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Candida albicans, the primary etiology of human mycoses, is well-adapted to catabolize proline to obtain energy to initiate morphological switching (yeast to hyphal) and for growth. We report that put1-/- and put2-/- strains, carrying defective Proline UTilization genes, display remarkable proline sensitivity with put2-/- mutants being hypersensitive due to the accumulation of the toxic intermediate pyrroline-5-carboxylate (P5C), which inhibits mitochondrial respiration. The put1-/- and put2-/- mutations attenuate virulence in Drosophila and murine candidemia models and decrease survival in human neutrophils and whole blood. Using intravital 2-photon microscopy and label-free non-linear imaging, we visualized the initial stages of C. albicans cells infecting a kidney in real-time, directly deep in the tissue of a living mouse, and observed morphological switching of wildtype but not of put2-/- cells. Multiple members of the Candida species complex, including C. auris, are capable of using proline as a sole energy source. Our results indicate that a tailored proline metabolic network tuned to the mammalian host environment is a key feature of opportunistic fungal pathogens. Author summary: Candida albicans is listed as one of four "critical priority" fungal pathogens by the World Health Organization (WHO). Intriguingly, C. albicans is a natural commensal organism thriving in symbiosis with other components of the human microflora. Given the opportunistic character of C. albicans, it is important to understand how nutrient availability within host microenvironments contribute to the transition from commensal to pathogenic growth. Here, we report that proline is catabolized by C. albicans as an important energy source, a characteristic that is conserved among other pathogenic Candida species, including the multidrug resistant C. glabrata and C. auris. Using different infection models and applying a state-of-the-art intravital imaging technique to visualize C. albicans cells infecting kidneys in a living mouse, we observed that strains unable to utilize proline exhibit significantly reduced virulence and filamentous hyphal growth. Genetic dissection of the Proline UTilization (PUT) pathway and deciphering the control mechanisms governing proline use led to the discovery that proline is toxic to cells unable to catabolize this amino acid. Our findings provide novel insights implicating proline metabolism as a key determinant of pathogenic fungal growth. [ABSTRACT FROM AUTHOR]

The fungal cell wall plays a crucial role in host-pathogen interactions. Its formation is the result of the coordinated activity of several extracellutar enzymes, which assemble the constituents, and remodel and hydrolyse them in the extracellular space. Canclida albicans Phrl and Phr2 proteins belong to family GH72 of the [beta]-(1,)-glucanosyltransferases and play a crucial role in cell wall assembly. PHR1 and PHR2, homologues of Saccharomyces cerevisiae GAS1, are differently regulated by extracellular pH. PHR1 is expressed when ambient pH is 5.5 or higher, whereas PHR2 has the reverse expression pattern. Their deletion causes a pH-conditional defect in morphogenesis and virulence. In this work we explored whether PHR1 deletion affects the ability of C. albicans to adhere to and invade human epithelia. PHR1 null mutants exhibited a marked reduction in adhesion to both abiotic surfaces and epithelial cell monolayers. In addition, the mutant was unable to penetrate and invade reconstituted human epithelia. Transcription profiling of selected hyphal-specific and adhesin-encoding genes indicated that in the PHR1 null mutant, HWP1 and ECE1 transcript levels were similarly reduced in both adhesion and suspension conditions. These results, combined with microscopy analysis of the septum position, suggest that PHR1 is not required for the induction of hyphal development but plays a key role in the maintenance of hyphal growth. Thus, the [beta]-(1,)-glucan processing catalysed by Phrl p is of fundamental importance in the maintenance of the morphological state on which the adhesive and invasive properties of C. albicans greatly depend. DOI 10.1099/mic.0.038000-0

