Functional analysis of the Candida albicans ECE1 Promoter.

The formation of hyphae is a key virulence attribute of Candida albicans as they are required for adhesion to and invasion of host cells, and ultimately deep-tissue dissemination. Hyphae also secrete the peptide toxin candidalysin, which is crucial for destruction of host cell membranes. The peptide is derived from a precursor protein encoded by the gene ECE1 which is strongly induced during hyphal growth. Previous studies revealed a very complex regulation of this gene involving several transcription factors. However, the promoter of the gene is still not characterized. Here, we present a functional analysis of the intergenic region upstream of the ECE1 gene. Rapid amplification of cDNA ends (RACE)-PCR was performed to identify the 5' untranslated region, which has a size of 49 bp regardless of the hyphae-inducing condition. By using green fluorescent protein (GFP) reporter constructs we further defined a minimal promoter length of 1,500 bp which was verified by RT-qPCR. Finally, we identified the TATA element required for the expression of the gene. It is located 106 to 109 bp upstream of the ECE1 start codon. Our results illustrate that despite a very short 5' UTR, a relatively long promoter is required to secure ECE1 transcription, indicating a complex regulatory machinery tightly controlling the expression of the gene. IMPORTANCE In recent years it was shown that secretion of the toxic peptide candidalysin from hyphae of the major human fungal pathogen Candida albicans contributes heavily to its virulence. The peptide is derived from a precursor protein which is encoded by the ECE1 gene whose transcription is known to be closely associated with formation of hyphae. Here, we used a GFP reporter system to determine the length of the ECE1 promoter and were able to show that it has a minimal size of 1,500 bp. Surprisingly, the gene has a very short 5' UTR of only 49 bp. In accordance with this, the TATA element required for transcription is located 106 to 109 bp upstream of the start codon. This indicates that ECE1 expression is controlled by a very long promoter allowing a complex network of transcription factors to contribute to the gene's regulation.

Impaired amino acid uptake leads to global metabolic imbalance of Candida albicans biofilms.

Candida albicans biofilm maturation is accompanied by enhanced expression of amino acid acquisition genes. Three state-of-the-art omics techniques were applied to detail the importance of active amino acid uptake during biofilm development. Comparative analyses of normoxic wild-type biofilms were performed under three metabolically challenging conditions: aging, hypoxia, and disabled amino acid uptake using a strain lacking the regulator of amino acid permeases Stp2. Aging-induced amino acid acquisition and stress responses to withstand the increasingly restricted environment. Hypoxia paralyzed overall energy metabolism with delayed amino acid consumption, but following prolonged adaptation, the metabolic fingerprints aligned with aged normoxic biofilms. The extracellular metabolome of stp2Δ biofilms revealed deficient uptake for 11 amino acids, resulting in extensive transcriptional and metabolic changes including induction of amino acid biosynthesis and carbohydrate and micronutrient uptake. Altogether, this study underscores the critical importance of a balanced amino acid homeostasis for C. albicans biofilm development.

Systematic Metabolic Profiling Identifies De Novo Sphingolipid Synthesis as Hypha Associated and Essential for Candida albicans Filamentation.

The yeast-to-hypha transition is a key virulence attribute of the opportunistic human fungal pathogen Candida albicans, since it is closely tied to infection-associated processes such as tissue invasion and escape from phagocytes. While the nature of hypha-associated gene expression required for fungal virulence has been thoroughly investigated, potential morphotype-dependent activity of metabolic pathways remained unclear. Here, we combined global transcriptome and metabolome analyses for the wild-type SC5314 and the hypha-defective hgc1Δ and cph1Δefg1Δ strains under three hypha-inducing (human serum, N-acetylglucosamine, and alkaline pH) and two yeast-promoting conditions to identify metabolic adaptions that accompany the filamentation process. We identified morphotype-related activities of distinct pathways and a metabolic core signature of 26 metabolites with consistent depletion or enrichment during the yeast-to-hypha transition. Most strikingly, we found a hypha-associated activation of de novo sphingolipid biosynthesis, indicating a connection of this pathway and filamentous growth. Consequently, pharmacological inhibition of this partially fungus-specific pathway resulted in strongly impaired filamentation, verifying the necessity of de novo sphingolipid biosynthesis for proper hypha formation. IMPORTANCE The reversible switch of Candida albicans between unicellular yeast and multicellular hyphal growth is accompanied by a well-studied hypha-associated gene expression, encoding virulence factors like adhesins, toxins, or nutrient scavengers. The investigation of this gene expression consequently led to fundamental insights into the pathogenesis of this fungus. In this study, we applied this concept to hypha-associated metabolic adaptations and identified morphotype-dependent activities of distinct pathways and a stimulus-independent metabolic signature of hyphae. Most strikingly, we found the induction of de novo sphingolipid biosynthesis as hypha associated and essential for the filamentation of C. albicans. These findings verified the presence of morphotype-specific metabolic traits in the fungus, which appear connected to the fungal virulence. Furthermore, the here-provided comprehensive description of the fungal metabolome will help to foster future research and lead to a better understanding of fungal physiology.

