Variations in candidalysin amino acid sequence influence toxicity and host responses.

Candida albicans causes millions of mucosal infections in humans annually. Hyphal overgrowth on mucosal surfaces is frequently associated with tissue damage caused by candidalysin, a secreted peptide toxin that destabilizes the plasma membrane of host cells thereby promoting disease and immunopathology. Candidalysin was first identified in C. albicans strain SC5314, but recent investigations have revealed candidalysin "variants" of differing amino acid sequence in isolates of C. albicans, and the related species C. dubliniensis, and C tropicalis, suggesting that sequence variation among candidalysins may be widespread in natural populations of these Candida species. Here, we analyzed ECE1 gene sequences from 182 C. albicans isolates, 10 C. dubliniensis isolates, and 78 C. tropicalis isolates and identified 10, 3, and 2 candidalysin variants in these species, respectively. Application of candidalysin variants to epithelial cells revealed differences in the ability to cause cellular damage, changes in metabolic activity, calcium influx, MAPK signalling, and cytokine secretion, while biophysical analyses indicated that variants exhibited differences in their ability to interact with and permeabilize a membrane. This study identifies candidalysin variants with differences in biological activity that are present in medically relevant Candida species. IMPORTANCE: Fungal infections are a significant burden to health. Candidalysin is a toxin produced by Candida albicans that damages host tissues, facilitating infection. Previously, we demonstrated that candidalysins exist in the related species C. dubliniensis and C. tropicalis, thereby identifying these molecules as a toxin family. Recent genomic analyses have highlighted the presence of a small number of candidalysin "variant" toxins, which have different amino acid sequences to those originally identified. Here, we screened genome sequences of isolates of C. albicans, C. dubliniensis, and C. tropicalis and identified candidalysin variants in all three species. When applied to epithelial cells, candidalysin variants differed in their ability to cause damage, activate intracellular signaling pathways, and induce innate immune responses, while biophysical analysis revealed differences in the ability of candidalysin variants to interact with lipid bilayers. These findings suggest that intraspecies variation in candidalysin amino acid sequence may influence fungal pathogenicity.

eIF2A represses cell wall biogenesis gene expression in Saccharomyces cerevisiae.

Translation initiation is a complex and highly regulated process that represents an important mechanism, controlling gene expression. eIF2A was proposed as an alternative initiation factor, however, its role and biological targets remain to be discovered. To further gain insight into the function of eIF2A in Saccharomyces cerevisiae, we identified mRNAs associated with the eIF2A complex and showed that 24% of the most enriched mRNAs encode proteins related to cell wall biogenesis and maintenance. In agreement with this result, we showed that an eIF2A deletion sensitized cells to cell wall damage induced by calcofluor white. eIF2A overexpression led to a growth defect, correlated with decreased synthesis of several cell wall proteins. In contrast, no changes were observed in the transcriptome, suggesting that eIF2A controls the expression of cell wall-related proteins at a translational level. The biochemical characterization of the eIF2A complex revealed that it strongly interacts with the RNA binding protein, Ssd1, which is a negative translational regulator, controlling the expression of cell wall-related genes. Interestingly, eIF2A and Ssd1 bind several common mRNA targets and we found that the binding of eIF2A to some targets was mediated by Ssd1. Surprisingly, we further showed that eIF2A is physically and functionally associated with the exonuclease Xrn1 and other mRNA degradation factors, suggesting an additional level of regulation. Altogether, our results highlight new aspects of this complex and redundant fine-tuned regulation of proteins expression related to the cell wall, a structure required to maintain cell shape and rigidity, providing protection against harmful environmental stress.

High-throughput functional profiling of the human fungal pathogen Candida albicans genome.

Candida albicans is a major fungal pathogen of humans. Although its genome has been sequenced more than two decades ago, there are still over 4300 uncharacterized C. albicans genes. We previously generated an ORFeome as well as a collection of destination vectors to facilitate overexpression of C. albicans ORFs. Here, we report the construction of ∼2500 overexpression mutants and their evaluation by in vitro spotting on rich medium and in a liquid pool experiment in rich medium, allowing the identification of genes whose overexpression has a fitness cost. The candidates were further validated at the individual strain level. This new resource allows large-scale screens in different growth conditions to be performed routinely. Altogether, based on the concept of identifying functionally related genes by cluster analysis, the availability of this overexpression mutant collection will facilitate the characterization of gene functions in C. albicans.