Herpes simplex virus-1 (HSV-1) is highly contagious, and there is a need for a therapeutic means to eradicate it. We have identified an siRNA (siHSV) that knocks down gene expression of the infected cell protein 0 (ICP0), which is important in the regulation of HSV infection. The selected siHSV was encapsulated in liposomes to overcome its poor stability, increase cell permeability, and prolonging siRNA circulation time. Several siRNAs against ICP0 have been designed and identified. We examined the role of various parameters, including formulation technique, lipids composition, and ratio. An optimal liposomal siHSV formulation (LipDOPE-siHSV) was characterized with desirable physiochemical properties, in terms of nano-size, low polydispersity index (PDI), neutral surface charge, high siHSV loading, spherical shape, high stability in physiologic conditions in vitro, and long-term shelf-life stability (>1 year, 4 °C). The liposomes exhibited profound internalization by human keratinocytes, no cytotoxicity in cell cultures, no detrimental effect on mice liver enzymes, and a gradual endo-lysosomal escape. Mice biodistribution studies in intact mice revealed accumulation, mainly in visceral organs but also in the trigeminal ganglion. The therapeutic potential of siHSV liposomes was demonstrated by significant antiviral activity both in the plaque reduction assay and in the 3D epidermis model, and the mechanism of action was validated by the reduction of ICP0 expression levels. [ABSTRACT FROM AUTHOR]

Techniques for continuously monitoring the formation of subgingival biofilm, in relation to the determination of species and their accumulation over time in gingivitis and periodontitis, are limited. In recent years, advancements in the field of optical spectroscopic techniques have provided an alternative for analyzing three-dimensional microbiological structures, replacing the traditional destructive or biofilm staining techniques. In this work, we have demonstrated that the use of confocal Raman spectroscopy coupled with multivariate analysis provides an approach to spatially differentiate bacteria in an in vitro model simulating a subgingival dual-species biofilm. The present study establishes a workflow to evaluate and differentiate bacterial species in a dual-species in vitro biofilm model, using confocal Raman microscopy (CRM). Biofilm models of Actinomyces denticolens and Streptococcus oralis were cultured using the "Zürich in vitro model" and were analyzed using CRM. Cluster analysis was used to spatially differentiate and map the biofilm model over a specified area. To confirm the clustering of species in the cultured biofilm, confocal laser scanning microscopy (CLSM) was coupled with fluorescent in vitro hybridization (FISH). Additionally, dense bacteria interface area (DBIA) samples, as an imitation of the clusters in a biofilm, were used to test the developed multivariate differentiation model. This confirmed model was successfully used to differentiate species in a dual-species biofilm and is comparable to morphology. The results show that the developed workflow was able to identify main clusters of bacteria based on spectral "fingerprint region" information from CRM. Using this workflow, we have demonstrated that CRM can spatially analyze two-species in vitro biofilms, therefore providing an alternative technique to map oral multi-species biofilm models. [ABSTRACT FROM AUTHOR]

Mannosylerythritol lipids (MELs) are glycolipid biosurfactants produced by fungi of the Ustilaginaceae family in the presence of hydrophobic carbon sources like plant oils. In the present study,we investigated the structural composition of MELs produced from castor oil using seven different microorganisms and compared them to MEL structures resulting fromother plant oils. Castor oil is an industrially relevant plant oil that presents as an alternative to currently employed edible plant oils like rapeseed or soybean oil. The main fatty acid in castor oil is the mono-hydroxylated ricinoleic acid, providing the possibility to produce novel MEL structures with interesting features. Analysis of the produced MELs from castor oil by different chromatographic and mass spectrometry techniques revealed that all seven microorganisms were generally able to integrate hydroxylated fatty acids into the MEL molecule, although at varying degrees. These novel MELs containing a hydroxy fatty acid (4-O-[2'-O-alka(e)noyl-3'-O-hydroxyalka(e)noyl-4'/6'-O-acetyl-β- D-mannopyranosyl]-erythritol) were more hydrophilic than conventional MEL and therefore showed a different elution behavior in chromatography. Large shares of novel hydroxy MELs (around 50% of total MELs) were found for the two MEL-B/C producing species Ustilago siamensis and Ustilago shanxiensis, but also for the MEL-A/B/C producer Moesziomyces aphidis (around 25%). In addition, triacylated hydroxylated MELs with a third long-chain fatty acid esterified to the free hydroxyl group of the hydroxy fatty acid were identified for some species. Overall, production of MEL from castor oil with the investigated organisms provided a complex mixture of various novel MEL structures that can be exploited for further research. [ABSTRACT FROM AUTHOR]

