A variant ECE1 allele contributes to reduced pathogenicity of Candida albicans during vulvovaginal candidiasis.

Vulvovaginal candidiasis (VVC), caused primarily by the human fungal pathogen Candida albicans, results in significant quality-of-life issues for women worldwide. Candidalysin, a toxin derived from a polypeptide (Ece1p) encoded by the ECE1 gene, plays a crucial role in driving immunopathology at the vaginal mucosa. This study aimed to determine if expression and/or processing of Ece1p differs across C. albicans isolates and whether this partly underlies differential pathogenicity observed clinically. Using a targeted sequencing approach, we determined that isolate 529L harbors a similarly expressed, yet distinct Ece1p isoform variant that encodes for a predicted functional candidalysin; this isoform was conserved amongst a collection of clinical isolates. Expression of the ECE1 open reading frame (ORF) from 529L in an SC5314-derived ece1Δ/Δ strain resulted in significantly reduced vaginopathogenicity as compared to an isogenic control expressing a wild-type (WT) ECE1 allele. However, in vitro challenge of vaginal epithelial cells with synthetic candidalysin demonstrated similar toxigenic activity amongst SC5314 and 529L isoforms. Creation of an isogenic panel of chimeric strains harboring swapped Ece1p peptides or HiBiT tags revealed reduced secretion with the ORF from 529L that was associated with reduced virulence. A genetic survey of 78 clinical isolates demonstrated a conserved pattern between Ece1p P2 and P3 sequences, suggesting that substrate specificity around Kex2p-mediated KR cleavage sites involved in protein processing may contribute to differential pathogenicity amongst clinical isolates. Therefore, we present a new mechanism for attenuation of C. albicans virulence at the ECE1 locus.

Remasking of Candida albicans ß-Glucan in Response to Environmental pH Is Regulated by Quorum Sensing.

Candida albicans is a commensal yeast of the human gut which is tolerated by the immune system but has the potential to become an opportunistic pathogen. One way in which C. albicans achieves this duality is through concealing or exposing cell wall pathogen-associated molecular patterns (PAMPs) in response to host-derived environment cues (pH, hypoxia, and lactate). This cell wall remodeling allows C. albicans to evade or hyperactivate the host's innate immune responses, leading to disease. Previously, we showed that adaptation of C. albicans to acidic environments, conditions encountered during colonization of the female reproductive tract, induces significant cell wall remodeling resulting in the exposure of two key fungal PAMPs (ß-glucan and chitin). Here, we report that this pH-dependent cell wall remodeling is time dependent, with the initial change in pH driving cell wall unmasking, which is then remasked at later time points. Remasking of ß-glucan was mediated via the cell density-dependent fungal quorum sensing molecule farnesol, while chitin remasking was mediated via a small, heat-stable, nonproteinaceous secreted molecule(s). Transcript profiling identified a core set of 42 genes significantly regulated by pH over time and identified the transcription factor Efg1 as a regulator of chitin exposure through regulation of CHT2 This dynamic cell wall remodeling influenced innate immune recognition of C. albicans, suggesting that during infection, C. albicans can manipulate the host innate immune responses.IMPORTANCECandida albicans is part of the microbiota of the skin and gastrointestinal and reproductive tracts of humans and has coevolved with us for millennia. During that period, C. albicans has developed strategies to modulate the host's innate immune responses, by regulating the exposure of key epitopes on the fungal cell surface. Here, we report that exposing C. albicans to an acidic environment, similar to the one of the stomach or vagina, increases the detection of the yeast by macrophages. However, this effect is transitory, as C. albicans is able to remask these epitopes (glucan and chitin). We found that glucan remasking is controlled by the production of farnesol, a molecule secreted by C. albicans in response to high cell densities. However, chitin-remasking mechanisms remain to be identified. By understanding the relationship between environmental sensing and modulation of the host-pathogen interaction, new opportunities for the development of innovative antifungal strategies are possible.

Titrating Gene Function in the Human Fungal Pathogen Candida albicans through Poly-Adenosine Tract Insertion.

