Federico Navarro-García

The Hog1 mitogen-activated protein kinase is essential in the oxidative stress response and chlamydospore formation in Candida albicans

ABSTRACT Candida albicans mutants with mutations in mitogen-activated protein (MAP) kinase HOG1 displayed an increased sensitivity to agents producing reactive oxygen species, such as oxidants (menadione, hydrogen peroxide, or potassium superoxide), and UV light. Consistent with this finding, C. albicans Hog1 was activated not only in response to an increase in external osmolarity, as happens with its Saccharomycescerevisiae homologue, but also in response to hydrogen peroxide. The Hog1-mediated response to oxidative stress was different from that of transcription factor Cap1, the homologue of S. cerevisiae Yap1, as shown by the different sensitivities to oxidants and the kinetics of cell death of cap1Δ, hog1, and hog1cap1Δ mutants. Deletion of CAP1 did not influence the level of Hog1 phosphorylation, and deletion of HOG1 did not affect Cap1 nuclear localization. Moreover, we show that the HOG1 gene plays a role in chlamydospore formation, another oxygen-related morphogenetic event, as demonstrated by the fact that hog1 cells were unable to generate these thick-walled structures in several media through a mechanism different from that of the EFG1 regulator. This is the first demonstration of the role of the Hog1-mediated MAP kinase pathway in resistance to oxidative stress in pathogenic fungi, and it allows us to propose a molecular model for the oxidative stress response in C. albicans.

Functional characterization of the MKC1 gene of Candida albicans, which encodes a mitogen-activated protein kinase homolog related to cell integrity.

Mitogen-activated protein (MAP) kinases represent a group of serine/threonine protein kinases playing a central role in signal transduction processes in eukaryotic cells. Using a strategy based on the complementation of the thermosensitive autolytic phenotype of slt2 null mutants, we have isolated a Candida albicans homolog of Saccharomyces cerevisiae MAP kinase gene SLT2 (MPK1), which is involved in the recently outlined PKC1-controlled signalling pathway. The isolated gene, named MKC1 (MAP kinase from C. albicans), coded for a putative protein, Mkc1p, of 58,320 Da that displayed all the characteristic domains of MAP kinases and was 55% identical to S. cerevisiae Slt2p (Mpk1p). The MKC1 gene was deleted in a diploid Candida strain, and heterozygous and homozygous strains, in both Ura+ and Ura- backgrounds, were obtained to facilitate the analysis of the function of the gene. Deletion of the two alleles of the MKC1 gene gave rise to viable cells that grew at 28 and 37 degrees C but, nevertheless, displayed a variety of phenotypic traits under more stringent conditions. These included a low growth yield and a loss of viability in cultures grown at 42 degrees C, a high sensitivity to thermal shocks at 55 degrees C, an enhanced susceptibility to caffeine that was osmotically remediable, and the formation of a weak cell wall with a very low resistance to complex lytic enzyme preparations. The analysis of the functions downstream of the MKC1 gene should contribute to understanding of the connection of growth and morphogenesis in pathogenic fungi.

A role for the MAP kinase gene MKC1 in cell wall construction and morphological transitions in Candida albicans.

The Candida albicans MKC1 gene encodes a mitogen-activated protein (MAP) kinase, which has been cloned by complementation of the lytic phenotype associated with Saccharomyces cerevisiae slt2 (mpk1) mutants. In this work, the physiological role of this MAP kinase in the pathogenic fungus C. albicans was characterized and a role for MKC1 in the biogenesis of the cell wall suggested based on the following criteria. First, C. albicans mkc1 delta/mkc1 delta strains displayed alterations in their cell surfaces under specific conditions as evidenced by scanning electron microscopy. Second, an increase in specific cell wall epitopes (O-glycosylated mannoprotein) was shown by confocal microscopy in mkc1 delta/mkc1 delta mutants. Third, the sensitivity to antifungals which inhibit (1,3)-beta-glucan and chitin synthesis was increased in these mutants. In addition, evidence for a role for the MKC1 gene in morphological transitions in C. albicans is presented based on the impairment of pseudohyphal formation of mkc1 delta/mkc1 delta strains on Spider medium and on the effect of its overexpression on Sacch. cerevisiae colony morphology on SLADH medium. Using the two-hybrid system, it was also demonstrated that MKC1 is able to interact specifically with Sacch. cerevisiae Mkk1p and Mkk2p, the MAP-kinase kinases of the PKC1-mediated route of Sacch. cerevisiae, and to activate transcription in Sacch. cerevisiae when bound to a DNA-binding element. These results suggest a role for this MAP kinase in the construction of the cell wall of C. albicans and indicate its potential relevance for the development of novel antifungals.