The study of oral disease progression, in relation to the accumulation of subgingival biofilm in gingivitis and periodontitis is limited, due to either the ability to monitor plaque in vitro. When compared, optical spectroscopic techniques offer advantages over traditional destructive or biofilm staining approaches, making it a suitable alternative for the analysis and continued development of three-dimensional structures. In this work, we have developed a confocal Raman spectroscopy analysis approach towards in vitro subgingival plaque models. The main objective of this study was to develop a method for differentiating multiple oral subgingival bacterial species in planktonic and biofilm conditions, using confocal Raman microscopy. Five common subgingival bacteria (Fusobacterium nucleatum, Streptococcus mutans, Veillonella dispar, Actinomyces naeslundii and Prevotella nigrescens) were used and differentiated using a 2-way orthogonal Partial Least Square with Discriminant Analysis (O2PLS-DA) for the collected spectral data. In addition to planktonic growth, mono-species biofilms cultured using the 'Zürich Model' were also analyzed. The developed method was successfully used to predict planktonic and mono-species biofilm species in a cross validation setup. The results show differences in the presence and absence of chemical bands within the Raman spectra. The O2PLS-DA model was able to successfully predict 100% of all tested planktonic samples and 90% of all mono-species biofilm samples. Using this approach we have shown that Confocal Raman microscopy can analyse and predict the identity of planktonic and mono-species biofilm species, thus enabling its potential as a technique to map oral multi-species biofilm models. [ABSTRACT FROM AUTHOR]

6 Foam generation ug and decay ud rates of samples 1-8 containing different fractions of a culture broth with U. maydis, as described in Table 1. Figure 6 shows: Foam generation u SB g sb and decay u SB d sb rates of samples 1-8 containing different fractions of a culture broth with I U. maydis i , as described in Table 1. [Extracted from the article]

Members of the Cysteine-rich secretory protein, Antigen 5 and Pathogenesis-related 1 (CAP) protein superfamily are important virulence factors in fungi but remain poorly characterized on molecular level. Here, we investigate the cellular localization and molecular function of Rbe1p and Rbt4p, two CAP family members from the human pathogen Candida albicans. We unexpectedly found that Rbe1p localizes to budding sites of yeast cells in a disulfide bond-dependent manner. Furthermore, we show that Rbe1p and Rbt4p bind free cholesterol in vitro and export cholesteryl acetate in vivo. These findings suggest a previously undescribed role for Rbe1p in cell wall-associated processes and a possible connection between the virulence attributes of fungal CAP proteins and sterol binding. [ABSTRACT FROM AUTHOR]

The human skin is commonly colonized by diverse fungal species. Some Candida species, especially C. albicans, do not only reside on the skin surface as commensals, but also cause infections by growing into the colonized tissue. However, defense mechanisms at the skin barrier level are very efficient, involving residential non-immune and immune cells as well as immune cells specifically recruited to the site of infection. Therefore, the skin is an effective barrier against fungal infection. While most studies about commensal and pathogenic interaction of Candida species with host epithelia focus on the interaction with mucosal surfaces such as the vaginal and gastrointestinal epithelia, less is known about the mechanisms underlying Candida interaction with the skin. In this review, we focus on the ecology and molecular pathogenesis of Candida species on the skin and give an overview of defense mechanisms against C. albicans in this context. We also discuss new research avenues in dermal infection, including the involvement of neurons, fibroblasts, and commensal bacteria in both mouse and human model systems. [ABSTRACT FROM AUTHOR]

Inactivated vaccines are commonly produced by incubating pathogens with chemicals such as formaldehyde or β-propiolactone. This is a time-consuming process, the inactivation efficiency displays high variability and extensive downstream procedures are often required. Moreover, application of chemicals alters the antigenic components of the viruses or bacteria, resulting in reduced antibody specificity and therefore stimulation of a less effective immune response. An alternative method for inactivation of pathogens is ionizing radiation. It acts very fast and predominantly damages nucleic acids, conserving most of the antigenic structures. However, currently used irradiation technologies (mostly gamma-rays and high energy electrons) require large and complex shielding constructions to protect the environment from radioactivity or X-rays generated during the process. This excludes them from direct integration into biological production facilities. Here, low-energy electron irradiation (LEEI) is presented as an alternative inactivation method for pathogens in liquid solutions. LEEI can be used in normal laboratories, including good manufacturing practice (GMP)- or high biosafety level (BSL)-environments, as only minor shielding is necessary. We show that LEEI efficiently inactivates different viruses (influenza A (H3N8), porcine reproductive and respiratory syndrome virus (PRRSV), equine herpesvirus 1 (EHV-1)) and bacteria (Escherichia coli) and maintains their antigenicity. Moreover, LEEI-inactivated influenza A viruses elicit protective immune responses in animals, as analyzed by virus neutralization assays and viral load determination upon challenge. These results have implications for novel ways of developing and manufacturing inactivated vaccines with improved efficacy. [ABSTRACT FROM AUTHOR]

