Genomic Insights into Pseudomonas protegens E1BL2 from Giant Jala Maize: A Novel Bioresource for Sustainable Agriculture and Efficient Management of Fungal Phytopathogens.

The relationships between plants and bacteria are essential in agroecosystems and bioinoculant development. The leaf endophytic Pseudomonas protegens E1BL2 was previously isolated from giant Jala maize, which is a native Zea mays landrace of Nayarit, Mexico. Using different Mexican maize landraces, this work evaluated the strain's plant growth promotion and biocontrol against eight phytopathogenic fungi in vitro and greenhouse conditions. Also, a plant field trial was conducted on irrigated fields using the hybrid maize Supremo. The grain productivity in this assay increased compared with the control treatment. The genome analysis of P. protegens E1BL2 showed putative genes involved in metabolite synthesis that facilitated its beneficial roles in plant health and environmental adaptation (bdhA, acoR, trpE, speE, potA); siderophores (ptaA, pchC); and extracellular enzymes relevant for PGPB mechanisms (cel3, chi14), protection against oxidative stress (hscA, htpG), nitrogen metabolism (nirD, nit1, hmpA), inductors of plant-induced systemic resistance (ISR) (flaA, flaG, rffA, rfaP), fungal biocontrol (phlD, prtD, prnD, hcnA-1), pest control (vgrG-1, higB-2, aprE, pslA, ppkA), and the establishment of plant-bacteria symbiosis (pgaA, pgaB, pgaC, exbD). Our findings suggest that P. protegens E1BL2 significantly promotes maize growth and offers biocontrol benefits, which highlights its potential as a bioinoculant.

Review and Current Perspectives on DNA Topoisomerase I and II Enzymes of Fungi as Study Models for the Development of New Antifungal Drugs.

Fungal infections represent a growing public health problem, mainly stemming from two phenomena. Firstly, certain diseases (e.g., AIDS and COVID-19) have emerged that weaken the immune system, leaving patients susceptible to opportunistic pathogens. Secondly, an increasing number of pathogenic fungi are developing multi-drug resistance. Consequently, there is a need for new antifungal drugs with novel therapeutic targets, such as type I and II DNA topoisomerase enzymes of fungal organisms. This contribution summarizes the available information in the literature on the biology, topology, structural characteristics, and genes of topoisomerase (Topo) I and II enzymes in humans, two other mammals, and 29 fungi (including Basidiomycetes and Ascomycetes). The evidence of these enzymes as alternative targets for antifungal therapy is presented, as is a broad spectrum of Topo I and II inhibitors. Research has revealed the genes responsible for encoding the Topo I and II enzymes of fungal organisms and the amino acid residues and nucleotide residues at the active sites of the enzymes that are involved in the binding mode of topoisomerase inhibitors. Such residues are highly conserved. According to molecular docking studies, antifungal Topo I and II inhibitors have good affinity for the active site of the respective enzymes. The evidence presented in the current review supports the proposal of the suitability of Topo I and II enzymes as molecular targets for new antifungal drugs, which may be used in the future in combined therapies for the treatment of infections caused by fungal organisms.

(Dimethylamino)methylene hydantoins as building blocks in the synthesis of oxoaplysinopsins and parabanic acids with antifungal activity.

A short, efficient, and stereoselective methodology is described for the synthesis of 5-((dimethylamino)methylene)hydantoins and their conversion into oxoaplysinopsins and parabanic acids. A highly convergent one-pot, two-step reaction between methyl N-arylglycinates, isocyanates, and DMFDMA under microwave irradiation provided the corresponding (dimethylamino)methylene hydantoins as a single E-stereoisomer in high overall yields. The synthesis of (S)-1-(1-phenylethyl) chiral hydantoins, which undergo a stereoselective addition of acetic anhydride, aza-heterocycles, and amines, received special attention. The reaction with indole delivered a series of novel oxoaplysinopsins. Meanwhile, parabanic acids were prepared by a new approach, treating (dimethylamino)methylene hydantoins with mCPBA to generate the oxidative fragmentation of the exocyclic methylene. The antifungal evaluation of the prepared products was carried out on a series of Candida spp., finding potent growth inhibition. According to previous docking studies, this activity is probably due to the inhibitory interaction of the derivatives with the active site of the fungal HMGR enzyme.