Cloning of the Candida albicans HIS1 gene by direct complementation of a C. albicans histidine auxotroph using an improved double-ARS shuttle vector ☆

ARS2 and ARS3 are two Candida albicans (Ca) DNA fragments with autonomous replicating activity that have been shown to promote non-integrative genetic transformation of both Ca and Saccharomyces cerevisiae (Sc). We have developed several shuttle vectors based on either ARS fragment, or the combination of both, and using the CaURA3 gene as a selection marker. The combination of ARS2 and ARS3 fragments in a single vector did not increase transformation frequencies but improved the stability of transformant plasmids in Ca cells, so that the degree of intracellular recombination was reduced. A Ca genomic DNA library was constructed on the double-ARS vector, pRM1, to be used for direct cloning in Ca by complementation of the histidine auxotrophy of strain CA9. By screening this library, we cloned CaHIS1, the Ca gene that encodes ATP phosphoribosyl transferase, one of the enzymes that participates in histidine biosynthesis. The deduced protein, CaHis1p, is 60.6% identical (73% similar) to ScHis1p (EC 2.4.2.17). The cloned gene is the first auxotrophic gene marker mapped to fragment I of chromosome 5 in the standard Ca genetic map. Our results represent the first demonstration of a direct cloning system in the opportunistic fungus Ca that does not require the use of an intermediate host such as Sc for plasmid rescue. This system could be used for the isolation of any gene affected in Ca mutants displaying a selectable or identifiable phenotype.

Chromosome reorganization in Candida albicans 1001 strain

The karotype of Candida albicans 1001, a pathogenic isolate, presents two additional chromosome bands when compared with C. albicans 1006 strain. These two bands were a 2600 kb chromosome located between chromosome group 1-R and chromosome 2 (named chromosome 2*) and a 710 kb small chromosome, called snc due to its similarity in size to the supernumerary chromosome in strain WO-1. A comparison of karyotypes of strains 1001, 1006 and WO-1 has enabled us to conclude that chromosomes 2 and 7 are involved in such a reorganization giving rise to the new chromosome bands of strain 1001. We describe a tentative physical map of C. albicans 1001 based on the previously outlined map of C. albicans strain 1006.

Strategies for the identification of virulence determinants in human pathogenic fungi

Abstract. The incidence of fungal infections is increasing in different countries. The current available therapy of these infections does not satisfy all requirements in terms of specificity and therapeutic index, a fact that has stimulated the scientific community to identify fungal virulence determinants. Several pathogenic fungi are opportunistic and, therefore, identification of virulence genes is difficult, given their close relationship with host cells. In recent years, the development of genetic tools in several pathogenic fungi has enabled the development of genetic strategies for their identification. These include several strategies based on the phenotypic analysis of strains or environmental conditions in which the expression of the putative gene(s) is either altered or deleted; and this is accomplished through the development of in vitro or in vivo systems. In the near future, this research will produce a better picture of fungal pathogenesis and therefore define novel promising targets in antifungal therapy.