A comparative study concerning the physicochemical, monomeric composition and biological characters among different fucoidan fractions is presented. Common purification techniques for fucoidan usually involve many steps. During these steps, the important structural features might be affected and consequently alter its biological activities. Three purified fractions were derived from Fucus vesiculosus water extract which, afterwards, were purified by a recently-developed dye affinity chromatography protocol. This protocol is based on dye-sulfated polysaccharide interactions. The first two fractions were obtained from crude precipitated fucoidan at different pH values of the adsorption phase: pH 1 and 6. This procedure resulted in fucoidan_1 and 6 fractions. The other, third, fraction: fucoidan_M, however, was obtained from a buffered crude extract at pH 1, eliminating the ethanol precipitation step. All of the three fractions were then further evaluated. Results revealed that fucoidan_M showed the highest sulfur content (S%), 12.11%, with the lowest average molecular weight, 48 kDa. Fucose, galactose, and uronic acid/glucose dimers were detected in all fractions, although, xylose was only detected in fucoidan_1 and 6. In a concentration of 10 μg·mL-1, Fucoidan_6 showed the highest heparin-like anticoagulant activity and could prolong the APTT and TT significantly to 66.03 ± 2.93 and 75.36 ± 1.37 s, respectively. In addition, fucoidan_M demonstrated the highest potency against HSV-1 with an IC50 of 2.41 μg·mL-1. The technique proved to be a candidate for fucoidan purifaction from its crude extract removing the precipitation step from common purification protocols and produced different fucoidan qualities resulted from the different incubation conditions with the immobilized thiazine toluidine blue O dye. [ABSTRACT FROM AUTHOR]

Since its development, microarray technology has evolved to a standard method in the biotechnological and medical field with a broad range of applications. Nevertheless, the underlying mechanism of the hybridization process of PCR-products to microarray capture probes is still not completely understood, and several observed phenomena cannot be explained with current models. We investigated the influence of several parameters on the hybridization reaction and identified ssDNA to play a major role in the process. An increase of the ssDNA content in a hybridization reaction strongly enhanced resulting signal intensities. A strong influence could also be observed when unlabeled ssDNA was added to the hybridization reaction. A reduction of the ssDNA content resulted in a massive decrease of the hybridization efficiency. According to these data, we developed a novel model for the hybridization mechanism. This model is based on the assumption that single stranded DNA is necessary as catalyst to induce the hybridization of dsDNA. The developed hybridization model is capable of giving explanations for several yet unresolved questions regarding the functionality of microarrays. Our findings not only deepen the understanding of the hybridization process, but also have immediate practical use in data interpretation and the development of new microarrays. [ABSTRACT FROM AUTHOR]

Protein-protein interactions play an essential role in almost any biological processes. Therefore, there is a particular need for methods which describe the interactions of a defined target protein in its physiological context. Here we report a method to photo-cross-link interacting proteins in S. cerevisiae by using the non-canonical amino acid p-azido-L-phenylalanine (pAzpa). Based on the expanded genetic code the photoreactive non-canonical amino acid pAzpa was site-specifically incorporated at eight positions into a domain of Aha1 that was previously described to bind Hsp90 in vitro to function as a cochaperone of Hsp90 and activates its ATPase activity. In vivo photo-cross-linking to the cognate binding partner of Aha1 was carried out by irradiation of mutant strains with UV light (365 nm) to induce covalent intermolecular bonds. Surprisingly, an interaction between Aha1 and Hsp90 was not detected, although, we could confirm binding of suppressed pAzpa containing Aha1 to Hsp90 by native co-immunoprecipitation. However, a homodimer consisting of two covalently crosslinked Aha1 monomers was identified by mass spectrometry. This homodimer could also be confirmed using p-benzoyl-L-phenylalanine, another photoreactive non-canonical amino acid. Crosslinking was highly specific as it was dependent on irradiation using UV light, the exact position of the non-canonical amino acid in the protein sequence as well as on the addition of the non-canonical amino acid to the growth medium. Therefore it seems possible that an interaction of Aha1 with Hsp90 takes place at different positions than previously described in vitro highlighting the importance of in vivo techniques to study protein-protein interactions. Accordingly, the expanded genetic code can easily be applied to other S. cerevisiae proteins to study their interaction under physiological relevant conditions in vivo. [ABSTRACT FROM AUTHOR]