Transcript profiling reveals the role of PDB1, a subunit of the pyruvate dehydrogenase complex, in Candida albicans biofilm formation.

Candida albicans, the most prevalent fungal pathogen in the human microbiota can form biofilms on implanted medical devices. These biofilms are tolerant to conventional antifungal drugs and the host immune system as compared to the free-floating planktonic cells. Several in vitro models of biofilm formation have been used to determine the C. albicans biofilm-forming process, regulatory networks, and their properties. Here, we performed a genome-wide transcript profiling with C. albicans cells grown in YPD medium both in planktonic and biofilm condition. Transcript profiling of YPD-grown biofilms was further compared with published Spider medium-grown biofilm transcriptome data. This comparative analysis highlighted the differentially expressed genes and the pathways altered during biofilm formation. In addition, we demonstrated that overexpression of the PDB1 gene encoding a subunit of the pyruvate dehydrogenase resulted in defective biofilm formation. Altogether, this comparative analysis of transcript profiles from two different studies provides a robust reading on biofilm-altered genes and pathways during C. albicans biofilm development.

Generating genomic platforms to study Candida albicans pathogenesis.

The advent of the genomic era has made elucidating gene function on a large scale a pressing challenge. ORFeome collections, whereby almost all ORFs of a given species are cloned and can be subsequently leveraged in multiple functional genomic approaches, represent valuable resources toward this endeavor. Here we provide novel, genome-scale tools for the study of Candida albicans, a commensal yeast that is also responsible for frequent superficial and disseminated infections in humans. We have generated an ORFeome collection composed of 5099 ORFs cloned in a Gateway™ donor vector, representing 83% of the currently annotated coding sequences of C. albicans. Sequencing data of the cloned ORFs are available in the CandidaOrfDB database at http://candidaorfeome.eu. We also engineered 49 expression vectors with a choice of promoters, tags and selection markers and demonstrated their applicability to the study of target ORFs transferred from the C. albicans ORFeome. In addition, the use of the ORFeome in the detection of protein-protein interaction was demonstrated. Mating-compatible strains as well as Gateway™-compatible two-hybrid vectors were engineered, validated and used in a proof of concept experiment. These unique and valuable resources should greatly facilitate future functional studies in C. albicans and the elucidation of mechanisms that underlie its pathogenicity.

Gene flow contributes to diversification of the major fungal pathogen Candida albicans.

Elucidating population structure and levels of genetic diversity and recombination is necessary to understand the evolution and adaptation of species. Candida albicans is the second most frequent agent of human fungal infections worldwide, causing high-mortality rates. Here we present the genomic sequences of 182 C. albicans isolates collected worldwide, including commensal isolates, as well as ones responsible for superficial and invasive infections, constituting the largest dataset to date for this major fungal pathogen. Although, C. albicans shows a predominantly clonal population structure, we find evidence of gene flow between previously known and newly identified genetic clusters, supporting the occurrence of (para)sexuality in nature. A highly clonal lineage, which experimentally shows reduced fitness, has undergone pseudogenization in genes required for virulence and morphogenesis, which may explain its niche restriction. Candida albicans thus takes advantage of both clonality and gene flow to diversify.

Candida albicans is not always the preferential yeast colonizing humans: a study in Wayampi Amerindians.

In industrialized countries Candida albicans is considered the predominant commensal yeast of the human intestine, with approximately 40% prevalence in healthy adults. We discovered a highly original colonization pattern that challenges this current perception by studying in a 4- year interval a cohort of 151 Amerindians living in a remote community (French Guiana), and animals from their environment. The prevalence of C. albicans was persistently low (3% and 7% of yeast carriers). By contrast, Candida krusei and Saccharomyces cerevisiae were detected in over 30% of carriers. We showed that C. krusei and S. cerevisiae carriage was of food or environmental origin, whereas C. albicans carriage was associated with specific risk factors (being female and living in a crowded household). We also showed using whole-genome sequence comparison that C. albicans strains can persist in the intestinal tract of a healthy individual over a 4-year period.

