transcriptional snapshot of a fungal genome during initiation of mammalian infection provides the global perspective required to direct much-needed diagnostic and therapeutic strategies and reveals genome organisation and subtelomeric diversity as potential driving forces in the evolution of pathogenicity in the genus Aspergillus.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Understanding how populations and communities respond to competition is a central concern of ecology. A seminal theoretical solution first formalised by Levins (and re‐derived in multiple fields) ...showed that, in theory, the form of a trade‐off should determine the outcome of competition. While this has become a central postulate in ecology it has evaded experimental verification, not least because of substantial technical obstacles. We here solve the experimental problems by employing synthetic ecology. We engineer strains of Escherichia coli with fixed resource allocations enabling accurate measurement of trade‐off shapes between bacterial survival and multiplication in multiple environments. A mathematical chemostat model predicts different, and experimentally verified, trajectories of gene frequency changes as a function of condition‐specific trade‐offs. The results support Levins' postulate and demonstrates that otherwise paradoxical alternative outcomes witnessed in subtly different conditions are predictable.
An integrative model of ion regulation in yeast Ke, Ruian; Ingram, Piers J; Haynes, Ken
PLOS computational biology/PLoS computational biology,
01/2013, Letnik:
9, Številka:
1
Journal Article
Recenzirano
Odprti dostop
Yeast cells are able to tolerate and adapt to a variety of environmental stresses. An essential aspect of stress adaptation is the regulation of monovalent ion concentrations. Ion regulation ...determines many fundamental physiological parameters, such as cell volume, membrane potential, and intracellular pH. It is achieved through the concerted activities of multiple cellular components, including ion transporters and signaling molecules, on both short and long time scales. Although each component has been studied in detail previously, it remains unclear how the physiological parameters are maintained and regulated by the concerted action of all components under a diverse range of stress conditions. In this study, we have constructed an integrated mathematical model of ion regulation in Saccharomyces cerevisiae to understand this coordinated adaptation process. Using this model, we first predict that the interaction between phosphorylated Hog1p and Tok1p at the plasma membrane inhibits Tok1p activity and consequently reduces Na(+) influx under NaCl stress. We further characterize the impacts of NaCl, sorbitol, KCl and alkaline pH stresses on the cellular physiology and the differences between the cellular responses to these stresses. We predict that the calcineurin pathway is essential for maintaining a non-toxic level of intracellular Na(+) in the long-term adaptation to NaCl stress, but that its activation is not required for maintaining a low level of Na(+) under other stresses investigated. We provide evidence that, in addition to extrusion of toxic ions, Ena1p plays an important role, in some cases alongside Nha1p, in re-establishing membrane potential after stress perturbation. To conclude, this model serves as a powerful tool for both understanding the complex system-level properties of the highly coordinated adaptation process and generating further hypotheses for experimental investigation.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The major fungal pathogen of humans, Candida albicans, mounts robust responses to oxidative stress that are critical for its virulence. These responses counteract the reactive oxygen species (ROS) ...that are generated by host immune cells in an attempt to kill the invading fungus. Knowledge of the dynamical processes that instigate C. albicans oxidative stress responses is required for a proper understanding of fungus-host interactions. Therefore, we have adopted an interdisciplinary approach to explore the dynamical responses of C. albicans to hydrogen peroxide (H2O2). Our deterministic mathematical model integrates two major oxidative stress signalling pathways (Cap1 and Hog1 pathways) with the three major antioxidant systems (catalase, glutathione and thioredoxin systems) and the pentose phosphate pathway, which provides reducing equivalents required for oxidative stress adaptation. The model encapsulates existing knowledge of these systems with new genomic, proteomic, transcriptomic, molecular and cellular datasets. Our integrative approach predicts the existence of alternative states for the key regulators Cap1 and Hog1, thereby suggesting novel regulatory behaviours during oxidative stress. The model reproduces both existing and new experimental observations under a variety of scenarios. Time- and dose-dependent predictions of the oxidative stress responses for both wild type and mutant cells have highlighted the different temporal contributions of the various antioxidant systems during oxidative stress adaptation, indicating that catalase plays a critical role immediately following stress imposition. This is the first model to encapsulate the dynamics of the transcriptional response alongside the redox kinetics of the major antioxidant systems during H2O2 stress in C. albicans.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Candida species are important pathogens of humans and the fourth most commonly isolated pathogen from nosocomial blood stream infections. Although Candida albicans is the principle causative agent of ...invasive candidosis, the incidence of
infections has rapidly grown. The reason for this increase is not fully understood, but it is clear that the species has a higher innate tolerance to commonly administered azole antifungals, in addition to being highly tolerant to stresses especially oxidative stress. Taking the approach that using the model organism,
, with its intrinsic sensitivity to oxidative stress, we hypothesized that by expressing mediators of stress resistance from C. glabrata in
, it would result in induced resistance. To test this we transformed, en-masse, the
ORFeome library into
. This resulted in 1,500 stress resistant colonies and the recovered plasmids of 118 ORFs. Sequencing of these plasmids revealed a total of 16 different C. glabrata ORFs. The recovery of genes encoding known stress protectant proteins such as GPD1, GPD2 and TRX3 was predicted and validated the integrity of the screen. Through this screen we identified a C. glabrata unique ORF that confers oxidative stress resistance. We set to characterise this gene herein, examining expression in oxidative stress sensitive strains, comet assays to measure DNA damage and synthetic genetic array analysis to identify genetic interaction maps in the presence and absence of oxidative stress.
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