Background: Although Candida albicans and Candida dubliniensis are most closely related, both species behave significantly different with respect to morphogenesis and virulence. In order to gain further insight into the divergent routes for morphogenetic adaptation in both species, we investigated qualitative along with quantitative differences in the transcriptomes of both organisms by cDNA deep sequencing. Results: Following genome-associated assembly of sequence reads we were able to generate experimentally verified databases containing 6016 and 5972 genes for C. albicans and C. dubliniensis, respectively. About 95% of the transcriptionally active regions (TARs) contain open reading frames while the remaining TARs most likely represent non-coding RNAs. Comparison of our annotations with publically available gene models for C. albicans and C. dubliniensis confirmed approximately 95% of already predicted genes, but also revealed so far unknown novel TARs in both species. Qualitative cross-species analysis of these databases revealed in addition to 5802 orthologs also 399 and 49 species-specific protein coding genes for C. albicans and C. dubliniensis, respectively. Furthermore, quantitative transcriptional profiling using RNA-Seq revealed significant differences in the expression of orthologs across both species. We defined a core subset of 84 hyphal-specific genes required for both species, as well as a set of 42 genes that seem to be specifically induced during hyphal morphogenesis in C. albicans. Conclusions: Species-specific adaptation in C. albicans and C. dubliniensis is governed by individual genetic repertoires but also by altered regulation of conserved orthologs on the transcriptional level. [ABSTRACT FROM AUTHOR]

Candida albicans and Candida dubliniensis are pathogenic fungi that are highly related but differ in virulence and in some phenotypic traits. During in vitro growth on certain nutrient-poor media, C. albicans and C. dubliniensis are the only yeast species which are able to produce chlamydospores, large thick-walled cells of unknown function. Interestingly, only C. dubliniensis forms pseudohyphae with abundant chlamydospores when grown on Staib medium, while C. albicans grows exclusively as a budding yeast. In order to further our understanding of chlamydospore development and assembly, we compared the global transcriptional profile of both species during growth in liquid Staib medium by RNA sequencing. We also included a C. albicans mutant in our study which lacks the morphogenetic transcriptional repressor Nrg1. This strain, which is characterized by its constitutive pseudohyphal growth, specifically produces masses of chlamydospores in Staib medium, similar to C. dubliniensis. This comparative approach identified a set of putatively chlamydospore-related genes. Two of the homologous C. albicans and C. dubliniensis genes (CSP1 and CSP2) which were most strongly upregulated during chlamydospore development were analysed in more detail. By use of the green fluorescent protein as a reporter, the encoded putative cell wall related proteins were found to exclusively localize to C. albicans and C. dubliniensis chlamydospores. Our findings uncover the first chlamydospore specific markers in Candida species and provide novel insights in the complex morphogenetic development of these important fungal pathogens. [ABSTRACT FROM AUTHOR]

Within the last decade, biotechnology gained pace in substituting petro-based products for the chemical industries. This is visible with the appearance of bio-based products in the market, from biosurfactants to bio-based polymers like polylactic acid to bio-ethylene. These technologies are mainly based on established fermentation technologies fostered by the use of renewable resources, culminating in the establishment of biorefineries that may be connected directly to the existing chemical infrastructure. Besides these large-scale technologies, the combination of molecular technologies, microfluidic devices, and enzymatic and cell-free conversions are currently developed to create new bioproduction systems enabling the production of compounds that may not be produced within a cell. This article summarizes some of the current ideas that are currently in development paving the way for a next generation of biotechnology. [ABSTRACT FROM AUTHOR]