Effects of TiS2 on Inhibiting Candida albicans Biofilm Formation and Its Compatibility with Human Gingival Fibroblasts in Titanium Implants.

Titanium is widely used in medical devices, such as dental and orthopedic implants, due to its excellent mechanical properties, low toxicity, and biocompatibility. However, the titanium surface has the risk of microbial biofilm formation, which results in infections from species such as Candida albicans (C. albicans). This kind of biofilm prevents antifungal therapy and complicates the treatment of infectious diseases associated with implanted devices. It is critical to developing a feasible surface to decrease microbial growth while not interfering with the growth of the host cells. This study reports the influence of titanium surface modification to titanium disulfide (TiS2) on inhibiting C. albicans biofilm formation while allowing the attachment of human gingival fibroblasts (HGFs) on their surface. The surface of titanium parts is directly converted to structured titanium and TiS2 using direct laser processing and crystal growth methods. C. albicans adhesion and colonization are then investigated on these surfaces by the colony counting assay and reactive oxygen species (ROS) assay, using 2',7'-dichlorofluorescin diacetate (DCFH-DA) and microscopy images. Also, the viability and adhesion of HGFs on these surfaces are investigated to show their adhesion and biocompatibility. Titanium samples with the TiS2 surface show both C. albicans biofilm inhibition and HGF attachment. This study provides insight into designing and manufacturing titanium biomedical implants.

Two-Dimensional-Material-Based Field-Effect Transistor Biosensor for Detecting COVID-19 Virus (SARS-CoV-2).

The emergence of rapidly expanding infectious diseases such as coronavirus (COVID-19) demands effective biosensors that can promptly detect and recognize the pathogens. Field-effect transistors based on semiconducting two-dimensional (2D) materials (2D-FETs) have been identified as potential candidates for rapid and label-free sensing applications. This is because any perturbation of such atomically thin 2D channels can significantly impact their electronic transport properties. Here, we report the use of FET based on semiconducting transition metal dichalcogenide (TMDC) WSe2 as a promising biosensor for the rapid and sensitive detection of SARS-CoV-2 in vitro. The sensor is created by functionalizing the WSe2 monolayers with a monoclonal antibody against the SARS-CoV-2 spike protein and exhibits a detection limit of down to 25 fg/µL in 0.01X phosphate-buffered saline (PBS). Comprehensive theoretical and experimental studies, including density functional theory, atomic force microscopy, Raman and photoluminescence spectroscopies, and electronic transport properties, were performed to characterize and explain the device performance. The results demonstrate that TMDC-based 2D-FETs can potentially serve as sensitive and selective biosensors for the rapid detection of infectious diseases.

Bacillus pumilus B12 Degrades Polylactic Acid and Degradation Is Affected by Changing Nutrient Conditions.

Poly-lactic acid (PLA) is increasingly used as a biodegradable alternative to traditional petroleum-based plastics. In this study, we identify a novel agricultural soil isolate of Bacillus pumilus (B12) that is capable of degrading high molecular weight PLA films. This degradation can be detected on a short timescale, with significant degradation detected within 48-h by the release of L-lactate monomers, allowing for a rapid identification ideal for experimental variation. The validity of using L-lactate as a proxy for degradation of PLA films is corroborated by loss of rigidity and appearance of fractures in PLA films, as measured by atomic force microscopy and scanning electron microscopy (SEM), respectively. Furthermore, we have observed a dose-dependent decrease in PLA degradation in response to an amino acid/nucleotide supplement mix that is driven mainly by the nucleotide base adenine. In addition, amendments of the media with specific carbon sources increase the rate of PLA degradation, while phosphate and potassium additions decrease the rate of PLA degradation by B. pumilus B12. These results suggest B. pumilus B12 is adapting its enzymatic expression based on environmental conditions and that these conditions can be used to study the regulation of this process. Together, this work lays a foundation for studying the bacterial degradation of biodegradable plastics.

Loss of carotenoids from membranes of Pantoea sp. YR343 results in altered lipid composition and changes in membrane biophysical properties.