Comparative Genomics of the First Resistant Candida auris Strain Isolated in Mexico: Phylogenomic and Pan-Genomic Analysis and Mutations Associated with Antifungal Resistance.

Candida auris is an emerging multidrug-resistant and opportunistic pathogenic yeast. Whole-genome sequencing analysis has defined five major clades, each from a distinct geographic region. The current study aimed to examine the genome of the C. auris 20-1498 strain, which is the first isolate of this fungus identified in Mexico. Based on whole-genome sequencing, the draft genome was found to contain 70 contigs. It had a total genome size of 12.86 Mbp, an N50 value of 1.6 Mbp, and an average guanine-cytosine (GC) content of 45.5%. Genome annotation revealed a total of 5432 genes encoding 5515 proteins. According to the genomic analysis, the C. auris 20-1498 strain belongs to clade IV (containing strains endemic to South America). Of the two genes (ERG11 and FKS1) associated with drug resistance in C. auris, a mutation was detected in K143R, a gene located in a mutation hotspot of ERG11 (lanosterol 14-α-demethylase), an antifungal drug target. The focus on whole-genome sequencing and the identification of mutations linked to the drug resistance of fungi could lead to the discovery of new therapeutic targets and new antifungal compounds.

Synthesis, In Silico Study, and In Vitro Antifungal Activity of New 5-(1,3-Diphenyl-1H-Pyrazol-4-yl)-4-Tosyl-4,5-Dihydrooxazoles.

The increase in multi-drug resistant Candida strains has caused a sharp rise in life-threatening fungal infections in immunosuppressed patients, including those with SARS-CoV-2. Novel antifungal drugs are needed to combat multi-drug-resistant yeasts. This study aimed to synthesize a new series of 2-oxazolines and evaluate the ligands in vitro for the inhibition of six Candida species and in silico for affinity to the CYP51 enzymes (obtained with molecular modeling and protein homology) of the same species. The 5-(1,3-diphenyl-1H-pyrazol-4-yl)-4-tosyl-4,5-dihydrooxazoles 6a-j were synthesized using the Van Leusen reaction between 1,3-diphenyl-4-formylpyrazoles 4a-j and TosMIC 5 in the presence of K2CO3 or KOH without heating, resulting in short reaction times, high compound purity, and high yields. The docking studies revealed good affinity for the active site of the CYP51 enzymes of the Candida species in the following order: 6a-j > 4a-j > fluconazole (the reference drug). The in vitro testing of the compounds against the Candida species showed lower MIC values for 6a-j than 4a-j, and for 4a-j than fluconazole, thus correlating well with the in silico findings. According to growth rescue assays, 6a-j and 4a-j (like fluconazole) inhibit ergosterol synthesis. The in silico toxicity assessment evidenced the safety of compounds 6a-j, which merit further research as possible antifungal drugs.

Interactions between the AraC/XylS-like transcriptional activator InvF of Salmonella Typhimurium, the RNA polymerase alpha subunit and the chaperone SicA.

Salmonella enterica serovar Typhimurium causes gastroenteritis and systemic infections in humans. For this bacterium the expression of a type III secretion system (T3SS) and effector proteins encoded in the Salmonella pathogenicity island-1 (SPI-1), is keystone for the virulence of this bacterium. Expression of these is controlled by a regulatory cascade starting with the transcriptional regulators HilD, HilC and RtsA that induce the expression of HilA, which then activates expression of the regulator InvF, a transcriptional regulator of the AraC/XylS family. InvF needs to interact with the chaperone SicA to activate transcription of SPI-1 genes including sicA, sopB, sptP, sopE, sopE2, and STM1239. InvF very likely acts as a classical activator; however, whether InvF interacts with the RNA polymerase alpha subunit RpoA has not been determined. Results from this study confirm the interaction between InvF with SicA and reveal that both proteins interact with the RNAP alpha subunit. Thus, our study further supports that the InvF/SicA complex acts as a classical activator. Additionally, we showed for the first time an interaction between a chaperone of T3SS effectors (SicA) and the RNAP.