A recent study demonstrated that the insertion of poly-adenosine (poly-A) tracts into an open reading frame can suppress expression of the encoded protein in both prokaryotic and eukaryotic species. Furthermore, the degree of suppression is proportional to the length of the poly-A insertion, which can therefore provide a reliable and predictable means to titrate a specific protein's expression. The goal of this study was to determine if this methodology can be applied to modulate the expression of proteins in the prevalent human fungal pathogen, Candida albicans Insertion of increasing numbers of AAA codons encoding lysine at the N terminus of the C. albicans lanosterol demethylase (Erg11p) progressively diminished expression without significantly reducing the levels of mRNA. This suggests that Erg11p expression was attenuated at the posttranscriptional level. A direct correlation between the number of AAA codons inserted and C. albicans susceptibility to the Erg11p inhibitor fluconazole was also noted, indicating a progressive loss of Erg11p activity. Finally, we constructed a series of C. albicans strains with 3 to 12 AAA codons inserted at the 5' end of the ARO1 gene, which encodes a pentafunctional enzyme catalyzing five sequential steps of the aromatic amino acid biosynthetic pathway. Increasing numbers of AAA codons progressively reduced the growth rate of C. albicans in standard laboratory medium, indicating a progressive loss of ARO biosynthetic activity. These data unequivocally demonstrate the potential utility of the poly-A insertion method to examine the phenotypic consequences of titrating target protein function in C. albicansIMPORTANCE Investigating a protein's functional importance at the whole-organism level usually involves altering its expression level or its specific activity and observing the consequences with respect to physiology or phenotype. Several approaches designed to partially or completely abolish the function of a gene, including its deletion from the genome and the use of systems that facilitate conditional expression, have been widely applied. However, each has significant limitations that are especially problematic in pathogenic microbes when it is desirable to determine if a particular gene is required for infection in an animal model. In this study, we sought to determine if an alternative approach-the insertion of poly-A repeats within the coding sequence of the gene-is sufficient to modulate its function in the prevalent human fungal pathogen C. albicans Our results confirm that this approach enables us to predictably and gradually titrate the expression level of a protein and thus to investigate the phenotypic consequences of various levels of gene/protein function.

An antibiofilm coating of 5-aryl-2-aminoimidazole covalently attached to a titanium surface.

Biofilms, especially those formed by Staphylococcus aureus, play a key role in the development of orthopedic implant infections. Eradication of these infections is challenging due to the elevated tolerance of biofilm cells against antimicrobial agents. In this study, we developed an antibiofilm coating consisting of 5-(4-bromophenyl)-N-cyclopentyl-1-octyl-1H-imidazol-2-amine, designated as LC0024, covalently bound to a titanium implant surface (LC0024-Ti). We showed in vitro that the LC0024-Ti surface reduces biofilm formation of S. aureus in a specific manner without reducing the planktonic cells above the biofilm, as evaluated by plate counting and fluorescence microscopy. The advantage of compounds that only inhibit biofilm formation without affecting the viability of the planktonic cells, is that reduced development of bacterial resistance is expected. To determine the antibiofilm activity of LC0024-Ti surfaces in vivo, a biomaterial-associated murine infection model was used. The results indicated a significant reduction in S. aureus biofilm formation (up to 96%) on the LC0024-Ti substrates compared to pristine titanium controls. Additionally, we found that the LC0024-Ti substrates did not affect the attachment and proliferation of human cells involved in osseointegration and bone repair. In summary, our results emphasize the clinical potential of covalent coatings of LC0024 on titanium implant surfaces to reduce the risk of orthopedic implant infections. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1908-1919, 2019.

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.

Loss of Upc2p-Inducible ERG3 Transcription Is Sufficient To Confer Niche-Specific Azole Resistance without Compromising Candida albicans Pathogenicity.

