ERG11 Polymorphism in Voriconazole-Resistant Candida tropicalis: Weak Role of ERG11 Expression, Ergosterol Content, and Membrane Permeability.

Mutations in ERG11 were detected by gene sequencing and amino acid alignment in 18 Candida tropicalis strains with different degrees of sensitivity to voriconazole (VRC). ERG11 expression, sterol content, and membrane permeability were also evaluated. We report three missense mutations in ERG11 that resulted in resistance to VRC. The transcriptional levels of ERG11 as well as the ergosterol content and membrane permeability demonstrated no correlation to only a slight correlation with the obtained MIC values, but the data did suggest a tendency toward such a correlation.

Expression of ERG11 and efflux pump genes CDR1, CDR2 and SNQ2 in voriconazole susceptible and resistant Candida glabrata strains.

Candida glabrata causes difficult to treat invasive candidiasis due to its antifungal resistance, mainly to azoles. The aim of the present work was to study the role of the genes ERG11, CDR1, CDR2, and SNQ2 on the resistance to voriconazole (VRC) in a set of C. glabrata strains with known in vitro and in vivo susceptibility to this drug. Eighteen clinical isolates of C. glabrata were exposed in vitro to VRC, and the expression of the cited genes was quantified by real time quantitative polymerase chain reaction (q-PCR). In addition, the ERG11 gene was amplified and sequenced to detect possible mutations. Ten synonymous mutations were found in 15 strains, two of them being reported for the first time; however, no amino acid changes were detected. ERG11 and CDR1 were the most expressed genes in all the strains tested, while the expression of CDR2 and SNQ2 was modest. Our results show that gene expression does not directly correlate with the VRC MIC. In addition, the expression profiles of ERG11 and efflux pump genes did not change consistently after exposure to VRC. Although individual analysis did not result in a clear correlation between MIC and gene expression, we did observe an increase in ERG11 and CDR1 expression in resistant strains. It is of interest that considering both in vitro and in vivo results, the slight increase in such gene expression correlates with the observed resistance to VRC.

Increased Efficacy of Oral Fixed-Dose Combination of Amphotericin B and AHCC® Natural Adjuvant against Aspergillosis.

Invasive pulmonary aspergillosis represents one of the most serious fungal infections among immunocompromised patients. In this study, we aimed to analyze the in vivo efficacy of prophylactic oral amphotericin B (AMB) encapsulated in modified chitosan-nanoparticles (Nanomerics' Molecular Envelope Technology (MET)) supplemented with a standardized extract of cultured Lentinula edodes mycelia (AHCC®) in a murine model of pulmonary aspergillosis. We determined fungal burden and survival of mice and additionally, we carried out a cytokine analysis in an attempt to understand the immunomodulation of the extract. Our results evidenced equivalent efficacy between orally administered AMB-MET and the intravenous liposomal AMB marketed formulation. Addition of the AHCC® supplement significantly improved efficacy in terms of burden reduction and survival increase of both oral and intravenous AMB therapies compared to the untreated control group. Moreover, a protective effect of the extract was observed in terms of weight loss. Regarding the cytokine profiles, the Th1 immune response was stimulated in treated animals when compared to the control group. This response was marked by an enhancement in the MCP-1, GM-CSF, VEGF, RANTES and IL-17 levels and a decrease in the IL-6, a biomarker related to the severity of the infection.

Clinical and Laboratory Development of Echinocandin Resistance in Candida glabrata: Molecular Characterization.

