Catalytic properties of Phr family members of cell wall glucan remodeling enzymes: implications for the adaptation of Candida albicans to ambient pH.

Fungal wall formation is a dynamic process involving several categories of enzymes. The GH72 family of ß(1,3)-glucanosyltransferases is essential for the determination of cell shape, for cell integrity and for virulence in pathogenic fungi. Candida albicans has five GH72 genes: PHR1 and PHR2 are pH dependent, the first being expressed at pH ≥ 6 and repressed at lower pH and the second regulated in the opposite manner, PGA4 is transcribed independently of pH whereas PHR3 and PGA5 have low expression levels. To characterize the catalytic properties of Phr1p-2p and probe the activity of Pga4p, we heterologously expressed these proteins and used a fluorescent assay based on the transfer of oligosaccharyl units from a donor to a sulforhodamine-labeled acceptor. Phr1p-2p used exclusively ß-1,3-glucan or cell wall glucan as donor and laminarin-derived oligosaccharides as acceptor. The acceptor efficiency increased with the length of the oligosaccharide. The temperature optimum was 30°C. The pH optimum was 5.8 for Phr1p and 3 for Phr2p. Overall, adaptation to pH of C. albicans appears to involve a fine interplay among the pH-dependent activity of Phr1p and Phr2p, the pH-regulated expression of their genes and protein stability. Unexpectedly, Pga4p was inactive suggesting that it turned into a structural mannoprotein.

A novel fluorescence assay and catalytic properties of Crh1 and Crh2 yeast cell wall transglycosylases.

The mechanical properties of fungal cell walls are largely determined by composition and mutual cross-linking of their macromolecular components. Previous work showed that the Crh proteins are required for the formation of cross-links between chitin and glucan at the Saccharomyces cerevisiae cell wall. In the present study, the proteins encoded by CRH1 and CRH2 were heterologously expressed in Pichia pastoris and a sensitive fluorescence in vitro soluble assay was devised for determination of their transglycosylating activities. Both proteins act as chitin transglycosylases; they use soluble chitin derivatives, such as carboxymethyl chitin, glycol-chitin and/or N-acetyl chito-oligosaccharides of DP (degree of polymerization)≥5 as the oligoglycosyl donors, and oligosaccharides derived from chitin, ß-(1,3)-glucan (laminarin) and ß-(1,6)-glucan (pustulan), fluorescently labelled with sulforhodamine or FITC as acceptors. The minimal number of intact hexopyranose units required by Crh1 and/or Crh2 in the molecule of the acceptor oligosaccharide was two and the effectivity of the acceptor increased with the increasing length of its oligosaccharide chain. Products of the transglycosylation reactions were hybrid molecules composed of the acceptor and portions of carboxymethyl chitin attached to its non-reducing end. Both proteins exhibited a weak chitinolytic activity in different assays whereby the ratio of endo- compared with exo-chitinase activity was approximately 4-fold higher in Crh1 than in Crh2. The pH optimum of both enzymes was 3.5 and the optimum temperature was 37°C. The results obtained in vitro with different fluorescently labelled oligosaccharides as artificial chitin acceptors corroborated well with those observed in vivo.

Dataset of Windows operating system forensics artefacts.

The dataset consists of records from the NTFS file system and event logs. In this study, we used images of devices from capture the flags competitions focused on the digital forensic of Windows operating systems and user activities. We created timelines of the security incident from the disk images using the Plaso tool, which we then processed and transformed the attributes of the timelines into binary values to simplify the application of data analysis and machine learning methods. The data are divided into 12 different files, and they are saved in CSV format.

Two Variants of a High-Throughput Fluorescent Microplate Assay of Polysaccharide Endotransglycosylases.

Polysaccharide endotransglycosylases (PETs) are the cell wall-modifying enzymes of fungi and plants. They catalyze random endo-splitting of the polysaccharide donor molecule and transfer of the newly formed reducing sugar residue to the nonreducing end of an acceptor molecule which can be a polysaccharide or an oligosaccharide. Owing to their important role in the cell wall formation, the inhibition of PETs represents an attractive strategy in the fight against fungal infections. We have elaborated two variants of a versatile high-throughput microplate fluorimetric assay that could be used for effective identification of PETs and screening of their inhibitors. Both assays use the respective polysaccharides as the donors and sulforhodamine-labeled oligosaccharides as the acceptors but differ from each other by mode of how the labeled polysaccharide products of transglycosylation are separated from the unreacted oligosaccharide acceptors. In the first variant, the reactions take place in a layer of agar gel laid on the bottoms of the wells of a microtitration plate. After the reaction, the high-Mr transglycosylation products are precipitated with 66 % ethanol and retained within the gel while the low-Mr products and the unreacted acceptors are washed out. In the second variant, the donor polysaccharides are adsorbed to the surface of a microplate well and remain adsorbed there also after becoming labeled in the course of the transglycosylation reaction whereas the unused low-Mr acceptors are washed out. As a proof of versatility, assays of heterologously expressed transglycosylases ScGas1, ScCrh1, and ScCrh2 from the yeast Saccharomyces cerevisiae, CaPhr1 and CaPhr2 from Candida albicans, and of a plant xyloglucan endotransglycosylase (XET) are demonstrated.