Bacterial membranes are complex mixtures of lipids and proteins, the combination of which confers biophysical properties that allows cells to respond to environmental conditions. Carotenoids are sterol analogs that are important for regulating membrane dynamics. The membrane of Pantoea sp. YR343 is characterized by the presence of the carotenoid zeaxanthin, and a carotenoid-deficient mutant, ΔcrtB, displays defects in root colonization, reduced secretion of indole-3-acetic acid, and defects in biofilm formation. Here we demonstrate that the loss of carotenoids results in changes to the membrane lipid composition in Pantoea sp. YR343, including increased amounts of unsaturated fatty acids in the ΔcrtB mutant membranes. These mutant cells displayed less fluid membranes in comparison to wild type cells as measured by fluorescence anisotropy of whole cells. Studies with artificial systems, however, have shown that carotenoids impart membrane rigidifying properties. Thus, we examined membrane fluidity using spheroplasts and vesicles composed of lipids extracted from either wild type or mutant cells. Interestingly, with the removal of the cell wall and membrane proteins, ΔcrtB vesicles were more fluid than vesicles made from lipids extracted from wild type cells. In addition, carotenoids appeared to stabilize membrane fluidity during rapidly changing temperatures. Taken together, these results suggest that Pantoea sp. YR343 compensates for the loss of carotenoids by changing lipid composition, which together with membrane proteins, results in reduced membrane fluidity. These changes may influence the abundance or function of membrane proteins that are responsible for the physiological changes observed in the ΔcrtB mutant cells.

Targeting the fungal cell wall: current therapies and implications for development of alternative antifungal agents.

Fungal infections are a worldwide problem associated with high morbidity and mortality. There are relatively few antifungal agents, and resistance has emerged within these pathogens for the newest antifungal drugs. As the fungal cell wall is critical for growth and development, it is one of the most important targets for drug development. In this review, the currently available cell wall inhibitors and suitable drug candidates for the treatment of fungal infections are explored. Future studies of the fungal cell wall and compounds that have detrimental effects on this important outer structural layer could aid in antifungal drug discovery and lead to the development of alternative cell wall inhibitors to fill gaps in clinical therapies for difficult-to-treat fungal infections.

Candida albicans Cannot Acquire Sufficient Ethanolamine from the Host To Support Virulence in the Absence of De Novo Phosphatidylethanolamine Synthesis.

Candida albicans mutants for phosphatidylserine (PS) synthase (cho1ΔΔ) and PS decarboxylase (psd1ΔΔ psd2ΔΔ) are compromised for virulence in mouse models of systemic infection and oropharyngeal candidiasis (OPC). Both of these enzymes are necessary to synthesize phosphatidylethanolamine (PE) by the de novo pathway, but these mutants are still capable of growth in culture media, as they can import ethanolamine from media to synthesize PE through the Kennedy pathway. Given that the host has ethanolamine in its serum, the exact mechanism by which virulence is lost in these mutants is not clear. There are two competing hypotheses to explain their loss of virulence. (i) PE from the Kennedy pathway cannot substitute for de novo-synthesized PE. (ii) The mutants cannot acquire sufficient ethanolamine from the host to support adequate PE synthesis. These hypotheses can be simultaneously tested if ethanolamine availability is increased for Candida while it is inside the host. We accomplish this by transcomplementation of C. albicans with the Arabidopsis thaliana serine decarboxylase gene (AtSDC), which converts cytoplasmic serine to ethanolamine. Expression of AtSDC in either mutant restores PE synthesis, even in the absence of exogenous ethanolamine. AtSDC also restores virulence to cho1ΔΔ and psd1ΔΔ psd2ΔΔ strains in systemic and OPC infections. Thus, in the absence of de novo PE synthesis, C. albicans cannot acquire sufficient ethanolamine from the host to support virulence. In addition, expression of AtSDC restores PS synthesis in the cho1ΔΔ mutant, which may be due to causing PS decarboxylase to run backwards and convert PE to PS.

