The microbial ecology of Candida albicans strains CHN1 and SC5314 in mice

Strain SC5314 is the most widely studied strain of Candida albicans. Despite C. albicans being the most commonly isolated yeast from the human gastrointestinal (GI) microbiome, strain SC5314 does not stably colonize the mouse GI tract long term, even after antibiotic disruption. In contrast, strain CHN1 will stably colonize the mouse GI tract long term. Comparative genomic analysis of strain CHN1 indicates that it belongs to a different evolutionary clade of C. albicans than strain SC5314. Previous studies from our laboratory have shown that colonization by strain CHN1 causes a change in the GI bacterial microbiome of mice and predisposes them to more robust Th2 immune responses. Despite this, little is known about the GI microbial ecology of SC5314 vs. CHN1 and subsequent host responses. Using a short-term antibiotic disruption model in C57BL/6 mice, we have been able to observe significantly different colonization kinetics between these two C. albicans strains, with CHN1 establishing stable long-term colonization. In contrast, colonization by SC5314 was lower, highly variable and cage-dependent. C. albicans colonization kinetics impacted the composition of the bacterial microbiome with a marked effect on the levels of Lactobacillus and Enterococcus. qPCR analysis of 46 host immune response genes did not detect significant differences in host gene expression between SC5134 and CHN1 colonized mice, except for chitinase expression. Thus, these studies suggest that yeast-bacteria interactions in the microbiome may be far more important in determining long-term colonization potential of C. albicans and secondary immunomodulatory effects.

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.

Candida albicans Biofilm Inhibition by Two Vaccinium macrocarpon (Cranberry) Urinary Metabolites: 5-(3′,4′-DihydroxyPhenyl)-γ-Valerolactone and 4-Hydroxybenzoic Acid

Candida spp. are pathobionts, as they can switch from commensals to pathogens, responsible for a variety of pathological processes. Adhesion to surfaces, morphological switch and biofilm-forming ability are the recognized virulence factors promoting yeast virulence. Sessile lifestyle also favors fungal persistence and antifungal tolerance. In this study, we investigated, in vitro, the efficacy of two urinary cranberry metabolites, 5-(3′,4′-dihydroxy phenyl)-γ-valerolactone (VAL) and 4-hydroxybenzoic acid (4-HBA), in inhibiting C. albicans adhesion and biofilm formation. Both the reference strain SC5314 and clinical isolates were used. We evaluated biomass reduction, by confocal microscopy and crystal violet assay, and the possible mechanisms mediating their inhibitory effects. Both VAL and 4-HBA were able to interfere with the yeast adhesion, by modulating the expression of key genes, HWP1 and ALS3. A significant dose-dependent reduction in biofilm biomass and metabolic activity was also recorded. Our data showed that the two cranberry metabolites VAL and 4-HBA could pave the way for drug development, for targeting the very early phases of biofilm formation and for preventing genitourinary Candida infections.

Candida Albicans ERG11 Gene Polymorphism: Impact on Its In Vivo Virulence and Susceptibility to Fluconazole According to the Galleria Mellonella Model

The aim of this study is to have an idea on the molecular mechanisms of C. albicans resistance to fluconazole in Burkina Faso, by studying the polymorphism of the ERG11 gene, and its implication in the C. albicans virulence and resistance in vivo according to the Galleria mellonella model; (2) Methods: Ten (10) clinical strains including, 5 resistant and 5 susceptible and 1 virulent and susceptible reference strain SC5314 are used. For the estimation of virulence, the larvae were inoculated with 10 μL of C. albicans cell suspension at variable concentrations: 2,5.105, 5.105, 1.106, and 5.106 CFU/larva of each strain. For the in vivo efficacy study, fluconazole was administered at 1, 4 and 16 mg/kg respectively to G. mellonella larvae, after infection by inoculum 5.106 CFU / larvae of each strain; (3) Results: Six (6) non-silent mutations in the ERG11 gene (K143R, F145L, G307S, S405F, G448E, V456I on ERG11p) were found in 4 resistant isolates. Larval mortality depended on fungal burden and strain. The inoculum 5.106 CFU caused 100% mortality in 2 days for the 2 CAAL-1 and CAAL-2 strains carrying the F145L mutation, in 3 days for the reference strain SC5314, in 4 days for the ensemble of resistant strains, and in 5 days for the ensemble of susceptible strains. The comparison of the mortality due to the reference strain SC5314 CFU / larva and the average mortality due to the two mutant F145L strains, shows a significant difference (P <0.05).Fluconazole significantly protected (P> 0.05) the larvae from infection by susceptible strains and the reference strain. However, 100% mortality in 6 days after injection of the resistant strains, was observed (4) Conclusions: Certain mutations in the ERG11 gene such as the F145L mutation are thought to be a source of increased virulence in Candida albicans. Fluconazole effectively protected larvae from infection by susceptible strains in vivo, unlike resistant strain