The pathogenic yeast Candida glabrata has become a public health issue due to the increasing number of echinocandin resistant clinical strains reported. In this study, acquisition and development of resistance to this antifungal class were studied in serial C. glabrata isolates from five patients admitted in two Spanish hospitals with a resistant profile against echinocandins associated with different mutations in hot-spot 1 of FKS2 gene. For two of these patients susceptible FKS wild-type isolates obtained prior to resistant ones were also investigated. Isolates were genotyped using multilocus sequence typing and microsatellite length polymorphism techniques, which yielded comparable results. Susceptible and resistant isolates from the same patient had the same genotype, being sequence type (ST) 3 the most prevalent among them. Isolates with different FKS mutations but the same ST were present in the same patient. MSH2 gene alterations were also studied to investigate their correlation with antifungal resistance acquisition but no association was found with antifungal resistance nor with specific genotypes. In vitro exposure to increasing concentrations of micafungin to susceptible isolates developed colonies carrying FKS mutations in agar plates containing a minimum concentration of 0.06 mg/L of micafungin after less than 48 h of exposure. We investigated the correlation between development of resistance and genotype in a set of susceptible strains after being in vitro exposed to micafungin and anidulafungin but no correlation was found. Mutant prevention concentration values and spontaneous growth frequencies after selection with both echinocandins were statistically similar, although FKS mutant colonies were more abundant after micafungin exposure (p < 0.001). Mutation S663P and F659 deletion were the most common ones found after selection with both echinocandins.

Lack of correlation of ECV and outcome in an in vivo murine model of systemic fusariosis.

The efficacy of liposomal amphotericin B and voriconazole was evaluated against the systemic infection by Fusarium oxysporum species complex or Fusarium keratoplasticum. Although MIC values were within the epidemiological cutoff values (ECVs) recently stablished for Fusarium spp., no efficacy was obtained, indicating that ECVs for Fusarium are not relevant for in vivo efficacy.

Understanding Mucor circinelloides pathogenesis by comparative genomics and phenotypical studies.

The increasing number of infections by species of Mucorales and their high mortality constitute an important concern for public health. This study aims to decipher the genetic basis of Mucor circinelloides pathogenicity, which displays virulence in a strain dependent manner. Assuming that genetic differences between strains may be linked to different pathotypes, we have conducted a study to explore genes responsible for virulence in M. circinelloides by whole genome sequencing of the avirulent strain NRRL3631 and comparison with the virulent strain CBS277.49. This genome analysis revealed 773 truncated, discontiguous and absent genes in the NRRL3631 strain. We also examined phenotypic traits resulting in reduced heat stress tolerance, chitosan content and lower susceptibility to toxic compounds (calcofluor white and sodium dodecyl sulphate) in the virulent strain, suggesting the influence of cell wall on pathogenesis. Based on these results, we focused on studying extracellular protein-coding genes by gene deletion and further pathotype characterization of mutants in murine models of pulmonary and systemic infection. Deletion of gene ID112092, which codes for a hypothetical extracellular protein of unknown function, resulted in significant reduction of virulence. Although pathogenesis is a multifactorial process, these findings highlight the crucial role of surface and secreted proteins in M. circinelloides virulence and should promote further studies of other differential genes.

RNAi-Based Functional Genomics Identifies New Virulence Determinants in Mucormycosis.

Mucorales are an emerging group of human pathogens that are responsible for the lethal disease mucormycosis. Unfortunately, functional studies on the genetic factors behind the virulence of these organisms are hampered by their limited genetic tractability, since they are reluctant to classical genetic tools like transposable elements or gene mapping. Here, we describe an RNAi-based functional genomic platform that allows the identification of new virulence factors through a forward genetic approach firstly described in Mucorales. This platform contains a whole-genome collection of Mucor circinelloides silenced transformants that presented a broad assortment of phenotypes related to the main physiological processes in fungi, including virulence, hyphae morphology, mycelial and yeast growth, carotenogenesis and asexual sporulation. Selection of transformants with reduced virulence allowed the identification of mcplD, which encodes a Phospholipase D, and mcmyo5, encoding a probably essential cargo transporter of the Myosin V family, as required for a fully virulent phenotype of M. circinelloides. Knock-out mutants for those genes showed reduced virulence in both Galleria mellonella and Mus musculus models, probably due to a delayed germination and polarized growth within macrophages. This study provides a robust approach to study virulence in Mucorales and as a proof of concept identified new virulence determinants in M. circinelloides that could represent promising targets for future antifungal therapies.