The vacuolar fusion regulated by HOPS complex promotes hyphal initiation and penetration in Candida albicans.

The transition between yeast and hyphae is crucial for regulating the commensalism and pathogenicity in Candida albicans. The mechanisms that affect the invasion of hyphae in solid media, whose deficiency is more related to the pathogenicity of C. albicans, have not been elucidated. Here, we found that the disruption of VAM6 or VPS41 which are components of the homotypic vacuolar fusion and protein sorting (HOPS) complex, or the Rab GTPase YPT72, all responsible for vacuole fusion, led to defects in hyphal growth in both liquid and solid media, but more pronounced on solid agar. The phenotypes of vac8Δ/Δ and GTR1OE-vam6Δ/Δ mutants indicated that these deficiencies are mainly caused by the reduced mechanical forces that drive agar and organs penetration, and confirmed that large vacuoles are required for hyphal mechanical penetration. In summary, our study revealed that large vacuoles generated by vacuolar fusion support hyphal penetration and provided a perspective to refocus attention on the role of solid agar in evaluating C. albicans invasion.

Alternative Oxidase: From Molecule and Function to Future Inhibitors.

In the respiratory chain of the majority of aerobic organisms, the enzyme alternative oxidase (AOX) functions as the terminal oxidase and has important roles in maintaining metabolic and signaling homeostasis in mitochondria. AOX endows the respiratory system with flexibility in the coupling among the carbon metabolism pathway, electron transport chain (ETC) activity, and ATP turnover. AOX allows electrons to bypass the main cytochrome pathway to restrict the generation of reactive oxygen species (ROS). The inhibition of AOX leads to oxidative damage and contributes to the loss of adaptability and viability in some pathogenic organisms. Although AOXs have recently been identified in several organisms, crystal structures and major functions still need to be explored. Recent work on the trypanosome alternative oxidase has provided a crystal structure of an AOX protein, which contributes to the structure-activity relationship of the inhibitors of AOX. Here, we review the current knowledge on the development, structure, and properties of AOXs, as well as their roles and mechanisms in plants, animals, algae, protists, fungi, and bacteria, with a special emphasis on the development of AOX inhibitors, which will improve the understanding of respiratory regulation in many organisms and provide references for subsequent studies of AOX-targeted inhibitors.

Design, synthesis and in vitro biological studies of novel triazoles with potent and broad-spectrum antifungal activity.

A series of novel triazole derivatives containing aryl-propanamide side chains was designed and synthesised. In vitro antifungal activity studies demonstrated that most of the compounds inhibited the growth of six human pathogenic fungi. In particular, parts of phenyl-propionamide-containing compounds had excellent, broad-spectrum antifungal activity against Candida albicans SC5314, Cryptococcus neoformans 22-21, Candida glabrata 537 and Candida parapsilosis 22-20 with MIC values in the range of ≤0.125 µg/mL-4.0 µg/mL. In addition, compounds A1, A2, A6, A12 and A15 showed inhibitory activities against fluconazole-resistant Candida albicans and Candida auris. Preliminary structure-activity relationships (SARs) are also summarised. Moreover, GC-MS analysis demonstrated that A1, A3, and A9 interfered with the C. albicans ergosterol biosynthesis pathway by inhibiting Cyp51. Molecular docking studies elucidated the binding modes of A3 and A9 with Cyp51. These compounds with low haemolytic activity and favourable ADME/T properties are promising for the development of novel antifungal agents.

Discovery of Novel Thiosemicarbazides Containing 1,3,5-Triazines Derivatives as Potential Synergists against Fluconazole-Resistant Candida albicans.