Inactivation of sterol Δ5,6-desaturase (Erg3p) in the prevalent fungal pathogen Candida albicans is one of several mechanisms that can confer resistance to the azole antifungal drugs. However, loss of Erg3p activity is also associated with deficiencies in stress tolerance, invasive hyphal growth, and attenuated virulence in a mouse model of disseminated infection. This may explain why relatively few erg3-deficient strains have been reported among azole-resistant clinical isolates. In this study, we examined the consequences of Erg3p inactivation upon C. albicans pathogenicity and azole susceptibility in mouse models of mucosal and disseminated infection. While a C. albicanserg3Δ/Δ mutant was unable to cause lethality in the disseminated model, it induced pathology in a mouse model of vaginal infection. The erg3Δ/Δ mutant was also more resistant to fluconazole treatment than the wild type in both models of infection. Thus, complete loss of Erg3p activity confers azole resistance but also niche-specific virulence deficiencies. Serendipitously, we discovered that loss of azole-inducible ERG3 transcription (rather than complete inactivation) is sufficient to confer in vitro fluconazole resistance, without compromising C. albicans stress tolerance, hyphal growth, or pathogenicity in either mouse model. It is also sufficient to confer fluconazole resistance in the mouse vaginal model, but not in the disseminated model of infection, and thus confers niche-specific azole resistance without compromising C. albicans pathogenicity at either site. Collectively, these results establish that modulating Erg3p expression or activity can have niche-specific consequences on both C. albicans pathogenicity and azole resistance.IMPORTANCE While conferring resistance to the azole antifungals in vitro, loss of sterol Δ5,6-desaturase (Erg3p) activity has also been shown to reduce C. albicans pathogenicity. Accordingly, it has been presumed that this mechanism may not be significant in the clinical setting. The results presented here challenge this assumption, revealing a more complex relationship between Erg3p activity, azole resistance, C. albicans pathogenicity, and the specific site of infection. Most importantly, we have shown that even modest changes in ERG3 transcription are sufficient to confer azole resistance without compromising C. albicans fitness or pathogenicity. Given that previous efforts to assess the importance of ERG3 as a determinant of clinical azole resistance have focused almost exclusively on detecting null mutants, its role may have been grossly underestimated. On the basis of our results, a more thorough investigation of the contribution of the ERG3 gene to azole resistance in the clinical setting is warranted.

Methionine is required for cAMP-PKA-mediated morphogenesis and virulence of Candida albicans.

Candida albicans is a major human fungal pathogen, causing superficial, as well as life-threatening invasive infections. Therefore, it has to adequately sense and respond to the host defense by expressing appropriate virulence attributes. The most important virulence factor of C. albicans is the yeast-to-hyphae morphogenetic switch, which can be induced by numerous environmental cues, including the amino acid methionine. Here, we show an essential role for methionine permease Mup1 in methionine-induced morphogenesis, biofilm formation, survival inside macrophages and virulence. Furthermore, we demonstrate that this process requires conversion of methionine into S-adenosyl methionine (SAM) and its decarboxylation by Spe2. The resulting amino-propyl group is then used for biosynthesis of polyamines, which have been shown to activate adenylate cyclase. Inhibition of the SPE2 SAM decarboxylase gene strongly impairs methionine-induced morphogenesis on specific media and significantly delays virulence in the mouse systemic infection model system. Further proof of the connection between methionine uptake and initial metabolism and the cAMP-PKA pathway was obtained by showing that both Mup1 and Spe2 are required for cAMP production in response to methionine. Our results suggest that amino acid transport and further metabolism are interesting therapeutic targets as inhibitors of this may prevent the morphogenetic switch, thereby preventing virulence.

A Bimolecular Fluorescence Complementation Tool for Identification of Protein-Protein Interactions in Candida albicans.

Investigation of protein-protein interactions (PPI) in Candida albicans is essential for understanding the regulation of the signal transduction network that triggers its pathogenic lifestyle. Unique features of C. albicans, such as its alternative codon usage and incomplete meiosis, have enforced the optimization of standard genetic methods as well as development of novel approaches. Since the existing methods for detection of PPI are limited for direct visualization of the interacting complex in vivo, we have established a bimolecular fluorescence complementation (BiFC) assay in C. albicans, a powerful technique for studying PPI. We have developed an optimized set of plasmids that allows for N- and C-terminal tagging of proteins with split yeast-enhanced monomeric Venus fragments, so that all eight combinations of fusion orientations can be analyzed. With the use of our BiFC assay we demonstrate three interaction complexes in vivo, which were also confirmed by two-hybrid analysis. Our Candida-optimized BiFC assay represents a useful molecular tool for PPI studies and shows great promise in expanding our knowledge of molecular mechanisms of protein functions.

In Vivo Indicators of Cytoplasmic, Vacuolar, and Extracellular pH Using pHluorin2 in Candida albicans.