GNP2 Encodes a High-Specificity Proline Permease in Candida albicans.

The tight association of Candida albicans with the human host has driven the evolution of mechanisms that permit metabolic flexibility. Amino acids, present in a free or peptide-bound form, are abundant carbon and nitrogen sources in many host niches. In C. albicans, the capacity to utilize certain amino acids, like proline, is directly connected to fungal morphogenesis and virulence. Yet the precise nature of proline sensing and uptake in this pathogenic fungus has not been investigated. Since C. albicans encodes 10 putative orthologs of the four Saccharomyces cerevisiae proline transporters, we tested deletion strains of the respective genes and identified Gnp2 (CR_09920W) as the main C. albicans proline permease. In addition, we found that this specialization of Gnp2 was reflected in its transcriptional regulation and further assigned distinct substrate specificities for the other orthologs, indicating functional differences of the C. albicans amino acid permeases compared to the model yeast. The physiological relevance of proline uptake is exemplified by the findings that strains lacking GNP2 were unable to filament in response to extracellular proline and had a reduced capacity to damage macrophages and impaired survival following phagocytosis. Furthermore, GNP2 deletion rendered the cells more sensitive to oxidative stress, illustrating new connections between amino acid uptake and stress adaptation in C. albicans. IMPORTANCE The utilization of various nutrients is of paramount importance for the ability of Candida albicans to successfully colonize and infect diverse host niches. In this context, amino acids are of special interest due to their ubiquitous availability, relevance for fungal growth, and direct influence on virulence traits like filamentation. In this study, we identify a specialized proline transporter in C. albicans encoded by GNP2. The corresponding amino acid permease is essential for proline-induced filamentation, oxidative stress resistance, and fungal survival following interaction with macrophages. Altogether, this work highlights the importance of amino acid uptake for metabolic and stress adaptation in this fungus.

Clinical Candida albicans Vaginal Isolates and a Laboratory Strain Show Divergent Behaviors during Macrophage Interactions.

Typically, established lab strains are widely used to study host-pathogen interactions. However, to better reflect the infection process, the experimental use of clinical isolates has come more into focus. Here, we analyzed the interaction of multiple vaginal isolates of the opportunistic fungal pathogen Candida albicans, the most common cause of vulvovaginal candidiasis in women, with key players of the host immune system: macrophages. We tested several strains isolated from asymptomatic or symptomatic women with acute and recurrent infections. While all clinical strains showed a response similar to the commonly used lab strain SC5314 in various in vitro assays, they displayed remarkable differences during interaction with macrophages. This coincided with significantly reduced ß-glucan exposure on the cell surface, which appeared to be a shared property among the tested vaginal strains for yeast extract/peptone/dextrose-grown cells, which is partly lost when the isolates faced vaginal niche-like nutrient conditions. However, macrophage damage, survival of phagocytosis, and filamentation capacities were highly strain-specific. These results highlight the high heterogeneity of C. albicans strains in host-pathogen interactions, which have to be taken into account to bridge the gap between laboratory-gained data and disease-related outcomes in an actual patient.IMPORTANCE Vulvovaginal candidiasis is one of the most common fungal infections in humans with Candida albicans as the major causative agent. This study is the first to compare clinical vaginal isolates of defined patient groups in their interaction with macrophages, highlighting the vastly different outcomes in comparison to a laboratory strain using commonly applied virulence-determining assays.

The Transcription Factor Stp2 Is Important for Candida albicans Biofilm Establishment and Sustainability.

