David M. Arana

Differential susceptibility of mitogen-activated protein kinase pathway mutants to oxidative-mediated killing by phagocytes in the fungal pathogen Candida albicans.

The role of four mitogen‐activated protein (MAP) kinase pathways in the survival of Candida albicans following infection of human phagocytes has been addressed through the analysis of mutants defective in their respective MAP kinase. While the contribution of the cell integrity (Mkc1‐mediated) or mating (Cek2‐mediated) pathways is relatively minor to survival, clear and opposite effects were observed for cek1 and hog1 mutants, despite the fact that these two MAP kinases are important virulence determinants in the mouse model of experimental infection. The Cek1‐mediated pathway is involved in sensitivity to phagocyte‐mediated killing, while the HOG pathway contributes to the survival of the fungal cells in this interaction. Furthermore, reporter genes have been developed to quantify oxidative and nitrosative stress. hog1 mutants show an oxidative and nitrosative stress response augmented – albeit non‐protective – when challenged with oxidants and NO donors in vitro or phagocytic cells (macrophages, neutrophils and the myelomonocytic cell line HL‐60), suggesting this as the cause of their reduced virulence in the murine model of infection. These data have important consequences for the development of novel antifungal therapies to combat against fungal infection.

Candida albicans β-Glucan Exposure Is Controlled by the Fungal CEK1-Mediated Mitogen-Activated Protein Kinase Pathway That Modulates Immune Responses Triggered through Dectin-1

ABSTRACT Innate immunity to Candida albicans depends upon the recognition of molecular patterns on the fungal cell wall. However, the masking of major components such as β-glucan seems to be a mechanism that fungi have evolved to avoid immune cell recognition through the dectin-1 receptor. Although the role of C. albicans mitogen-activated protein kinase (MAPK) pathways as virulence determinants has been established previously with animal models, the mechanism involved in this behavior is largely unknown. In this study we demonstrate that a disruption of the C. albicans extracellular signal-regulated kinase (ERK)-like 1 (CEK1)-mediated MAPK pathway causes enhanced cell wall β-glucan exposure, triggering immune responses more efficiently than the wild type, as measured by dectin-1-mediated specific binding and human dendritic cell (hDC)- and macrophage-mediated phagocytosis, killing, and activation of intracellular signaling pathways. At the molecular level, the disruption of CEK1 resulted in altered spleen tyrosine kinase (Syk), Raf-1, and ERK1/2 activations together with IκB degradation on hDCs and increased dectin-1-dependent activator protein 1 (AP-1) activation on transfected cells. In addition, concurring with these altered pathways, we detected increased reactive oxygen species production and cytokine secretion. In conclusion, the CEK1-mediated MAPK pathway is involved in β-glucan exposure in a fungal pathogen, hence influencing dectin-1-dependent immune cell recognition, thus establishing this fungal intracellular signaling route as a promising novel therapeutic target.

Fungi sensing environmental stress

Cells need to adapt to the external environment in order to survive. Signal transduction pathways are crucial mechanisms that allow cells to sense and respond to extracellular stimuli. Among the signal transduction pathways, we point out the cascades mediated by mitogen-activated protein kinases (MAPKs). The MAPKs are conserved from yeast to human and play relevant roles in the physiology of the cell. In pathogenic fungi these MAPK pathways control virulence factors. This review describes the MAPK cascades described in Candida albicans, the most frequently isolated fungus, from fungal systemic infections among individuals in developed countries.

The Sko1 protein represses the yeast-to-hypha transition and regulates the oxidative stress response in Candida albicans

Cells respond to environmental changes triggering adaptive responses which are, in part, mediated by a transcriptional response. These responses are complex and are dependent on different transcription factors. The present work reports the implication of the Sko1 protein in several processes relevant to the physiology of Candida albicans. First, Sko1 acts as transcriptional repressor of genes involved in pathogenesis and hyphal formation, which results in increased expression of the hyphal related genes ECE1 and HWP1 without significant changes in the virulence using a mouse model of systemic infection. Second Sko1 is involved in the response to oxidative stress and sko1 mutants increase the sensitivity of hog1 to the myelomonocytic cell line HL-60. Genome-wide transcriptional analysis after hydrogen peroxide treatment revealed that sko1 mutants were able to generate an adaptive response similar to wild type strains, although important differences were detected in the magnitude of the transcriptional response. Collectively, these results implicate Sko1 as an important mediator of the oxidative stress response in C. albicans.

Fluconazole at subinhibitory concentrations induces the oxidative- and nitrosative-responsive genes TRR1, GRE2 and YHB1, and enhances the resistance of Candida albicans to phagocytes

OBJECTIVES To analyse the oxidative and nitrosative stress response in Candida albicans generated by fluconazole at subinhibitory concentrations, and the functional consequences of such a response for the interaction with phagocytic cells. METHODS The C. albicans CAI-4 strain carrying transcriptional fusions of the TRR1p, YHB1p and GRE2p genes to the Renilla reniformis luciferase LUC gene was pre-treated with subinhibitory concentrations of fluconazole and incubated with oxidants (diamide and hydrogen peroxide) or with the myelomonocytic cell line HL-60. RESULTS Fluconazole induced oxidative and nitrosative stress in a time- and dose-dependent manner as determined using oxidative- and nitrosative-specific gene reporters. At subinhibitory concentrations, fluconazole was able to induce protection in vitro to subsequent challenges with oxidants in both liquid and solid media, and also induced partial protection against the oxidative-mediated killing mechanisms of the myelocytic HL-60 cells. CONCLUSIONS Subinhibitory concentrations of fluconazole protect against oxidants and killing mediated by phagocytes.