The clinical prevalence of antifungal drug resistance has been increasing over recent years, resulting in the failure of treatments. In an attempt to overcome this critical problem, we sought novel synergistic enhancers to restore the effectiveness of fluconazole against resistant Candida albicans. Based on the structural optimization of hit compound 8 from our in-house library, a series of novel 1,3,5-triazines derivatives was designed, synthesized, and biologically evaluated for synergistic activity in combination with fluconazole. Among them, compounds 10a-o, which contain thiosemicarbazides side chains, exhibited excellent in vitro synergistic antifungal potency (MIC80 = 0.125-2.0 µg/mL, FICI range from 0.127 to 0.25). Interestingly, compound 10l exhibited moderate C. albicans activity as monotherapy with an MIC80 value of 4.0 µg/mL, and also on several Cryptococcus strains (MIC80 ranging from ≤ 0.125-0.5 µg/mL) and C. glabrata (MIC80 ≤ 0.125 µg/mL). These effects were fungal-selective, with much lower levels of cytotoxicity towards human umbilical vein endothelial cells. Here, we report a series of thiosemicarbazides containing 1,3,5-triazines derivatives as potent synergists with fluconazole, and have preliminarily validated compound 10l as a promising antifungal lead for further investigation.

Design, synthesis, and in vitro evaluation of novel antifungal triazoles containing substituted 1,2,3-triazole-methoxyl side chains.

In order to develop new triazole derivatives, we optimized the lead compound a6 by structural modifications to obtain a series of (2R,3R)-3-((1-substituted-1H-1,2,3-triazol-4-yl) methoxy)-2-(2,4-difluorophenyl)-1-(1H-1,2,4-triazol-1-yl) butan-2-ol, compounds 5-36. Most of the target compounds exhibited excellent in vitro antifungal activity against Candida albicans 10231 and Candida glabrata 537 with MIC ≤ 0.125 µg/mL. Of particular note, compounds 6, 22, 28, 30 and 36 were highly active against Candida neoformans 32609 with MIC ≤ 0.125 µg/mL and showed broad-spectrum antifungal activity including against fluconazole-resistant Candida auris 891. In addition, compounds 6 and 22 demonstrated inhibitory effects on filamentation in the azole-resistant C. albicans isolate. Moreover, compounds 6 and 22 were minimally toxic to HUVECs and possessed weak inhibitory effects on the human CYP3A4 and CYP2D6. SARs and docking study further indicated that ortho-substituted groups in the terminal phenyl ring can promote the compounds to improve their antifungal activity.

Application of the Mutant Libraries for Candida albicans Functional Genomics.

Candida albicans is a typical opportunistic pathogen in humans that causes serious health risks in clinical fungal infections. The construction of mutant libraries has made remarkable developments in the study of C. albicans molecular and cellular biology with the ongoing advancements of gene editing, which include the application of CRISPR-Cas9 and novel high-efficient transposon. Large-scale genetic screens and genome-wide functional analysis accelerated the investigation of new genetic regulatory mechanisms associated with the pathogenicity and resistance to environmental stress in C. albicans. More importantly, sensitivity screening based on C. albicans mutant libraries is critical for the target identification of novel antifungal compounds, which leads to the discovery of Sec7p, Tfp1p, Gwt1p, Gln4p, and Erg11p. This review summarizes the main types of C. albicans mutant libraries and interprets their applications in morphogenesis, biofilm formation, fungus-host interactions, antifungal drug resistance, and target identification.

Development of a three-compartment toxicokinetic model for T-2 toxin in shrimp by blindfold particle swarm optimization algorithm.

Tricothecenes-2 toxin (T-2) is a major mycotoxin that is widely distributed in aquatic feeds and poses a huge challenge to the aquatic industry, but there is scant information on the toxicokinetics of T-2 in aquatic animals. Here, we describe the development of a three-compartment toxicokinetic model for the absorption, distribution, metabolism and elimination (ADME) of T-2 in shrimp. The three compartments were central (the hemolymph), slow metabolizing and fast metabolizing compartments to account for the varying ADME rates of T-2 in different shrimp organs. The toxicokinetic model was solved by the blindfold particle swarm optimization algorithm, and the values for the model equation parameters were obtained by applying the experimental data of T-2 concentrations in shrimp. The model had a good fit with the experimental data. It was revealed through the model that after i.m. administration, T-2 was rapidly absorbed into the hemolymph and distributed into shrimp organs. The hepatopancreas and intestine belonged to the fast and muscle to the slow metabolizing compartments, respectively, while the hemolymph had no capacity to metabolize T-2. The T-2 elimination rates in the hepatopancreas and intestine were similar and quite high while that in the muscle was very low. The methods used in developing and solving the model could be used for similar toxicokinetic and pharmacokinetic studies of other animals.