Inhibitors of 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase Decrease the Growth, Ergosterol Synthesis and Generation of petite Mutants in Candida glabrata and Candida albicans.

Candida glabrata and Candida albicans, the most frequently isolated candidiasis species in the world, have developed mechanisms of resistance to treatment with azoles. Among the clinically used antifungal drugs are statins and other compounds that inhibit 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), resulting in decreased growth and ergosterol levels in yeasts. Ergosterol is a key element for the formation of the yeast cell membrane. However, statins often cause DNA damage to yeast cells, facilitating mutation and drug resistance. The aim of the current contribution was to synthesize seven series of compounds as inhibitors of the HMGR enzyme of Candida ssp., and to evaluate their effect on cellular growth, ergosterol synthesis and generation of petite mutants of C. glabrata and C. albicans. Compared to the reference drugs (fluconazole and simvastatin), some HMGR inhibitors caused lower growth and ergosterol synthesis in the yeast species and generated fewer petite mutants. Moreover, heterologous expression was achieved in Pichia pastoris, and compounds 1a, 1b, 6g and 7a inhibited the activity of recombinant CgHMGR and showed better binding energy values than for α-asarone and simvastatin. Thus, we believe these are good candidates for future antifungal drug development.

Phenotypic and Genomic Characterization of Streptomyces pakalii sp. nov., a Novel Species with Anti-Biofilm and Anti-Quorum Sensing Activity in ESKAPE Bacteria.

The increasing number of infections caused by antimicrobial multi-resistant microorganisms has led to the search for new microorganisms capable of producing novel antibiotics. This work proposes Streptomyces pakalii sp. nov. as a new member of the Streptomycetaceae family. The strain ENCB-J15 was isolated from the jungle soil in Palenque National Park, Chiapas, Mexico. The strain formed pale brown, dry, tough, and buried colonies in the agar with no diffusible pigment in GAE (glucose-asparagine-yeast extract) medium. Scanning electron micrographs showed typical mycelium with long chains of smooth and oval-shaped spores (3-10 m). The strain grew in all of the International Streptomyces Project (ISP)'s media at 28-37 °C with a pH of 6-9 and 0-10% NaCl. S. pakalii ENCB-J15 assimilated diverse carbon as well as organic and inorganic nitrogen sources. The strain also exhibited significant inhibitory activity against the prodigiosin synthesis of Serratia marcescens and the inhibition of the formation and destruction of biofilms of ESKAPE strains of Acinetobacter baumannii and Klebsiella pneumoniae. The draft genome sequencing of ENCB-J15 revealed a 7.6 Mb genome with a high G + C content (71.6%), 6833 total genes, and 6746 genes encoding putative proteins. A total of 26 accessory clusters of proteins associated with carbon sources and amino acid catabolism, DNA modification, and the antibiotic biosynthetic process were annotated. The 16S rRNA gene phylogeny, core-proteome phylogenomic tree, and virtual genome fingerprints support that S. pakalii ENCB-J15 is a new species related to Streptomyces badius and Streptomyces globisporus. Similarly, its average nucleotide identity (ANI) (96.4%), average amino acid identity (AAI) (96.06%), and virtual DNA-DNA hybridization (67.3%) provide evidence to recognize it as a new species. Comparative genomics revealed that S. pakalli and its closest related species maintain a well-conserved genomic synteny. This work proposes Streptomyces pakalii sp. nov. as a novel species that expresses anti-biofilm and anti-quorum sensing activities.

Pyrrole-Based Enaminones as Building Blocks for the Synthesis of Indolizines and Pyrrolo[1,2-a]pyrazines Showing Potent Antifungal Activity.

