Elva T. Aréchiga-Carvajal

The RIM101/pacC homologue from the basidiomycete Ustilago maydis is functional in multiple pH-sensitive phenomena.

ABSTRACT A homologue of the gene encoding the transcription factor Rim101 (PacC), involved in pH signal transduction in fungi, was identified in the pathogenic basidiomycete Ustilago maydis. The gene (RIM101) encodes a protein of 827 amino acid residues, which shows highest similarity to PacC proteins from Fusarium oxysporum and Aspergillus niger. The gene had the capacity to restore protease activity to rim101 mutants from Yarrowia lipolytica, confirming its homologous function, and was expressed at both acid and neutral pH. Null Δrim101 mutants were not affected in the in vitro pH-induced dimorphic transition, their growth rate, resistance to hypertonic sorbitol or KCl stress, and pathogenicity. However, similar to pacC (rim101) mutants in other fungi, they displayed a pleiotropic phenotype with alterations in morphogenesis, impairment in protease secretion, and increased sensitivity to Na+ and Li+ ions. Other phenotypic characteristics not previously reported in fungal pacC (rim101) mutants (morphological changes, increased sensitivity to lytic enzymes, and augmented polysaccharide secretion) were also observed in U. maydis mutants. All these modifications were alleviated by transformation with the wild-type gene, confirming that all were the result of mutation in RIM101. These data indicate that the Pal/Rim pathway is functional in U. maydis (and probably in other basidiomycetes) and plays complex roles in pH-sensing phenomena, as occurs in ascomycetes and deuteromycetes.

Citrus Extracts as Inhibitors of Quorum Sensing, Biofilm Formation and Motility of Campylobacter jejuni

Quorum Sensing (QS), a signaling system present in bacteria, influences the expression of a variety of virulence factors. This study investigated the ability of citrus extracts to inhibit the activity of AI-2 molecules that mediate QS in Campylobacter jejuni and the effects of these extracts on motility, biofilm formation and expression of flaA-B. Cultures of C. jejuni were exposed to extracts of Citrus limon, Citrus medica and Citrus aurantium peels at various concentrations. Swarm motility tests were performed in Muller Hinton agar; biofilm formation was determined colorimetrically in microtiter plates; AI-2 activity was measured with a bioluminescence assay using V. harveyi; and flaA-B expression was determined by qRT-PCR. Treatment with C. limon or C. medica peel extract reduced swarm motility 44–59%, whereas treatment with C. aurantium extract reduced swarm motility 35–40%. Biofilm formation was reduced 60–75 % by these extracts, depending on extract concentration and /or strain tested. All three citrus extracts decreased AI-2 activity by about 90 %, and at 75 % of MBC, significantly (P ≤ 0.05) reduced expression of flaA-B. These findings provide preliminary metabolic and molecular insights into the effects of edible antimicrobials on QS and virulence factors of C. jejuni.

Transcriptomic analysis of the role of Rim101/PacC in the adaptation of Ustilago maydis to an alkaline environment.

Alkaline pH triggers an adaptation mechanism in fungi that is mediated by Rim101/PacCp, a zinc finger transcription factor. To identify the genes under its control in Ustilago maydis, we performed microarray analyses, comparing gene expression in a wild-type strain versus a rim101/pacC mutation strain of the fungus. In this study we obtained evidence of the large number of genes regulated mostly directly, but also indirectly (probably through regulation of other transcription factors), by Rim101/PacCp, including proteins involved in a large number of physiological activities of the fungus. Our analyses suggest that the response to alkaline conditions under the control of the Pal/Rim pathway involves changes in the cell wall and plasma membrane through alterations in their lipid, protein and polysaccharide composition, changes in cell polarity, actin cytoskeleton organization, and budding patterns. Also as expected, adaptation involves regulation by Rim101/PacC of genes involved in meiotic functions, such as recombination and segregation, and expression of genes involved in ion and nutrient transport, as well as general vacuole functions.

Cryptococcus spp. isolation from excreta of pigeons (Columba livia) in and around Monterrey, Mexico

The presence of Cryptococcus spp. has been reported in Mexico’s capital city; however, to our knowledge there are no reports of its presence in the state of Nuevo León located in northeast Mexico. This is presumed to be because the hot and dry climate in this region does not favor cryptococcal proliferation. This study confirmed the presence of C. neoformans and C. albidus in 20% (10/50) of randomly selected fecal samples of pigeons (Columba livia) in the Monterrey metropolitan area. The presence of this yeast in the state of Nuevo León is proof of its adaptation to the typically hot climate of the area and is consistent with recent reviews of cryptococcosis cases in several local hospitals. The two species were identified and characterized through microbiological tests and molecular identification by DNA extraction and PCR amplification of highly conserved 18S ribosomal DNA using ITS1 and ITS2 as target regions. The PCR products were sequenced and compared with those reported in GenBank.