Roadmap for annotating transposable elements in eukaryote genomes.

Current high-throughput techniques have made it feasible to sequence even the genomes of non-model organisms. However, the annotation process now represents a bottleneck to genome analysis, especially when dealing with transposable elements (TE). Combined approaches, using both de novo and knowledge-based methods to detect TEs, are likely to produce reasonably comprehensive and sensitive results. This chapter provides a roadmap for researchers involved in genome projects to address this issue. At each step of the TE annotation process, from the identification of TE families to the annotation of TE copies, we outline the tools and good practices to be used.

In search of lost trajectories: Recovering the diversification of transposable elements.

Transposable elements (TEs) are DNA sequences that have the capacity to move and duplicate within genomes, and occasionally between them. They are present in almost all species and are especially prevalent in eukaryotes where they can account for most of the genomic content. As a result of their dynamics and their mere presence, TEs can profoundly shape genomes and gene expression. With the current pace of sequencing technology improvement, a rapidly increasing number of genomes, particularly from non-model species, are being sequenced. However, the complete annotation of these genomes and especially of the TEs they contain, still poses fundamental difficulties. In a recent article, we presented a combined method that automatically annotates TEs with accuracy and sensitivity, and takes their diversification dynamics into account in the de novo annotation process. Here, we further discuss several additional aspects of our results, notably in the light of our knowledge of TE dynamics and the conceptual model behind the TE detection algorithms currently in use. In addition, we propose a new approach that uses simulations to improve algorithm performance.

Correlation of LNCR rasiRNAs expression with heterochromatin formation during development of the holocentric insect Spodoptera frugiperda.

Repeat-associated small interfering RNAs (rasiRNAs) are derived from various genomic repetitive elements and ensure genomic stability by silencing endogenous transposable elements. Here we describe a novel subset of 46 rasiRNAs named LNCR rasiRNAs due to their homology with one long non-coding RNA (LNCR) of Spodoptera frugiperda. LNCR operates as the intermediate of an unclassified transposable element (TE-LNCR). TE-LNCR is a very invasive transposable element, present in high copy numbers in the S. frugiperda genome. LNCR rasiRNAs are single-stranded RNAs without a prominent nucleotide motif, which are organized in two distinct, strand-specific clusters. The expression of LNCR and LNCR rasiRNAs is developmentally regulated. Formation of heterochromatin in the genomic region where three copies of the TE-LNCR are embedded was followed by chromatin immunoprecipitation (ChIP) and we observed this chromatin undergo dynamic changes during development. In summary, increased LNCR expression in certain developmental stages is followed by the appearance of a variety of LNCR rasiRNAs which appears to correlate with subsequent accumulation of a heterochromatic histone mark and silencing of the genomic region with TE-LNCR. These results support the notion that a repeat-associated small interfering RNA pathway is linked to heterochromatin formation and/or maintenance during development to establish repression of the TE-LNCR transposable element. This study provides insights into the rasiRNA silencing pathway and its role in the formation of fluctuating heterochromatin during the development of one holocentric organism.

The ERPIN server: an interface to profile-based RNA motif identification.

ERPIN is an RNA motif identification program that takes an RNA sequence alignment as an input and identifies related sequences using a profile-based dynamic programming algorithm. ERPIN differs from other RNA motif search programs in its ability to capture subtle biases in the training set and produce highly specific and sensitive searches, while keeping CPU requirements at a practical level. In its latest version, ERPIN also computes E-values, which tell biologists how likely they are to encounter a specific sequence match by chance-a useful indication of biological significance. We present here the ERPIN online search interface (http://tagc.univ-mrs.fr/erpin/). This web server automatically performs ERPIN searches for different RNA genes or motifs, using predefined training sets and search parameters. With a couple of clicks, users can analyze an entire bacterial genome or a genomic segment of up to 5Mb for the presence of tRNAs, 5S rRNAs, SRP RNA, C/D box snoRNAs, hammerhead motifs, miRNAs and other motifs. Search results are displayed with sequence, score, position, E-value and secondary structure graphics. An example of a complete genome scan is provided, as well as an evaluation of run times and specificity/sensitivity information for all available motifs.