The role of the cell wall in fungal pathogenesis

Fungal infections are a serious health problem. In recent years, basic research is focusing on the identification of fungal virulence factors as promising targets for the development of novel antifungals. The wall, as the most external cellular component, plays a crucial role in the interaction with host cells mediating processes such as adhesion or phagocytosis that are essential during infection. Specific components of the cell wall (called PAMPs) interact with specific receptors in the immune cell (called PRRs), triggering responses whose molecular mechanisms are being elucidated. We review here the main structural carbohydrate components of the fungal wall (glucan, mannan and chitin), how their biogenesis takes place in fungi and the specific receptors that they interact with. Different model fungal pathogens are chosen to illustrate the functional consequences of this interaction. Finally, the identification of the key components will have important consequences in the future and will allow better approaches to treat fungal infections.

Reporting antimicrobial susceptibilities and resistance phenotypes in Acinetobacter spp: a nationwide proficiency study

Objectives To evaluate the proficiency of Spanish microbiology laboratories with respect to the antimicrobial susceptibility testing (AST) of Acinetobacter spp. Methods Eight Acinetobacter spp. with different resistance mechanisms were sent to 48 Spanish centres which were asked to report: (i) the AST system used; (ii) MICs; (iii) breakpoints used (CLSI versus EUCAST); (iv) clinical category; and (v) resistance mechanisms inferred. Minor, major and very major errors (mE, ME and VME, respectively) were determined. Results The greatest percentages of discrepancies were: (i) by AST method: 18.5% Etest, 14.3% Vitek 2 and Sensititre; (ii) by breakpoints: 20.5% (CLSI) and 10.8% (EUCAST); and (iii) by antimicrobial agent: ampicillin/sulbactam (56.2% CLSI), minocycline (40.7% CLSI), tobramycin (38.7% CLSI, 16.8% EUCAST), imipenem (27.8% CLSI, 30.0% EUCAST) and meropenem (25.4% CLSI, 20.8% EUCAST). Categorical error rates: (i) by AST method ranged from 30.0% (Phoenix) to 100% (Sensititre and disc diffusion) for mE, 0.0% (Etest, Sensititre, disc diffusion) to 40% (Phoenix) for ME, and 0.0% (Sensititre and disc diffusion) to 30% (Phoenix) for VME; (ii) by breakpoints: mE (80.1% CLSI, 58.4% EUCAST), ME (3.5% CLSI, 12.4% EUCAST) and VME (16.4% CLSI, 29.2% EUCAST); and (iii) by antimicrobial agent: mE (100% levofloxacin/CLSI, 100% levofloxacin and meropenem/EUCAST), ME (35.3% colistin/CLSI, 25.0% colistin/EUCAST) and VME (64.7% colistin/CLSI, 86.7% gentamicin/EUCAST). Conclusions Clinical microbiology laboratories must improve their ability to determine antimicrobial susceptibilities of Acinetobacter spp. isolates. Higher discrepancies using CLSI when compared with EUCAST are mainly due to mE and to a much lesser extent to ME or VME.

Chapter 16 Signalling and defences against oxidative stress in Candida albicans

Abstract Oxidative stress is one the major mechanisms by which immune cells are able to deal with microbial infections. Pathogens have therefore developed a set of antioxidant mechanisms, both non-enzymatic and enzymatic in nature. This process is adaptive and in the human pathogenic fungus Candida albicans different MAP kinase-mediated signal transduction pathways have been shown to play an essential role in transmitting the signal to downstream effectors, in addition to their roles in other processes such as morphogenesis and virulence. The cell integrity pathway, mediated by the Mkcl MAP kinase, is activated in response to a wide range of conditions, including oxidative, saline and cell wall stress. The HOG pathway, mediated by the Hogl MAP kinase, is also activated in response to different stress situations (osmotic and oxidative) and is able to trigger a transcriptional specific response, which is distinguishable from that due to other mediators such as the Yapl-homologue Capl or the Skn7 regulator. Manipulation of the oxidative stress response, either in the host or in the microbe, may therefore be an important therapeutic mechanism to control fungal infections.

In vitro models to analyse fungal infection

According to the molecular Koch’s postulates (Falkow, 1988), putative virulence traits can be identified in a pathogen because deletion of the gene encoding a virulence factor in an otherwise wild-type strain generates a mutant with reduced pathogenicity in a certain model of experimental infection. Recent advances in molecular genetics have led to the generation of such altered strains in several clinically relevant fungi, including Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus, and Histoplasma capsulatum. This has, in turn allowed the identification of several virulence genes involved in important physiological processes in the pathogen. These processes include, among others, the biogenesis of the cell wall, the acquisition of nutrients, the production of extracellular enzymes, and the tolerance to stress. In experimental infection models these mutants frequently display attenuated or abolished virulence. While this methodology provides global information on whether a gene is involved in virulence or not, it does not define the specific step(s) in the pathogenic process that is (are) impaired by the molecular lesion. During the pathoenic cycle fungi interact with various types of host cells, which may lead to dissemination from the original entry site and deep-seated infection of inner organs (Figure 2.1). This interaction is characterized by successive events at the cellular level. Fungi first attach and then enter the host cells, where they may persist or even proliferate before they leave and infect other host cells and tissues. Each of these steps may be crucial for the development of the disease, and virulence factors may contribute in each of these steps by different molecular mechanisms (Figure 2.2). In vitro models of infection provide a defined experimental set-up to characterize the host– pathogen interplay at a cellular level and allow us to ascertain more precisely the molecular lesion present in the mutant and the corresponding step of the pathogenic process specifically altered. This chapter reviews the main in vitro models for epithelial, endothelial, and immune system cells. It outlines the available techniques to characterize and to quantify host cell–pathogen interactions following a structure as preset by the