Environmental or chemically induced stresses often trigger physiological responses that regulate intracellular pH. As such, the capacity to detect pH changes in real time and within live cells is of fundamental importance to essentially all aspects of biology. In this respect, pHluorin, a pH-sensitive variant of green fluorescent protein, has provided an invaluable tool to detect such responses. Here, we report the adaptation of pHluorin2 (PHL2), a substantially brighter variant of pHluorin, for use with the human fungal pathogen Candida albicans. As well as a cytoplasmic PHL2 indicator, we describe a version that specifically localizes within the fungal vacuole, an acidified subcellular compartment with important functions in nutrient storage and pH homeostasis. In addition, by means of a glycophosphatidylinositol-anchored PHL2-fusion protein, we generated a cell surface pH sensor. We demonstrated the utility of these tools in several applications, including accurate intracellular and extracellular pH measurements in individual cells via flow cytometry and in cell populations via a convenient plate reader-based protocol. The PHL2 tools can also be used for endpoint as well as time course experiments and to conduct chemical screens to identify drugs that alter normal pH homeostasis. These tools enable observation of the highly dynamic intracellular pH shifts that occur throughout the fungal growth cycle, as well as in response to various chemical treatments. IMPORTANCECandida albicans is an opportunistic fungal pathogen that colonizes the reproductive and gastrointestinal tracts of its human host. It can also invade the bloodstream and deeper organs of immunosuppressed individuals, and thus it encounters enormous variations in external pH in vivo. Accordingly, survival within such diverse niches necessitates robust adaptive responses to regulate intracellular pH. However, the impact of antifungal drugs upon these adaptive responses, and on intracellular pH in general, is not well characterized. Furthermore, the tools and methods currently available to directly monitor intracellular pH in C. albicans, as well as other fungal pathogens, have significant limitations. To address these issues, we developed a new and improved set of pH sensors based on the pH-responsive fluorescent protein pHluorin. This includes a cytoplasmic sensor, a probe that localizes inside the fungal vacuole (an acidified compartment that plays a central role in intracellular pH homeostasis), and a cell surface probe that can detect changes in extracellular pH. These tools can be used to monitor pH within single C. albicans cells or in cell populations in real time through convenient and high-throughput assays.

An Azole-Tolerant Endosomal Trafficking Mutant of Candida albicans Is Susceptible to Azole Treatment in a Mouse Model of Vaginal Candidiasis.

We recently reported that a Candida albicans endosomal trafficking mutant continues to grow after treatment with the azole antifungals. Herein, we report that the vps21Δ/Δ mutant does not have a survival advantage over wild-type isolates after fluconazole treatment in a mouse model of vaginal candidiasis. Furthermore, loss of VPS21 does not synergize with established mechanisms of azole resistance, such as overexpression of efflux pumps or of Erg11p, the target enzyme of the azoles. In summary, although loss of VPS21 function enhances C. albicans survival after azole treatment in vitro, it does not seem to affect azole susceptibility in vivo.

Identification of small molecules that disrupt vacuolar function in the pathogen Candida albicans.

The fungal vacuole is a large acidified organelle that performs a variety of cellular functions. At least a sub-set of these functions are crucial for pathogenic species of fungi, such as Candida albicans, to survive within and invade mammalian tissue as mutants with severe defects in vacuolar biogenesis are avirulent. We therefore sought to identify chemical probes that disrupt the normal function and/or integrity of the fungal vacuole to provide tools for the functional analysis of this organelle as well as potential experimental therapeutics. A convenient indicator of vacuolar integrity based upon the intracellular accumulation of an endogenously produced pigment was adapted to identify Vacuole Disrupting chemical Agents (VDAs). Several chemical libraries were screened and a set of 29 compounds demonstrated to reproducibly cause loss of pigmentation, including 9 azole antifungals, a statin and 3 NSAIDs. Quantitative analysis of vacuolar morphology revealed that (excluding the azoles) a sub-set of 14 VDAs significantly alter vacuolar number, size and/or shape. Many C. albicans mutants with impaired vacuolar function are deficient in the formation of hyphal elements, a process essential for its pathogenicity. Accordingly, all 14 VDAs negatively impact C. albicans hyphal morphogenesis. Fungal selectivity was observed for approximately half of the VDA compounds identified, since they did not alter the morphology of the equivalent mammalian organelle, the lysosome. Collectively, these compounds comprise of a new collection of chemical probes that directly or indirectly perturb normal vacuolar function in C. albicans.

Characterization of the Candida albicans Amino Acid Permease Family: Gap2 Is the Only General Amino Acid Permease and Gap4 Is an S-Adenosylmethionine (SAM) Transporter Required for SAM-Induced Morphogenesis.

