Stress signalling and fungal pathogenesis in Candida species

PhD ThesisCandida albicans and Candida glabrata are major pathogens of humans, causing 8% of all hospital acquired systemic infections worldwide. Moreover, such systemic infections are associated with alarmingly high morbidity and mortality rates. One of the major immune defence mechanisms mounted by the host against fungal infections involves phagocytosis by innate immune cells. Phagocytic immune cells employ a suite of antimicrobial mechanisms in order to kill invading pathogens, such as the generation of reactive oxygen species (ROS), cationic fluxes, nutrient deprivation, extremes of pH, and the release of antimicrobial peptides. Being successful pathogens, C. albicans and C. glabrata have acquired multiple defence strategies to allow survival in the host, and in vitro demonstrate high levels of resistance to many of the stresses likely to be encountered following phagocytosis. However, these fungi can only cause systemic infections when host immune responses are compromised. A major question, therefore, is what underlies the potency of innate immune defences in healthy individuals to prevent fungal infections? I address this question in this thesis, and investigate the interplay between fungal stress responses and immune defences of the host. Recent studies have indicated that it is exposure to combinations of stresses encountered following phagocytosis that effectively kills C. albicans. Specifically, the combination of oxidative and cationic stresses leads to a dramatic increase in intracellular ROS levels, which kills this fungus much more effectively than the corresponding single stresses in vitro. In this work I show that combinatorial oxidative and cationic stresses, or high concentrations of ROS, delay the activation of the oxidative stress-responsive Cap1 transcription factor in C. albicans. Cap1 is oxidised in response to H2O2, which masks the nuclear export sequence from the Crm1 nuclear export factor. This allows for the nuclear accumulation of the transcriptional factor and induction of Cap1-dependent antioxidant genes. In this work I demonstrate that combinatorial stress, or high ROS levels, trigger the generation of a transcriptionally inactive, partially oxidised, Cap1OX-1 form. However, whilst Cap1OX-1 readily accumulates in the nucleus and binds to target genes following high H2O2 stress, the nuclear accumulation of Cap1OX-1 following combinatorial H2O2 and NaCl stress is delayed due to a cationic stress-enhanced interaction with the Crm1 nuclear export factor. These findings define novel mechanisms that delay activation of the Cap1 transcription factor, thus preventing the rapid activation of stress responses vital for the survival of C. albicans within the host, and which probably underlines the potency of the innate immune cells in immunocompetent hosts. C. glabrata is more resistant to ROS than C. albicans, and recent work from the Haynes laboratory has identified four ORFs (designated CRI-1-4) which contribute to this enhanced ROS resistance. Orthologues of CRI1-4 are seemingly not present in other fungal species, and their expression can confer oxidative and combinatorial stress resistance in the model yeast Saccharomyces cerevisiae. In this work I show that the antioxidant properties of CRI genes are not due to their role in reducing intracellular ROS levels in C. glabrata and that, in contrast to S. cerevisiae, ectopic expression in C. albicans has no impact on stress resistance. This suggests that the mechanism behind the CRI1-4 stress protection is restricted to C. glabrata and closely related fungi. To further explore the relationship between fungal stress resistance and virulence, the Caenorhabditis elegans infection model was employed. Previous unpublished work from J. Quinn laboratory revealed that key fungal stress regulators were only needed for C. albicans virulence in immunocompetent but not immunocompromised worms. This fits with the concept that survival of the pathogen against robust immune responses requires activation of key signalling pathways. C. elegans is also a well-established model used to study the process of aging. Here I use the C. elegans model of infection to study age-dependent increases in susceptibility to C. albicans-mediated killing. Significantly, as seen with immunocompromised worms, robust stress responses are only needed for C. albicans to cause infection in young but not old animals. These results indicate that age-dependent susceptibility to fungal infections is related to the immune status of the host, and that C. albicans stress responses are only important for virulence in young, immunocompetent animals. Taken together, the findings presented in this thesis provide insight into the mechanisms underlying the differential ability of C. albicans and C. glabrata to survive combinations of stresses encountered following phagocytosis, and that agedependent effect on host immune function may determine the importance of stress responses in mediating the virulence of C. albicans

Mechanisms Underlying the Delayed Activation of the Cap1 Transcription Factor in Candida albicans following Combinatorial Oxidative and Cationic Stress Important for Phagocytic Potency