Fate of classical faecal bacterial markers and ampicillin-resistant bacteria in agricultural soils under Mediterranean climate after urban sludge amendment

The use of sewage sludge or biosolids as agricultural amendments may pose environmental and human health risks related to pathogen or antibiotic-resistant microorganism transmission from soils to vegetables or to water through runoff. Since the survival of those microorganisms in amended soils has been poorly studied under Mediterranean climatic conditions, we followed the variation of soil fecal bacterial markers and ampicillin-resistant bacteria for two years with samplings every four months in a split block design with three replica in a crop soil where two different types of biosolids (aerobically or anaerobically digested) at three doses (low, 40; intermediate, 80; and high, 160Mg·ha(-1)) were applied. Low amounts of biosolids produced similar decay rates of coliform populations than in control soil (-0.19 and -0.27log10CFUs·g(-1)drysoilmonth(-1) versus -0.22) while in the case of intermediate and high doses were close to zero and their populations remained 24months later in the range of 4-5log10CFUs·g(-1)ds. Enterococci populations decayed at different rates when using aerobic than anaerobic biosolids although high doses had higher rates than control (-0.09 and -0.13log10CFUs·g(-1)dsmonth(-1) for aerobic and anaerobic, respectively, vs -0.07). At the end of the experiment, counts in high aerobic and low and intermediate anaerobic plots were 1 log10 higher than in control (4.21, 4.03, 4.2 and 3.11log10CFUs·g(-1) ds, respectively). Biosolid application increased the number of Clostridium spores in all plots at least 1 log10 with respect to control with a different dynamic of decay for low and intermediate doses of aerobic and anaerobic sludge. Ampicillin-resistant bacteria increased in amended soils 4months after amendment and remained at least 1 log10 higher 24months later, especially in aerobic and low and intermediate anaerobic plots due to small rates of decay (in the range of -0.001 to -0.008log10CFUs·g(-1)dsmonth(-1) vs -0.016 for control). Aerobic plots had relative populations of ampicillin-resistant bacteria higher than anaerobic plots with different positive trends. Dose (22%) and time (13%) explained most of the variation of the bacterial populations. Dynamics of fecal markers did not correlate with ampicillin-resistant bacteria thus making necessary to evaluate specifically this trait to avoid possible risks for human and environmental health.

Cloning and sequence of a 3·835 kbp DNA fragment containing the HIS4 gene and a fragment of a PEX5‐like gene from Candida albicans

We have isolated the Candida albicans HIS4 (CaHIS4) gene by complementation of a his4‐34 Saccharomyces cerevisiae mutant. The sequenced DNA fragment contains a putative ORF of 2514 bp, whose translation product shares a global identity of 44% and 55% to the His4 protein homologs of S. cerevisiae and Kluyveromyces lactis, respectively. Analysis of CaHIS4 sequence suggests that, similarly to S. cerevisiae HIS4, it codes for a polypeptide having three separate enzymatic activities (phosphoribosyl‐AMP cyclohydrolase, phosphoribosyl‐ATP pyrophosphohydrolase and histidinol dehydrogenase) which reside in different domains of the protein. A C. albicans his4 strain is complemented with this gene when using a C. albicans‐S. cerevisiae‐Escherichia coli shuttle vector, thus enabling the construction of a host system for C. albicans genetic manipulation. In addition, upstream of the sequenced CaHIS4 sequence, we have found the 3′‐terminal half of a gene encoding a PEX5‐like protein. The EMBL/DDJB/GenBank Accession Number of this sequence is AJ003115.

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 Debaryomyces hansenii (Dh) strains isolated from a wide variety of cheeses were identified as producing killer toxins active against Candida albicans and Candida tropicalis. We have analyzed the killer activity of these toxins in C. albicans mutants defective in MAPK signaling pathways and found that the lack of the MAPK Hog1 (but not Cek1 or Mkc1) renders cells hypersensitive to Dh 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 HOG1 gene showed that both activities were relevant for the survival of C. albicans to Dh 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 D. hansenii 242 strain (Dh-242) induced a slight intracellular increase in Reactive Oxygen Species (ROS), overexpression of cytosolic catalase did not protect C. albicans 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 Dh-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.