The HOG MAPK pathway in Candida albicans: more than an osmosensing pathway.

In 1993, Brewster and Gustin described the existence of a kinase whose activity was essential for Saccharomyces cerevisiae to grow in environments with high osmolarity. This led to the discovery of the HOG pathway, a MAP kinase (MAPK) pathway that has been revealed to be crucial to respond to a wide range of stress conditions frequently encountered by fungi in their common habitats. MAPK signaling is initiated at the plasma membrane, where triggering stimuli lead to a phosphorylation cascade that ultimately activates transcription factors to ensure an appropriate adaptive response. In pathogenic fungi, the HOG pathway gains special significance as it is involved in traits related to pathogenicity; these include biofilm formation, adhesion to surfaces, and morphogenetic and epigenetic transitions. It also plays a role in controlling both the pathogen and the commensal state program. Understanding the signals leading to its activation, the elements of the pathways and the targets of the pathway are therefore of primary importance in the design of novel antifungals

Implementation of a CRISPR-Based System for Gene Regulation in Candida albicans.

Clustered regularly interspaced short palindromic repeat (CRISPR) methodology is not only an efficient tool in gene editing but also an attractive platform to facilitate DNA, RNA, and protein interactions. We describe here the implementation of a CRISPR-based system to regulate expression in the clinically important yeast By fusing an allele of Cas9 devoid of nuclease activity to a transcriptional repressor (Nrg1) or activator (Gal4), we were able to show specific repression or activation of the tester gene , encoding the cytosolic catalase. We generated strains where a 1.6-kbp upstream regulatory region of controls the expression of the green fluorescent protein (GFP) and demonstrated the functionality of the constructs by quantitative PCR (qPCR), flow cytometry, and analysis of sensitivity/resistance to hydrogen peroxide. Activation and repression were strongly dependent on the position of the complex in this regulatory region. We also improved transcriptional activation using an RNA scaffolding strategy to allow interaction of inactive variants of Cas9 (dCas9) with the RNA binding protein MCP (monocyte chemoattractant protein) fused to the VP64 activator. The strategy shown here may facilitate the analysis of complex regulatory traits in this fungal pathogen. CRISPR technology is a new and efficient way to edit genomes, but it is also an appealing way to regulate gene expression. We have implemented CRISPR as a gene expression platform in using fusions between a Cas9 inactive enzyme and specific repressors or activators and demonstrated its functionality. This will allow future manipulation of complex virulence pathways in this important fungal pathogen

The Glyoxylate Cycle Is Involved in White-Opaque Switching in Candida albicans

Candida albicans is a commensal yeast that inhabits the gastrointestinal tract of humans. The master regulator of the white-opaque transition WOR1 has been implicated in the adaptation to this commensal status. A proteomic analysis of cells overexpressing this transcription factor (WOR1OE) suggested an altered metabolism of carbon sources and a phenotypic analysis confirmed this alteration. The WOR1OE cells are deficient in using trehalose and xylose and are unable to use 2C sources, which is consistent with a reduction in the amount of Icl1, the isocitrate lyase enzyme. The icl1∆/∆ mutants overexpressing WOR1 are deficient in the production of phloxine B positive cells, a main characteristic of opaque cells, a phenotype also observed in mating type hemizygous mtla1∆ icl1∆/∆ cells, suggesting the involvement of Icl1 in the adaptation to the commensal state. In fact, icl1∆/∆ cells have reduced fitness in mouse gastrointestinal tract as compared with essentially isogenic heterozygous ICL1/icl1∆, but overproduction of WOR1 in an icl1∆/∆ mutant does not restore fitness. These results implicate the glyoxylate shunt in the adaptation to commensalism of C. albicans by mechanisms that are partially independent of WOR1

TUP1-mediated filamentation in Candida albicans leads to inability to colonize the mouse gut.

AIM To investigate the role of Candida albicans TUP1-mediated filamentation in the colonization of the mice gut. MATERIALS & METHODS We used molecular genetics to generate a strain where filamentation is regulated by altering the expression of the TUP1 gene with tetracyclines. RESULTS The colonization rates reached with the TUP1-RFP strain were lower compared with wild-type strain and completely absent after induction of filamentation. No differences in the susceptibility to bile salts nor in the adhesion to the mouse intestine epithelium were observed. CONCLUSION Blockage of C. albicans in a filamentous form impedes gut cell colonization in the mouse

Climatic control on palaeohydrology and cyclical sediment distribution in the Plio-Quaternary deposits of the Guadix Basin (Betic Cordillera, Spain)