ACKNOWLEDGMENTS We are grateful to Brian Morgan and Elizabeth Veal for insightful discussions, Mélanie Ikeh for experimental assistance, and Scott Moye-Rowley (University of Iowa) for the gift of the anti-Cap1 antibody. This work was funded by the NIHR Newcastle Biomedical Research Centre (I.K.), a BBSRC DTG studentship (M.J.P.), the Wellcome Trust (grants 089930 and 097377 to J.Q. and 080088 and 097377 to A.J.P.B.), the BBSRC (grants BB/K016393/1 to J.Q. and BB/F00513X/1 and BB/K017365/1 to A.J.P.B.), the European Research Council (STRIFE Advanced grant ERC-2009-AdG-249793 to A.J.P.B.), the ANR (grant CANDIHUB, ANR-14-CE14-0018-01, to C.D.), and the French Government’s Investissement d’Avenir program (grant IBEID, ANR-10-LABX-62-IBEID, to C.D.). FUNDING INFORMATION This work, including the efforts of Alistair J.P. Brown, was funded by Wellcome Trust (097377 and 080088). This work, including the efforts of Janet Quinn, was funded by Wellcome Trust (097377 and 089930). This work, including the efforts of Alistair J.P. Brown, was funded by EC European Research Council (ERC) (ERC-2009-AdG-249793). This work, including the efforts of Alistair J.P. Brown, was funded by Biotechnology and Biological Sciences Research Council (BBSRC) (BB/F00513X/1 and BB/K017365/1). This work, including the efforts of Janet Quinn, was funded by Biotechnology and Biological Sciences Research Council (BBSRC) (BB/K016393/1). This work, including the efforts of Christophe d’Enfert, was funded by Agence Nationale de la Recherche (ANR) (ANR-14-CE14-0018-01 and ANR-10-LABX-62-IBEID).Peer reviewedPublisher PD

Oxidative Stress Responses in the Human Fungal Pathogen, Candida albicans

Candida albicans is a major fungal pathogen of humans, causing approximately 400,000 life-threatening systemic infections world-wide each year in severely immunocompromised patients. An important fungicidal mechanism employed by innate immune cells involves the generation of toxic reactive oxygen species (ROS), such as superoxide and hydrogen peroxide. Consequently, there is much interest in the strategies employed by C. albicans to evade the oxidative killing by macrophages and neutrophils. Our understanding of how C. albicans senses and responds to ROS has significantly increased in recent years. Key findings include the observations that hydrogen peroxide triggers the filamentation of this polymorphic fungus and that a superoxide dismutase enzyme with a novel mode of action is expressed at the cell surface of C. albicans. Furthermore, recent studies have indicated that combinations of the chemical stresses generated by phagocytes can actively prevent C. albicans oxidative stress responses through a mechanism termed the stress pathway interference. In this review, we present an up-date of our current understanding of the role and regulation of oxidative stress responses in this important human fungal pathogen

New insights in cell wall adhesins in [i]Candida albicans[/i]

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The LHCb upgrade I

International audienceThe LHCb upgrade represents a major change of the experiment. The detectors have been almost completely renewed to allow running at an instantaneous luminosity five times larger than that of the previous running periods. Readout of all detectors into an all-software trigger is central to the new design, facilitating the reconstruction of events at the maximum LHC interaction rate, and their selection in real time. The experiment's tracking system has been completely upgraded with a new pixel vertex detector, a silicon tracker upstream of the dipole magnet and three scintillating fibre tracking stations downstream of the magnet. The whole photon detection system of the RICH detectors has been renewed and the readout electronics of the calorimeter and muon systems have been fully overhauled. The first stage of the all-software trigger is implemented on a GPU farm. The output of the trigger provides a combination of totally reconstructed physics objects, such as tracks and vertices, ready for final analysis, and of entire events which need further offline reprocessing. This scheme required a complete revision of the computing model and rewriting of the experiment's software