Elucidating Duramycin's Bacterial Selectivity and Mode of Action on the Bacterial Cell Envelope.

The use of naturally occurring antimicrobial peptides provides a promising route to selectively target pathogenic agents and to shape microbiome structure. Lantibiotics, such as duramycin, are one class of bacterially produced peptidic natural products that can selectively inhibit the growth of other bacteria. However, despite longstanding characterization efforts, the microbial selectivity and mode of action of duramycin are still obscure. We describe here a suite of biological, chemical, and physical characterizations that shed new light on the selective and mechanistic aspects of duramycin activity. Bacterial screening assays have been performed using duramycin and Populus-derived bacterial isolates to determine species selectivity. Lipidomic profiles of selected resistant and sensitive strains show that the sensitivity of Gram-positive bacteria depends on the presence of phosphatidylethanolamine (PE) in the cell membrane. Further the surface and interface morphology were studied by high resolution atomic force microscopy and showed a progression of cellular changes in the cell envelope after treatment with duramycin for the susceptible bacterial strains. Together, these molecular and cellular level analyses provide insight into duramycin's mode of action and a better understanding of its selectivity.

Influence of phosphatidylserine and phosphatidylethanolamine on farnesol tolerance in Candida albicans.

Candida albicans is among the most common human fungal pathogens. The ability to undergo the morphological transition from yeast to hyphal growth is critical for its pathogenesis. Farnesol, a precursor in the isoprenoid/sterol pathway, is a quorum-sensing molecule produced by C. albicans that inhibits hyphal growth in this polymorphic fungus. Interestingly, C. albicans can tolerate farnesol concentrations that are toxic to other fungi. We hypothesized that changes in phospholipid composition are one of the factors contributing to farnesol tolerance in C. albicans. In this study, we found that loss of enzymes that synthesize the phospholipids phosphatidylserine (PS) and/or phosphatidylethanolamine (PE) compromise the tolerance of C. albicans to farnesol. Compared with wild type, the phospholipid mutant cho1∆/∆ (loss of PS and decreased PE synthesis) shows greater inhibition of growth, loss of ATP production, increased consumption of oxygen, and increased formation of reactive oxygen species in the presence of farnesol. The cho1∆/∆ mutant also exhibits decreased sensitivity to mitochondrial ATPase inhibition, suggesting that cells lacking PS and/or downstream PE rely less on mitochondrial function for ATP synthesis. These data reveal that PS and PE play roles in farnesol tolerance and maintaining mitochondrial respiratory function.

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform.

Root hairs increase root surface area for better water uptake and nutrient absorption by the plant. Because they are small in size and often obscured by their natural environment, root hair morphology and function are difficult to study and often excluded from plant research. In recent years, microfluidic platforms have offered a way to visualize root systems at high resolution without disturbing the roots during transfer to an imaging system. The microfluidic platform presented here builds on previous plant-on-a-chip research by incorporating a two-layer device to confine the Arabidopsis thaliana main root to the same optical plane as the root hairs. This design enables the quantification of root hairs on a cellular and organelle level and also prevents z-axis drifting during the addition of experimental treatments. We describe how to store the devices in a contained and hydrated environment, without the need for fluidic pumps, while maintaining a gnotobiotic environment for the seedling. After the optical imaging experiment, the device may be disassembled and used as a substrate for atomic force or scanning electron microscopy while keeping fine root structures intact.

ß-(1,3)-Glucan Unmasking in Some Candida albicans Mutants Correlates with Increases in Cell Wall Surface Roughness and Decreases in Cell Wall Elasticity.