Candida species diversity and antifungal susceptibility patterns in oral samples of HIV/AIDS patients in Baja California, Mexico

Candidiasis is the most common opportunistic fungal infection in HIV patients. The aims of this study were to identify the prevalence of carriers of Candida, Candida species diversity, and in vitro susceptibility to antifungal drugs. In 297 HIV/AIDS patients in Baja California, Mexico, Candida strains were identified by molecular methods (PCR-RFLP) from isolates of oral rinses of patients in Tijuana, Mexicali, and Ensenada. 56.3% of patients were colonized or infected with Candida. In Tijuana, there was a significantly higher percentage of carriers (75.5%). Out of the 181 strains that were isolated, 71.8% were Candida albicans and 28.2% were non-albicans species. The most common non-albicans species was Candida tropicalis (12.2%), followed by Candida glabrata (8.3%), Candida parapsilosis (2.2%), Candida krusei (1.7%), and Candida guilliermondii (1.1%). Candida dubliniensis was not isolated. Two associated species were found in 11 patients. In Mexicali and Ensenada, there was a lower proportion of Candida carriers compared to other regions in Mexico and worldwide, however, in Tijuana, a border town with many peculiarities, a higher carrier rate was found. In this population, only a high viral load was associated with oral Candida carriers. Other factors such as gender, use of antiretroviral therapy, CD4+ T-lymphocyte levels, time since diagnosis, and alcohol/ tobacco consumption, were not associated with Candida carriers.

A molecular probe for Basidiomycota: the spermidine synthase‐saccharopine dehydrogenase chimeric gene

By means of an in silico analysis, we demonstrated that a previously described chimeric gene (Spe-Sdh) encoding spermidine synthase, a key enzyme involved in the synthesis of polyamines, and saccharopine dehydrogenase, an enzyme involved in lysine synthesis in fungi, were present exclusively in members of all Basidiomycota subphyla, but not in any other group of living organisms. We used this feature to design degenerated primers to amplify a specific fragment of the Spe-Sdh gene by PCR, as a tool to unequivocally identify Basidiomycota isolates. The specificity of this procedure was tested using different fungal species. As expected, positive results were obtained only with Basidiomycota species, whereas no amplification was achieved with species belonging to other fungal phyla.

Adaptation of Ustilago maydis to extreme pH values: A transcriptomic analysis.

Fungi are capable to adapt to environments with different pH values. Here we used microarrays to analyze the transcriptomic response of the Basidiomycota Ustilago maydis when transferred from a neutral pH medium to acidic, or alkaline media. Yeast and hyphal monomorphic mutants were used as controls, permitting the identification of 301 genes differentially regulated during the transfer from neutral to an acidic medium, of which 162 were up‐regulated and 139 down‐regulated. When cells were transferred to an alkaline medium, we identified 797 differentially regulated genes, 335 up‐regulated, and 462 down‐regulated. The category showing the highest number of regulated genes during the change to either pH, besides “unclassified,” was “metabolism,” indicating that a very important factor for adaptation is a change in the metabolic machinery. These data reveal that adaptation of U. maydis to environments with different pH involves a severe modification of the transcription machinery to cope with the new conditions, and that the stress by an alkaline environment is more drastic than a change to an acidic medium. The data also revealed that only a minor proportion of the identified genes are under the apparent control of the Pal/Rim pathway, indicating that pH adaptation of this fungus involves other than this cannonical pathway.

Transcriptomic analysis of basidiocarp development in Ustilago maydis (DC) Cda.

Previously, we demonstrated that when Ustilago maydis (DC) Cda., a phytopathogenic basidiomycete and the causal agent of corn smut, is grown in the vicinity of maize embryogenic calli in a medium supplemented with the herbicide Dicamba, it developed gastroid-like basidiocarps. To elucidate the molecular mechanisms involved in the basidiocarp development by the fungus, we proceeded to analyze the transcriptome of the process, identifying a total of 2002 and 1064 differentially expressed genes at two developmental stages, young and mature basidiocarps, respectively. Function of these genes was analyzed with the use of different databases. MIPS analysis revealed that in the stage of young basidiocarp, among the ca. two thousand differentially expressed genes, there were some previously described for basidiocarp development in other fungal species. Additional elements that operated at this stage included, among others, genes encoding the transcription factors FOXO3, MIG3, PRO1, TEC1, copper and MFS transporters, and cytochromes P450. During mature basidiocarp development, important up-regulated genes included those encoding hydrophobins, laccases, and ferric reductase (FRE/NOX). The demonstration that a mapkk mutant was unable to form basidiocarps, indicated the importance of the MAPK signaling pathway in this developmental process.