The LHCb upgrade I

International audienceThe LHCb upgrade represents a major change of the experiment. The detectors have been almost completely renewed to allow running at an instantaneous luminosity five times larger than that of the previous running periods. Readout of all detectors into an all-software trigger is central to the new design, facilitating the reconstruction of events at the maximum LHC interaction rate, and their selection in real time. The experiment's tracking system has been completely upgraded with a new pixel vertex detector, a silicon tracker upstream of the dipole magnet and three scintillating fibre tracking stations downstream of the magnet. The whole photon detection system of the RICH detectors has been renewed and the readout electronics of the calorimeter and muon systems have been fully overhauled. The first stage of the all-software trigger is implemented on a GPU farm. The output of the trigger provides a combination of totally reconstructed physics objects, such as tracks and vertices, ready for final analysis, and of entire events which need further offline reprocessing. This scheme required a complete revision of the computing model and rewriting of the experiment's software

The LHCb upgrade I

The LHCb upgrade represents a major change of the experiment. The detectors have been almost completely renewed to allow running at an instantaneous luminosity five times larger than that of the previous running periods. Readout of all detectors into an all-software trigger is central to the new design, facilitating the reconstruction of events at the maximum LHC interaction rate, and their selection in real time. The experiment's tracking system has been completely upgraded with a new pixel vertex detector, a silicon tracker upstream of the dipole magnet and three scintillating fibre tracking stations downstream of the magnet. The whole photon detection system of the RICH detectors has been renewed and the readout electronics of the calorimeter and muon systems have been fully overhauled. The first stage of the all-software trigger is implemented on a GPU farm. The output of the trigger provides a combination of totally reconstructed physics objects, such as tracks and vertices, ready for final analysis, and of entire events which need further offline reprocessing. This scheme required a complete revision of the computing model and rewriting of the experiment's software

The LHCb upgrade I

International audienceThe LHCb upgrade represents a major change of the experiment. The detectors have been almost completely renewed to allow running at an instantaneous luminosity five times larger than that of the previous running periods. Readout of all detectors into an all-software trigger is central to the new design, facilitating the reconstruction of events at the maximum LHC interaction rate, and their selection in real time. The experiment's tracking system has been completely upgraded with a new pixel vertex detector, a silicon tracker upstream of the dipole magnet and three scintillating fibre tracking stations downstream of the magnet. The whole photon detection system of the RICH detectors has been renewed and the readout electronics of the calorimeter and muon systems have been fully overhauled. The first stage of the all-software trigger is implemented on a GPU farm. The output of the trigger provides a combination of totally reconstructed physics objects, such as tracks and vertices, ready for final analysis, and of entire events which need further offline reprocessing. This scheme required a complete revision of the computing model and rewriting of the experiment's software

The LHCb upgrade I

International audienceThe LHCb upgrade represents a major change of the experiment. The detectors have been almost completely renewed to allow running at an instantaneous luminosity five times larger than that of the previous running periods. Readout of all detectors into an all-software trigger is central to the new design, facilitating the reconstruction of events at the maximum LHC interaction rate, and their selection in real time. The experiment's tracking system has been completely upgraded with a new pixel vertex detector, a silicon tracker upstream of the dipole magnet and three scintillating fibre tracking stations downstream of the magnet. The whole photon detection system of the RICH detectors has been renewed and the readout electronics of the calorimeter and muon systems have been fully overhauled. The first stage of the all-software trigger is implemented on a GPU farm. The output of the trigger provides a combination of totally reconstructed physics objects, such as tracks and vertices, ready for final analysis, and of entire events which need further offline reprocessing. This scheme required a complete revision of the computing model and rewriting of the experiment's software

The LHCb upgrade I

International audienceThe LHCb upgrade represents a major change of the experiment. The detectors have been almost completely renewed to allow running at an instantaneous luminosity five times larger than that of the previous running periods. Readout of all detectors into an all-software trigger is central to the new design, facilitating the reconstruction of events at the maximum LHC interaction rate, and their selection in real time. The experiment's tracking system has been completely upgraded with a new pixel vertex detector, a silicon tracker upstream of the dipole magnet and three scintillating fibre tracking stations downstream of the magnet. The whole photon detection system of the RICH detectors has been renewed and the readout electronics of the calorimeter and muon systems have been fully overhauled. The first stage of the all-software trigger is implemented on a GPU farm. The output of the trigger provides a combination of totally reconstructed physics objects, such as tracks and vertices, ready for final analysis, and of entire events which need further offline reprocessing. This scheme required a complete revision of the computing model and rewriting of the experiment's software