Automated quantification of Candida albicans biofilm-related phenotypes reveals additive contributions to biofilm production

Biofilms are organized communities of microbial cells that promote persistence among bacterial and fungal species. Biofilm formation by host-associated Candida species of fungi occurs on both tissue surfaces and implanted devices, contributing to host colonization and disease. In C. albicans, biofilms are built sequentially by adherence of yeast to a surface, invasion into the substrate, the formation of aerial hyphal projections, and the secretion of extracellular matrix. Measurement of these biofilm-related phenotypes remains highly qualitative and often subjective. Here, we designed an informatics pipeline for quantifying filamentation, adhesion, and invasion of Candida species on solid agar media and utilized this approach to determine the importance of these component phenotypes to C. albicans biofilm production. Characterization of 23 C. albicans clinical isolates across three media and two temperatures revealed a wide range of phenotypic responses among isolates in any single condition. Media profoundly altered all biofilm-related phenotypes among these isolates, whereas temperature minimally impacted these traits. Importantly, the extent of biofilm formation correlated significantly with the additive score for its component phenotypes under some conditions, experimentally linking the strength of each component to biofilm mass. In addition, the response of the genome reference strain, SC5314, across these conditions was an extreme outlier compared to all other strains, suggesting it may not be representative of the species. Taken together, development of a high-throughput, unbiased approach to quantifying Candida biofilm-related phenotypes linked variability in these phenotypes to biofilm production and can facilitate genetic dissection of these critical processes to pathogenesis in the host.

Natural Variation in Clinical Isolates of Candida albicans Modulates Neutrophil Responses

ABSTRACT Neutropenia predisposes patients to life-threatening infection with Candida albicans, a commensal and opportunistic fungal pathogen. How phenotypic variation in C. albicans isolates dictates neutrophil responses is poorly understood. By using a panel of clinical C. albicans strains, here we report that the prototype strain SC5314 induces the most potent accumulation of reactive oxygen species (ROS) and neutrophil extracellular traps (NETs) by human neutrophils of all tested isolates. ROS and NET accumulation positively correlated with the degree of hyphal formation by the isolates, the hypha being the fungal morphotype that promotes pathogenesis. However, there was no correlation of ROS and NET accumulation with fungal killing by neutrophils. Fungal killing was also not correlated with phagocytosis levels or oxidative stress susceptibility of the isolates. The bloodstream isolate P94015 cannot make hyphae and was previously shown to be hyperfit in the murine gut commensalism model. Our results show that P94015 displays poor phagocytosis by neutrophils, the least ROS and NET accumulation of all tested isolates, and resistance to neutrophil-mediated killing. Our data suggest that reduced susceptibility to neutrophils is likely to be independent from a previously described genetic mutation in P94015 that promotes commensalism. Reduced clearance by neutrophils could benefit commensal fitness of C. albicans and could also have promoted the virulence of P94015 in the human patient in the absence of hyphal morphogenesis. Collectively, our study provides new insights into neutrophil interactions with C. albicans and suggests that studying diverse isolates informs knowledge of the relevant aspects of this key immune interaction. IMPORTANCE Neutrophils are the key immune cell type for host defenses against infections with Candida albicans. C. albicans strains isolated from patients display large phenotypic diversity, but how this diversity impacts host-pathogen interactions with neutrophils is incompletely defined. Here, we show that important neutrophil responses, such as accumulation of reactive oxygen species and neutrophil extracellular traps, as well as the levels of phagocytosis and killing of the pathogen, differ when comparing diverse C. albicans isolates. A bloodstream patient isolate previously described as more suited to commensalism than pathogenesis in animal models is relatively “silent” to neutrophils and resistant to killing. Our findings illuminate the relationships between fungal morphogenesis, neutrophil responses, and C. albicans survival. Our findings suggest that host phenotypes of a commensally adapted strain could be driven by resistance to immune clearance and indicate that we should extend our studies beyond the “prototype” strain SC5314 for deeper understanding of Candida-neutrophil interactions.