Amino acids are key sources of nitrogen for growth of Candida albicans. In order to detect and take up these amino acids from a broad range of different and changing nitrogen sources inside the host, this fungus must be able to adapt via its expression of genes for amino acid uptake and further metabolism. We analyzed six C. albicans putative general amino acid permeases based on their homology to the Saccharomyces cerevisiae Gap1 general amino acid permease. We generated single- and multiple-deletion strains and found that, based on growth assays and transcriptional or posttranscriptional regulation, Gap2 is the functional orthologue to ScGap1, with broad substrate specificity. Expression analysis showed that expression of all GAP genes is under control of the Csy1 amino acid sensor, which is different from the situation in S. cerevisiae, where the expression of ScGAP1 is not regulated by Ssy1. We show that Gap4 is the functional orthologue of ScSam3, the only S-adenosylmethionine (SAM) transporter in S. cerevisiae, and we report that Gap4 is required for SAM-induced morphogenesis. IMPORTANCECandida albicans is a commensal organism that can thrive in many niches in its human host. The environmental conditions at these different niches differ quite a bit, and this fungus must be able to sense these changes and adapt its metabolism to them. Apart from glucose and other sugars, the uptake of amino acids is very important. This is underscored by the fact that the C. albicans genome encodes 6 orthologues of the Saccharomyces. cerevisiae general amino acid permease Gap1 and many other amino acid transporters. In this work, we characterize these six permeases and we show that C. albicans Gap2 is the functional orthologue of ScGap1 and that C. albicans Gap4 is an orthologue of ScSam3, an S-adenosylmethionine (SAM) transporter. Furthermore, we show that Gap4 is required for SAM-induced morphogenesis, an important virulence factor of C. albicans.

Endosomal Trafficking Defects Can Induce Calcium-Dependent Azole Tolerance in Candida albicans.

The azole antifungals arrest fungal growth through inhibition of ergosterol biosynthesis. We recently reported that a Candida albicans vps21Δ/Δ mutant, deficient in membrane trafficking through the late endosome/prevacuolar compartment (PVC), continues to grow in the presence of the azoles despite the depletion of cellular ergosterol. Here, we report that the vps21Δ/Δ mutant exhibits less plasma membrane damage upon azole treatment than the wild type, as measured by the release of a cytoplasmic luciferase reporter into the culture supernatant. Our results also reveal that the vps21Δ/Δ mutant has abnormal levels of intracellular Ca2+ and, in the presence of fluconazole, enhanced expression of a calcineurin-responsive RTA2-GFP reporter. Furthermore, the azole tolerance phenotype of the vps21Δ/Δ mutant is dependent upon both extracellular calcium levels and calcineurin activity. These findings underscore the importance of endosomal trafficking in determining the cellular consequences of azole treatment and indicate that this may occur through modulation of calcium- and calcineurin-dependent responses.

Modulation of the Substitution Pattern of 5-Aryl-2-Aminoimidazoles Allows Fine-Tuning of Their Antibiofilm Activity Spectrum and Toxicity.

We previously synthesized several series of compounds, based on the 5-aryl-2-aminoimidazole scaffold, that showed activity preventing the formation of Salmonella enterica serovar Typhimurium and Pseudomonas aeruginosa biofilms. Here, we further studied the activity spectrum of a number of the most active N1- and 2N-substituted 5-aryl-2-aminoimidazoles against a broad panel of biofilms formed by monospecies and mixed species of bacteria and fungi. An N1-substituted compound showed very strong activity against the biofilms formed by Gram-negative and Gram-positive bacteria and the fungus Candida albicans but was previously shown to be toxic against various eukaryotic cell lines. In contrast, 2N-substituted compounds were nontoxic and active against biofilms formed by Gram-negative bacteria and C. albicans but had reduced activity against biofilms formed by Gram-positive bacteria. In an attempt to develop nontoxic compounds with potent activity against biofilms formed by Gram-positive bacteria for application in antibiofilm coatings for medical implants, we synthesized novel compounds with substituents at both the N1 and 2N positions and tested these compounds for antibiofilm activity and toxicity. Interestingly, most of these N1-,2N-disubstituted 5-aryl-2-aminoimidazoles showed very strong activity against biofilms formed by Gram-positive bacteria and C. albicans in various setups with biofilms formed by monospecies and mixed species but lost activity against biofilms formed by Gram-negative bacteria. In light of application of these compounds as anti-infective coatings on orthopedic implants, toxicity against two bone cell lines and the functionality of these cells were tested. The N1-,2N-disubstituted 5-aryl-2-aminoimidazoles in general did not affect the viability of bone cells and even induced calcium deposition. This indicates that modulating the substitution pattern on positions N1 and 2N of the 5-aryl-2-aminoimidazole scaffold allows fine-tuning of both the antibiofilm activity spectrum and toxicity.