The fungal pathogen Candida albicans forms polymorphic biofilms where hyphal morphogenesis and metabolic adaptation are tightly coordinated by a complex intertwined network of transcription factors. The sensing and metabolism of amino acids play important roles during various phases of biofilm development - from adhesion to maturation. Stp2 is a transcription factor that activates the expression of amino acid permease genes and is required for environmental alkalinization and hyphal growth in vitro and during macrophage phagocytosis. While it is well established that Stp2 is activated in response to external amino acids, its role in biofilm formation remains unknown. In addition to widely used techniques, we applied newly developed approaches for automated image analysis to quantify Stp2-regulated filamentation and biofilm growth. Our results show that in the stp2Δ deletion mutant adherence to abiotic surfaces and initial germ tube formation were strongly impaired, but formed mature biofilms with cell density and morphological structures comparable to the control strains. Stp2-dependent nutrient adaptation appeared to play an important role in biofilm development: stp2Δ biofilms formed under continuous nutrient flow displayed an overall reduction in biofilm formation, whereas under steady conditions the mutant strain formed biofilms with lower metabolic activity, resulting in increased cell survival and biofilm longevity. A deletion of STP2 led to increased rapamycin susceptibility and transcriptional activation of GCN4, the transcriptional regulator of the general amino acid control pathway, demonstrating a connection of Stp2 to other nutrient-responsive pathways. In summary, the transcription factor Stp2 is important for C. albicans biofilm formation, where it contributes to adherence and induction of morphogenesis, and mediates nutrient adaption and cell longevity in mature biofilms.

Ahr1 and Tup1 Contribute to the Transcriptional Control of Virulence-Associated Genes in Candida albicans.

The capacity of Candida albicans to reversibly change its morphology between yeast and filamentous stages is crucial for its virulence. Formation of hyphae correlates with the upregulation of genes ALS3 and ECE1, which are involved in pathogenicity processes such as invasion, iron acquisition, and host cell damage. The global repressor Tup1 and its cofactor Nrg1 are considered to be the main antagonists of hyphal development in C. albicans However, our experiments revealed that Tup1, but not Nrg1, was required for full expression of ALS3 and ECE1 In contrast to NRG1, overexpression of TUP1 was found to inhibit neither filamentous growth nor transcription of ALS3 and ECE1 In addition, we identified the transcription factor Ahr1 as being required for full expression of both genes. A hyperactive version of Ahr1 bound directly to the promoters of ALS3 and ECE1 and induced their transcription even in the absence of environmental stimuli. This regulation worked even in the absence of the crucial hyphal growth regulators Cph1 and Efg1 but was dependent on the presence of Tup1. Overall, our results show that Ahr1 and Tup1 are key contributors in the complex regulation of virulence-associated genes in the different C. albicans morphologies.IMPORTANCECandida albicans is a major human fungal pathogen and the leading cause of systemic Candida infections. In recent years, Als3 and Ece1 were identified as important factors for fungal virulence. Transcription of both corresponding genes is closely associated with hyphal growth. Here, we describe how Tup1, normally a global repressor of gene expression as well as of filamentation, and the transcription factor Ahr1 contribute to full expression of ALS3 and ECE1 in C. albicans hyphae. Both regulators are required for high mRNA amounts of the two genes to ensure functional relevant protein synthesis and localization. These observations identified a new aspect of regulation in the complex transcriptional control of virulence-associated genes in C. albicans.

Tracking the elusive 5' exonuclease activity of Chlamydomonas reinhardtii RNase J.

KEY MESSAGE: Chlamydomonas RNase J is the first member of this enzyme family that has endo- but no intrinsic 5' exoribonucleolytic activity. This questions its proposed role in chloroplast mRNA maturation. RNA maturation and stability in the chloroplast are controlled by nuclear-encoded ribonucleases and RNA binding proteins. Notably, mRNA 5' end maturation is thought to be achieved by the combined action of a 5' exoribonuclease and specific pentatricopeptide repeat proteins (PPR) that block the progression of the nuclease. In Arabidopsis the 5' exo- and endoribonuclease RNase J has been implicated in this process. Here, we verified the chloroplast localization of the orthologous Chlamydomonas (Cr) RNase J and studied its activity, both in vitro and in vivo in a heterologous B. subtilis system. Our data show that Cr RNase J has endo- but no significant intrinsic 5' exonuclease activity that would be compatible with its proposed role in mRNA maturation. This is the first example of an RNase J ortholog that does not possess a 5' exonuclease activity. A yeast two-hybrid screen revealed a number of potential interaction partners but three of the most promising candidates tested, failed to induce the latent exonuclease activity of Cr RNase J. We still favor the hypothesis that Cr RNase J plays an important role in RNA metabolism, but our findings suggest that it rather acts as an endoribonuclease in the chloroplast.