The Combination of Plasma-Processed Air (PPA) and Plasma-Treated Water (PTW) Causes Synergistic Inactivation of Candida albicans SC5314

Microwave-induced plasma was used for the generation of plasma-processed air (PPA) and plasma-treated water (PTW). By this way, the plasma was able to functionalize the compressed air and the used water to antimicrobial effective agents. Their fungicidal effects by single and combined application were investigated on Candida albicans strain SC5314. The monoculture of C. albicans was cultivated on specimens with polymeric surface structures (PE-stripes). The additive as well as the synergistic fungicidal potential of PPA and PTW was investigated by different process windows of plasma exposure time (5–50 s) and sample treatment time with PPA/PTW (1–5 min). For a single PTW or PPA treatment, an increase in the reduction factor with the indicated treatment time was observed (maximum reduction factor of 1.1 and 1.6, respectively). In comparison, the combined application of PTW and then PPA resulted in antagonistic, additive and synergistic effects, depending on the combination. An application of the synergistically acting processes of PTW for cleaning and PPA for drying can be an innovative alternative to the sanitary processes currently used in production plants.

Identification of Recessive Lethal Alleles in the Diploid Genome of aCandida albicansLaboratory Strain Unveils a Potential Role of Repetitive Sequences in Buffering Their Deleterious Impact

ABSTRACTThe heterozygous diploid genome ofCandida albicansis highly plastic, with frequent loss of heterozygosity (LOH) events. In the SC5314 laboratory strain, while LOH events are ubiquitous, a chromosome homozygosis bias is observed for certain chromosomes, whereby only one of the two homologs can occur in the homozygous state. This suggests the occurrence of recessive lethal allele(s) (RLA) preventing large-scale LOH events on these chromosomes from being stably maintained. To verify the presence of an RLA on chromosome 7 (Chr7), we utilized a system that allows (i) DNA double-strand break (DSB) induction on Chr7 by the I-SceI endonuclease and (ii) detection of the resulting long-range homozygosis. I-SceI successfully induced a DNA DSB on both Chr7 homologs, generally repaired by gene conversion. Notably, cells homozygous for the right arm of Chr7B were not recovered, confirming the presence of RLA(s) in this region. Genome data mining for RLA candidates identified a premature nonsense-generating single nucleotide polymorphism (SNP) within the HapB allele of C7_03400c whoseSaccharomycescerevisiaeortholog encodes the essential Mtr4 RNA helicase. Complementation with a wild-type copy ofMTR4rescued cells homozygous for the right arm of Chr7B, demonstrating that themtr4K880*RLA is responsible for the Chr7 homozygosis bias in strain SC5314. Furthermore, we observed that the major repeat sequences (MRS) on Chr7 acted as hot spots for interhomolog recombination. Such recombination events provideC. albicanswith increased opportunities to survive DNA DSBs whose repair can lead to homozygosis of recessive lethal or deleterious alleles. This might explain the maintenance of MRS in this species.IMPORTANCECandida albicansis a major fungal pathogen, whose mode of reproduction is mainly clonal. Its genome is highly tolerant to rearrangements, in particular loss of heterozygosity events, known to unmask recessive lethal and deleterious alleles in heterozygous diploid organisms such asC. albicans. By combining a site-specific DSB-inducing system and mining genome sequencing data of 182 C. albicansisolates, we were able to ascribe the chromosome 7 homozygosis bias of theC. albicanslaboratory strain SC5314 to an heterozygous SNP introducing a premature STOP codon in theMTR4gene. We have also proposed genome-wide candidates for new recessive lethal alleles. We additionally observed that the major repeat sequences (MRS) on chromosome 7 acted as hot spots for interhomolog recombination. Maintaining MRS inC. albicanscould favor haplotype exchange, of vital importance to LOH events, leading to homozygosis of recessive lethal or deleterious alleles that inevitably accumulate upon clonality.

Genome Sequence of a Staphylococcus epidermidis Strain (GTH12) Associated with Candida albicans SC5314 Cultured under Hypoxia at 37°C in Glycerol for 12 Weeks

Polymicrobial infections with mixed-species biofilms are important health problems because of increased antimicrobial resistance and worse patient outcomes than with monomicrobial infections. Here, we present the whole-genome sequence of Staphylococcus epidermidis strain GTH12, which was cocultured with the yeast Candida albicans SC5314 (generating C. albicans strain SC5314 GTH12), thus providing genomic information on polymicrobial infections.

Whole-Genome Sequences and Annotation of the Opportunistic Pathogen Candida albicans Strain SC5314 Grown under Two Different Environmental Conditions

ABSTRACT The genetic variability of the opportunistic pathogen Candida albicans is an important adaptive mechanism. Here, we present the whole-genome sequences of the C. albicans SC5314 strain under two different growth conditions, providing useful information for comparative genomic studies and further intraspecific analysis.