Covalent immobilization of antimicrobial agents on titanium prevents Staphylococcus aureus and Candida albicans colonization and biofilm formation.

OBJECTIVES: Biofilm-associated implant infections represent a serious public health problem. Covalent immobilization of antimicrobial agents on titanium (Ti), thereby inhibiting biofilm formation of microbial pathogens, is a solution to this problem. METHODS: Vancomycin (VAN) and caspofungin (CAS) were covalently bound on Ti substrates using an improved processing technique adapted to large-scale coating of implants. Resistance of the VAN-coated Ti (VAN-Ti) and CAS-coated Ti (CAS-Ti) substrates against in vitro biofilm formation of the bacterium Staphylococcus aureus and the fungal pathogen Candida albicans was determined by plate counting and visualized by confocal laser scanning microscopy. The efficacy of the coated Ti substrates was also tested in vivo using an adapted biomaterial-associated murine infection model in which control-Ti, VAN-Ti or CAS-Ti substrates were implanted subcutaneously and subsequently challenged with the respective pathogens. The osseointegration potential of VAN-Ti and CAS-Ti was examined in vitro using human bone marrow-derived stromal cells, and for VAN-Ti also in a rat osseointegration model. RESULTS: In vitro biofilm formation of S. aureus and C. albicans on VAN-Ti and CAS-Ti substrates, respectively, was significantly reduced compared with biofilm formation on control-Ti. In vivo, we observed over 99.9% reduction in biofilm formation of S. aureus on VAN-Ti substrates and 89% reduction in biofilm formation of C. albicans on CAS-Ti substrates, compared with control-Ti substrates. The coated substrates supported osseointegration in vitro and in vivo. CONCLUSIONS: These data demonstrate the clinical potential of covalently bound VAN and CAS on Ti to reduce microbial biofilm formation without jeopardizing osseointegration.

Detecting nanoscale vibrations as signature of life.

The existence of life in extreme conditions, in particular in extraterrestrial environments, is certainly one of the most intriguing scientific questions of our time. In this report, we demonstrate the use of an innovative nanoscale motion sensor in life-searching experiments in Earth-bound and interplanetary missions. This technique exploits the sensitivity of nanomechanical oscillators to transduce the small fluctuations that characterize living systems. The intensity of such movements is an indication of the viability of living specimens and conveys information related to their metabolic activity. Here, we show that the nanomotion detector can assess the viability of a vast range of biological specimens and that it could be the perfect complement to conventional chemical life-detection assays. Indeed, by combining chemical and dynamical measurements, we could achieve an unprecedented depth in the characterization of life in extreme and extraterrestrial environments.

Tight control of trehalose content is required for efficient heat-induced cell elongation in Candida albicans.

The ability to form hyphae in the human pathogenic fungus Candida albicans is a prerequisite for virulence. It contributes to tissue infection, biofilm formation, as well as escape from phagocytes. Cell elongation triggered by human body temperature involves the essential heat shock protein Hsp90, which negatively governs a filamentation program dependent upon the Ras-protein kinase A (PKA) pathway. Tight regulation of Hsp90 function is required to ensure fast appropriate response and maintenance of a wide range of regulatory and signaling proteins. Client protein activation by Hsp90 relies on a conformational change of the chaperone, whose ATPase activity is competitively inhibited by geldanamycin. We demonstrate a novel regulatory mechanism of heat- and Hsp90-dependent induced morphogenesis, whereby the nonreducing disaccharide trehalose acts as a negative regulator of Hsp90 release. By means of a mutant strain deleted for Gpr1, the G protein-coupled receptor upstream of PKA, we demonstrate that elevated trehalose content in that strain, resulting from misregulation of enzymatic activities involved in trehalose metabolism, disrupts the filamentation program in response to heat. Addition of geldanamycin does not result in hyphal extensions at 30 °C in the gpr1Δ/gpr1Δ mutant as it does in wild type cells. In addition, validamycin, a specific inhibitor of trehalase, the trehalose-degrading enzyme, inhibits cell elongation in response to heat and geldanamycin. These results place Gpr1 as a regulator of trehalose metabolism in C. albicans and illustrate that trehalose modulates Hsp90-dependent activation of client proteins and signaling pathways leading to filamentation in the human fungal pathogen.