Candida albicans is among the most common human fungal pathogens, causing a broad range of infections, including life-threatening systemic infections. The cell wall of C. albicans is the interface between the fungus and the innate immune system. The cell wall is composed of an outer layer enriched in mannosylated glycoproteins (mannan) and an inner layer enriched in ß-(1,3)-glucan and chitin. Detection of C. albicans by Dectin-1, a C-type signaling lectin specific for ß-(1,3)-glucan, is important for the innate immune system to recognize systemic fungal infections. Increased exposure of ß-(1,3)-glucan to the immune system occurs when the mannan layer is altered or removed in a process called unmasking. Nanoscale changes to the cell wall during unmasking were explored in live cells with atomic force microscopy (AFM). Two mutants, the cho1Δ/Δ and kre5Δ/Δ mutants, were selected as representatives that exhibit modest and strong unmasking, respectively. Comparisons of the cho1Δ/Δ and kre5Δ/Δ mutants to the wild type reveal morphological changes in their cell walls that correlate with decreases in cell wall elasticity. In addition, AFM tips functionalized with Dectin-1 revealed that the forces of binding of Dectin-1 to all of the strains were similar, but the frequency of binding was highest for the kre5Δ/Δ mutant, decreased for the cho1Δ/Δ mutant, and rare for the wild type. These data show that nanoscale changes in surface topology are correlated with increased Dectin-1 adhesion and decreased cell wall elasticity. AFM, using tips functionalized with immunologically relevant molecules, can map epitopes of the cell wall and increase our understanding of pathogen recognition by the immune system.

Biotin Auxotrophy and Biotin Enhanced Germ Tube Formation in Candida albicans.

Due to the increased number of immunocompromised patients, infections with the pathogen Candida albicans have significantly increased in recent years. C. albicans transition from yeast to germ tubes is one of the essential factors for virulence. In this study we noted that Lee's medium, commonly used to induce filamentation, contained 500-fold more biotin than needed for growth and 40-fold more biotin than is typically added to growth media. Thus, we investigated the effects of excess biotin on growth rate and filamentation by C. albicans in different media. At 37 °C, excess biotin (4 µM) enhanced germ tube formation (GTF) ca. 10-fold in both Lee's medium and a defined glucose-proline medium, and ca. 4-fold in 1% serum. Two biotin precursors, desthiobiotin and 7-keto-8-aminopelargonic acid (KAPA), also stimulated GTF. During these studies we also noted an inverse correlation between the number of times the inoculum had been washed and the concentration of serum needed to stimulate GTF. C. albicans cells that had been washed eight times achieved 80% GTF with only 0.1% sheep serum. The mechanism by which 1-4 µM biotin enhances GTF is still unknown except to note that equivalent levels of biotin are needed to create an internal supply of stored biotin and biotinylated histones. Biotin did not restore filamentation for any of the four known filamentation defective mutants tested. C. albicans is auxotrophic for biotin and this biotin auxotrophy was fulfilled by biotin, desthiobiotin, or KAPA. However, biotin auxotrophy is not temperature dependent or influenced by the presence of 5% CO2. Biotin starvation upregulated the biotin biosynthetic genes BIO2, BIO3, and BIO4 by 11-, 1500-, and 150-fold, respectively, and BIO2p is predicted to be mitochondrion-localized. Based on our findings, we suggest that biotin has two roles in the physiology of C. albicans, one as an enzymatic cofactor and another as a morphological regulator. Finally, we found no evidence supporting prior claims that C. albicans only forms hyphae at very low biotin (0.1 nM) growth conditions.

A glutathione-independent glyoxalase of the DJ-1 superfamily plays an important role in managing metabolically generated methylglyoxal in Candida albicans.

