Candida albicans chitinase 3 with potential as a vaccine antigen: production, purification, and characterisation.

Chitinases are widely studied enzymes that have already found widespread application. Their continued development and valorisation will be driven by the identification of new and improved variants and/or novel applications bringing benefits to industry and society. We previously identified a novel application for chitinases wherein the Candida albicans cell wall surface chitinase 3 (Cht3) was shown to have potential in vaccine applications as a subunit antigen against fungal infections. In the present study, this enzyme was investigated further, developing production and purification protocols, enriching our understanding of its properties, and advancing its application potential. Cht3 was heterologously expressed in Pichia pastoris and a 4-step purification protocol developed and optimised: this involves activated carbon treatment, hydrophobic interaction chromatography, ammonium sulphate precipitation, and gel filtration chromatography. The recombinant enzyme was shown to be mainly O-glycosylated and to retain the epitopes of the native protein. Functional studies showed it to be highly specific, displaying activity on chitin, chitosan, and chito-oligosaccharides larger than chitotriose only. Furthermore, it was shown to be a stable enzyme, exhibiting activity, and stability over broad pH and temperature ranges. This study represents an important step forward in our understanding of Cht3 and contributes to its development for application.

Effects of Zn-ZnO Core-Shell Nanoparticles on Antimicrobial Mechanisms and Immune Cell Activation.

The deposition of zinc-zinc oxide nanoparticles (Zn-ZnO NPs) onto porous Ta2O5 surfaces enriched with calcium phosphate by DC magnetron sputtering was investigated to improve the surface antimicrobial activity without triggering an inflammatory response. Different sizes and amounts of Zn NPs obtained by two optimized different depositions and an additional thin carbon (C) layer deposited over the NPs were explored. The deposition of the Zn NPs and the C layer mitigates the surface porosity, increasing the surface hydrophobicity and decreasing the surface roughness. The possible antimicrobial effect and immune system activation of Zn-ZnO NPs were investigated in Candida albicans and macrophage cells, respectively. It was found that the developed surfaces displayed a fungistatic behavior, as they impair the growth of C. albicans between 5 and 24 h of culture. This behavior was more evident on the surfaces with bigger NPs and the highest amounts of Zn. The same trend was observed in both reactive oxygen species (ROS) generation and loss of C. albicans' membrane integrity. After 24 h of culture, cell toxicity was also dependent on the amount of the NPs. Cell toxicity was observed in surfaces with the highest amount of Zn NPs and with the C layer, while cells were able to grow without any signs of cytotoxicity in the porous surfaces with the lowest amount of NPs. The same Zn-dose-dependent behavior was noticed in the TNF-α production. The Zn-containing surfaces show a vastly inferior cytokine secretion than the lipopolysaccharide (LPS)-stimulated cells, indicating that the modified surfaces do not induce an inflammatory response from macrophage cells. This study provides insights for understanding the Zn amount threshold that allows a simultaneous inhibition of the fungi growth with no toxic effect and the main antimicrobial mechanisms of Zn-ZnO NPs, contributing to future clinical applications.

Design of a lipid nano-delivery system containing recombinant Candida albicans chitinase 3 as a potential vaccine against fungal infections.

Opportunistic fungi cause lethal systemic infections and impose high medical costs to health systems. The World Health Organization has recognized the importance of fungal infections, including them in its global priority list guiding research, development, and discovery of new therapeutic approaches. Fungal vaccine development has been proposed as one of the treatment and prevention strategies in the last decade. In this study, we present the design of a lipid antigen delivery system based on Dioctadecyldimethylammonium bromide: Monoolein (DODAB: MO) containing recombinant Candida albicans Chitinase 3 (Cht3) for modulation the immune response against fungal infections. Several DODAB:MO liposomes containing Cht3 were prepared and those prepared by the incubation method and containing 5 µg/mL Cht3 were selected due to their favorable size, ζ-potential and stability, suited for antigen delivery applications. The encapsulation of Cht3 in these liposomes resulted in a significant increase in cellular uptake compared to empty liposomes, demonstrating their efficacy in delivering the antigen. Moreover, the liposomes proved to be safe for use in immunization procedures. Subcutaneous administration of Cht3 liposomes elicited a Th1/Th17 immune response profile, associated with the production of high levels of antibodies against Cht3. These antibodies recognized both the native and the recombinant forms of the protein, opsonizing mother-yeast at the cell scars, which has the potential to disrupt cell separation and hinder yeast growth. The findings suggest that the designed lipid antigen delivery system shows promise as a potential candidate for enhancing immune responses against fungal infections, offering a valuable strategy for future fungal vaccine development.

Immobilization of Streptavidin on a Plasmonic Au-TiO2 Thin Film towards an LSPR Biosensing Platform.