A cyclical pattern can be observed in the central sector of the Guadix Basin (southern Spain) in the Late Pliocene-Quaternary alluvial fan deposits prograding into its axial valley. A climatic significance has been attributed to this cyclicity on the basis of sedimentological and preliminary isotopic studies. The progradation phases of the alluvial fans are here attributed to more arid time intervals in which the vegetation cover would be less developed, erosion and sediment supply would be higher, and base level would be lower. In contrast, the time intervals during which the fluvial system sediments dominated the area are inferred to be wetter and base level higher, with vegetation cover retaining the soils and preventing erosion. Permanent water supply to the river would therefore facilitate the aggradation of the floodplain and prevent progradation of the fans. Starting from a litho-, bio- and magnetostratigraphical frame provided for the area, an age is assigned to the alternation of the reddish sediments of the transverse alluvial fans and the greyish to white fluvio-lacustrine sediments of the axial drainage system. A cyclicity of ca. 100 ky has been identified in most of the alluvial fan progradation phases, falling within Milankovitch high-frequency eccentricity periodicities. Correlation of the phases with insolation curves is accordingly discussed as a possible origin for the cyclicity. Finally, the results offer new insights into early hominin occupation patterns in the region, through the identification of predictable resources of permanent fresh water that would have remained available throughout the recorded time span (that includes the Early–Middle Pleistocene transition) even during times of aridification.The study was supported by the Project CGL2009-07830/BTE and the Working Group RNM-369JA

Identification of Clinical Isolates of Candida albicans with Increased Fitness in Colonization of the Murine Gut

The commensal and opportunistic pathogen Candida albicans is an important cause of fungal diseases in humans, with the gastrointestinal tract being an important reservoir for its infections. The study of the mechanisms promoting the C. albicans commensal state has attracted considerable attention over the last few years, and several studies have focused on the identification of the intestinal human mycobiota and the characterization of Candida genes involved in its establishment as a commensal. In this work, we have barcoded 114 clinical C. albicans isolates to identify strains with an enhanced fitness in a murine gastrointestinal commensalism model. The 114 barcoded clinical isolates were pooled in four groups of 28 to 30 strains that were inoculated by gavage in mice previously treated with antibacterial therapy. Eight strains that either exhibited higher colonization load and/or remained in the gut after antibiotic removal were selected. The phenotypic analysis of these strains compared to an RFP-tagged SC5314 wild type strain did not reveal any specific trait associated with its increased colonization; all strains were able to filament and six of the eight strains displayed invasive growth on Spider medium. Analysis of one of these strains, CaORAL3, revealed that although mice required previous bacterial microbiota reduction with antibiotics to be able to be colonized, removal of this procedure could take place the same day (or even before) Candida inoculation. This strain was able to colonize the intestine of mice already colonized with Candida without antibiotic treatment in co-housing experiments. CaORAL3 was also able to be established as a commensal in mice previously colonized by another (CaHG43) or the same (CaORAL3) C. albicans strain. Therefore, we have identified C. albicans isolates that display higher colonization load than the standard strain SC5314 which will surely facilitate the analysis of the factors that regulate fungal colonization

Non-canonical Activities of Hog1 Control Sensitivity of Candida albicans to Killer Toxins From Debaryomyces hansenii

Certain yeasts secrete peptides known as killer toxins or mycocins with a deleterious effect on sensitive yeasts or filamentous fungi, a common phenomenon in environmental species. In a recent work, different () strains isolated from a wide variety of cheeses were identified as producing killer toxins active against and . We have analyzed the killer activity of these toxins in mutants defective in MAPK signaling pathways and found that the lack of the MAPK Hog1 (but not Cek1 or Mkc1) renders cells hypersensitive to mycocins while mutants lacking other upstream elements of the pathway behave as the wild type strain. Point mutations in the phosphorylation site (T174A-176F) or in the kinase domain (K52R) of gene showed that both activities were relevant for the survival of to killer toxins. Moreover, Hog1 phosphorylation was also required to sense and adapt to osmotic and oxidative stress while the kinase activity was somehow dispensable. Although the addition of supernatant from the killer toxin- producing 242 strain (-242) induced a slight intracellular increase in Reactive Oxygen Species (ROS), overexpression of cytosolic catalase did not protect against this mycocin. This supernatant induced an increase in intracellular glycerol concentration suggesting that this toxin triggers an osmotic stress. We also provide evidence of a correlation between sensitivity to -242 killer toxin and resistance to Congo red, suggesting cell wall specific alterations in sensitive strains

Educating in antimicrobial resistance awareness: adaptation of the Small World Initiative program to service-learning.

The Small World Initiative (SWI) and Tiny Earth are a consolidated and successful education programs rooted in the USA that tackle the antibiotic crisis by a crowdsourcing strategy. Based on active learning, it challenges young students to discover novel bioactive-producing microorganisms from environmental soil samples. Besides its pedagogical efficiency to impart microbiology content in academic curricula, SWI promotes vocations in research and development in Experimental Sciences and, at the same time, disseminates the antibiotic awareness guidelines of the World Health Organization. We have adapted the SWI program to the Spanish academic environment by a pioneering hierarchic strategy based on service-learning that involves two education levels (higher education and high school) with different degrees of responsibility. Throughout the academic year, 23 SWI teams, each consisting of 3-7 undergraduate students led by one faculty member, coordinated off-campus programs in 22 local high schools, involving 597 high school students as researchers. Post-survey-based evaluation of the program reveals a satisfactory achievement of goals: acquiring scientific abilities and general or personal competences by university students, as well as promoting academic decisions to inspire vocations for science- and technology-oriented degrees in younger students, and successfully communicating scientific culture in antimicrobial resistance to a young stratum of society