Methylglyoxal is a cytotoxic reactive carbonyl compound produced by central metabolism. Dedicated glyoxalases convert methylglyoxal to d-lactate using multiple catalytic strategies. In this study, the DJ-1 superfamily member ORF 19.251/GLX3 from Candida albicans is shown to possess glyoxalase activity, making this the first demonstrated glutathione-independent glyoxalase in fungi. The crystal structure of Glx3p indicates that the protein is a monomer containing the catalytic triad Cys(136)-His(137)-Glu(168). Purified Glx3p has an in vitro methylglyoxalase activity (Km = 5.5 mM and kcat = 7.8 s(-1)) that is significantly greater than that of more distantly related members of the DJ-1 superfamily. A close Glx3p homolog from Saccharomyces cerevisiae (YDR533C/Hsp31) also has glyoxalase activity, suggesting that fungal members of the Hsp31 clade of the DJ-1 superfamily are all probable glutathione-independent glyoxalases. A homozygous glx3 null mutant in C. albicans strain SC5314 displays greater sensitivity to millimolar levels of exogenous methylglyoxal, elevated levels of intracellular methylglyoxal, and carbon source-dependent growth defects, especially when grown on glycerol. These phenotypic defects are complemented by restoration of the wild-type GLX3 locus. The growth defect of Glx3-deficient cells in glycerol is also partially complemented by added inorganic phosphate, which is not observed for wild-type or glucose-grown cells. Therefore, C. albicans Glx3 and its fungal homologs are physiologically relevant glutathione-independent glyoxalases that are not redundant with the previously characterized glutathione-dependent GLO1/GLO2 system. In addition to its role in detoxifying glyoxals, Glx3 and its close homologs may have other important roles in stress response.

Histone biotinylation in Candida albicans.

Candida albicans is an opportunistic fungal pathogen in humans. It is a polymorphic fungus: it can live as yeasts, hyphae, or pseudohyphae. Biotin is required for cell growth and fatty acid metabolism because it is used as a cofactor for carboxylases such as acetyl-CoA carboxylase, and pyruvate carboxylase. In addition, we have discovered that biotin is used to modify histones in C. albicans. Biotinylation was detected by Western blots using a monoclonal antibiotin HRP-conjugated antibody as well as with qTOF and LC/MS/MS mass spectrometry. As a precaution, the antibiotin antibody was dialyzed against neutravidin prior to use. During this study, we observed that three histones, H2A, H2B, and H4, were biotinylated at many lysine residues in an apparently nonsite-specific manner. Roughly, equivalent levels of acetylation, methylation, and phosphorylation were found in histones from biotin-replete and biotin-starved cells, but histone biotinylation was only observed for cells grown in excess biotin. The function of histone biotinylation in C. albicans is still unknown but, because C. albicans is a natural biotin auxotroph, a storage reservoir for biotin is attractive. Techniques used to detect histone biotinylation in C. albicans did not detect any histone biotinylation in Saccharomyces cerevisiae.

Activity and toxicity of farnesol towards Candida albicans are dependent on growth conditions.

Farnesol interacts with Candida albicans as both a quorum-sensing molecule and toxic agent, but confusion abounds regarding which conditions promote these distinct responses. Farnesol sensitivity was measured when inoculum cell history and size, temperature, and growth media were altered. Parameters for farnesol tolerance/sensitivity were defined, validating previous studies and identifying new variables, such as energy availability. This study clearly defines what farnesol concentrations are lethal to C. albicans, based on environmental conditions.

Cysteine pKa depression by a protonated glutamic acid in human DJ-1.

Human DJ-1, a disease-associated protein that protects cells from oxidative stress, contains an oxidation-sensitive cysteine (C106) that is essential for its cytoprotective activity. The origin of C106 reactivity is obscure, due in part to the absence of an experimentally determined p K a value for this residue. We have used atomic-resolution X-ray crystallography and UV spectroscopy to show that C106 has a depressed p K a of 5.4 +/- 0.1 and that the C106 thiolate accepts a hydrogen bond from a protonated glutamic acid side chain (E18). X-ray crystal structures and cysteine p K a analysis of several site-directed substitutions at residue 18 demonstrate that the protonated carboxylic acid side chain of E18 is required for the maximal stabilization of the C106 thiolate. A nearby arginine residue (R48) participates in a guanidinium stacking interaction with R28 from the other monomer in the DJ-1 dimer and elevates the p K a of C106 by binding an anion that electrostatically suppresses thiol ionization. Our results show that the ionizable residues (E18, R48, and R28) surrounding C106 affect its p K a in a way that is contrary to expectations based on the typical ionization behavior of glutamic acid and arginine. Lastly, a search of the Protein Data Bank (PDB) produces several candidate hydrogen-bonded aspartic/glutamic acid-cysteine interactions, which we propose are particularly common in the DJ-1 superfamily.