Optical biosensors based on localized surface plasmon resonance (LSPR) are the future of label-free detection methods. This work reports the development of plasmonic thin films, containing Au nanoparticles dispersed in a TiO2 matrix, as platforms for LSPR biosensors. Post-deposition treatments were employed, namely annealing at 400 °C, to develop an LSPR band, and Ar plasma, to improve the sensitivity of the Au-TiO2 thin film. Streptavidin and biotin conjugated with horseradish peroxidase (HRP) were chosen as the model receptor-analyte, to prove the efficiency of the immobilization method and to demonstrate the potential of the LSPR-based biosensor. The Au-TiO2 thin films were activated with O2 plasma, to promote the streptavidin immobilization as a biorecognition element, by increasing the surface hydrophilicity (contact angle drop to 7°). The interaction between biotin and the immobilized streptavidin was confirmed by the detection of HRP activity (average absorbance 1.9 ± 0.6), following a protocol based on enzyme-linked immunosorbent assay (ELISA). Furthermore, an LSPR wavelength shift was detectable (0.8 ± 0.1 nm), resulting from a plasmonic thin-film platform with a refractive index sensitivity estimated to be 33 nm/RIU. The detection of the analyte using these two different methods proves that the functionalization protocol was successful and the Au-TiO2 thin films have the potential to be used as an LSPR platform for label-free biosensors.

Development and Characterization of Monoolein-Based Liposomes of Carvacrol, Cinnamaldehyde, Citral, or Thymol with Anti-Candida Activities.

There is an increasing need for novel drugs and new strategies for the therapy of invasive candidiasis. This study aimed to develop and characterize liposome-based nanoparticles of carvacrol, cinnamaldehyde, citral, and thymol with anti-Candida activities. Dioctadecyldimethylammonium bromide- and monoolein-based liposomes in a 1:2 molar ratio were prepared using a lipid-film hydration method. Liposomes were assembled with equal volumes of liposomal stock dispersion and stock solutions of carvacrol, cinnamaldehyde, citral, or thymol in dimethyl sulfoxide. Cytotoxicity was tested on RAW 264.7 macrophages. In vitro antifungal activity of liposomes with phytocompounds was evaluated according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) methodology using clinical isolates of Candida albicans, Candida auris, Candida dubliniensis, and Candida tropicalis Finally, the ability of macrophage cells to kill Candida isolates after addition of phytocompounds and their nanoparticles was determined. Nanoparticles with 64 µg/ml of cinnamaldehyde, 256 µg/ml of citral, and 128 µg/ml of thymol had the best characteristics among the formulations tested. The highest encapsulation efficiencies were achieved with citral (78% to 83%) and carvacrol (66% to 71%) liposomes. Carvacrol and thymol in liposome-based nanoparticles were nontoxic regardless of the concentration. Moreover, carvacrol and thymol maintained their antifungal activity after encapsulation, and there was a significant reduction (∼41%) of yeast survival when macrophages were incubated with carvacrol or thymol liposomes. In conclusion, carvacrol and thymol liposomes possess high stability, low cytotoxicity, and antifungal activity that act synergistically with macrophages.

The Role of Candida albicans Transcription Factor RLM1 in Response to Carbon Adaptation.

Candida albicans is the main causative agent of candidiasis and one of the most frequent causes of nosocomial infections worldwide. In order to establish an infection, this pathogen supports effective stress responses to counter host defenses and adapts to changes in the availability of important nutrients, such as alternative carbon sources. These stress responses have clear implications on the composition and structure of Candida cell wall. Therefore, we studied the impact of lactate, a physiologically relevant carbon source, on the activity of C. albicans RLM1 transcriptional factor. RLM1 is involved in the cell wall integrity pathway and plays an important role in regulating the flow of carbohydrates into cell wall biosynthesis pathways. The role of C. albicans RLM1 in response to lactate adaptation was assessed in respect to several virulence factors, such as the ability to grow under cell wall damaging agents, filament, adhere or form biofilm, as well as to immune recognition. The data showed that growth of C. albicans cells in the presence of lactate induces the secretion of tartaric acid, which has the potential to modulate the TCA cycle on both the yeast and the host cells. In addition, we found that adaptation of C. albicans cells to lactate reduces their internalization by immune cells and consequent % of killing, which could be correlated with a lower exposure of the cell wall ß-glucans. In addition, absence of RLM1 has a minor impact on internalization, compared with the wild-type and complemented strains, but it reduces the higher efficiency of lactate grown cells at damaging phagocytic cells and induces a high amount of IL-10, rendering these cells more tolerable to the immune system. The data suggests that RLM1 mediates cell wall remodeling during carbon adaptation, impacting their interaction with immune cells.

High variability within Candida albicans transcription factor RLM1: Isolates from vulvovaginal infections show a clear bias toward high molecular weight alleles.

Previous studies have correlated the severity of recurrent vulvovaginal Candida infections (VVC) and balanitis in patients from China with the presence of some dominant genotypes at the ORF RLM1. Here we tested VVC vs non-VVC isolates from Portugal, Brazil and Greece and, although the same genotypes were identified in VVC isolates, they were present in only five out of 150 strains. However, this analysis showed that VVC isolates presented a higher percentage of genotypes with similar high molecular weight alleles, in comparison with strains isolated from other biological sources.