As a new approach, pyrrolo[1,2-a]pyrazines were synthesized through the cyclization of 2-formylpyrrole-based enaminones in the presence of ammonium acetate. The enaminones were prepared with a straightforward method, reacting the corresponding alkyl 2-(2-formyl-1H-pyrrol-1-yl)acetates, 2-(2-formyl-1H-pyrrol-1-yl)acetonitrile, and 2-(2-formyl-1H-pyrrol-1-yl)acetophenones with DMFDMA. Analogous enaminones elaborated from alkyl (E)-3-(1H-pyrrol-2-yl)acrylates were treated with a Lewis acid to afford indolizines. The antifungal activity of the series of substituted pyrroles, pyrrole-based enaminones, pyrrolo[1,2-a]pyrazines, and indolizines was evaluated on six Candida spp., including two multidrug-resistant ones. Compared to the reference drugs, most test compounds produced a more robust antifungal effect. Docking analysis suggests that the inhibition of yeast growth was probably mediated by the interaction of the compounds with the catalytic site of HMGR of the Candida species.

Unlocking the hidden potential of Mexican teosinte seeds: revealing plant growth-promoting bacterial and fungal biocontrol agents.

The bacterial component of plant holobiont maintains valuable interactions that contribute to plants' growth, adaptation, stress tolerance, and antagonism to some phytopathogens. Teosinte is the grass plant recognized as the progenitor of modern maize, domesticated by pre-Hispanic civilizations around 9,000 years ago. Three teosinte species are recognized: Zea diploperennis, Zea perennis, and Zea mays. In this work, the bacterial diversity of three species of Mexican teosinte seeds was explored by massive sequencing of 16S rRNA amplicons. Streptomyces, Acinetobacter, Olivibacter, Erwinia, Bacillus, Pseudomonas, Cellvibrio, Achromobacter, Devosia, Lysobacter, Sphingopyxis, Stenotrophomonas, Ochrobactrum, Delftia, Lactobacillus, among others, were the bacterial genera mainly represented. The bacterial alpha diversity in the seeds of Z. diploperennis was the highest, while the alpha diversity in Z. mays subsp. mexicana race was the lowest observed among the species and races. The Mexican teosintes analyzed had a core bacteriome of 38 bacterial genera, including several recognized plant growth promoters or fungal biocontrol agents such as Agrobacterium, Burkholderia, Erwinia, Lactobacillus, Ochrobactrum, Paenibacillus, Pseudomonas, Sphingomonas, Streptomyces, among other. Metabolic inference analysis by PICRUSt2 of bacterial genera showed several pathways related to plant growth promotion (PGP), biological control, and environmental adaptation. The implications of these findings are far-reaching, as they highlight the existence of an exceptional bacterial germplasm reservoir teeming with potential plant growth promotion bacteria (PGPB). This reserve holds the key to cultivating innovative bioinoculants and formidable fungal antagonistic strains, thereby paving the way for a more sustainable and eco-friendly approach to agriculture. Embracing these novel NGS-based techniques and understanding the profound impact of the vertical transference of microorganisms from seeds could revolutionize the future of agriculture and develop a new era of symbiotic harmony between plants and microbes.

Streptomyces albidoflavus Q antifungal metabolites inhibit the ergosterol biosynthesis pathway and yeast growth in fluconazole-resistant Candida glabrata: phylogenomic and metabolomic analyses.

There is an urgent need to develop new antifungals due to the increasing prevalence of multidrug-resistant fungal infections and the recent emergence of COVID-19-associated candidiasis. A good study model for evaluating new antifungal compounds is Candida glabrata, an opportunistic fungal pathogen with intrinsic resistance to azoles (the most common clinical drugs for treating fungal infections). The aim of the current contribution was to conduct in vitro tests of antifungal metabolites produced by the bacteria Streptomyces albidoflavus Q, identify their molecular structures, and utilize several techniques to provide evidence of their therapeutic target. S. albidoflavus was isolated from maize rhizospheric soil in Mexico and identified by phylogenomic analysis using a 92-gene core. Of the 66 metabolites identified in S. albidoflavus Q by a liquid chromatography-high resolution mass spectrometry (LC-HRMS) metabolomic analysis of the lyophilized supernatant, six were selected by the Way2drug server based on their in silico binding to the likely target, 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR, the key enzyme in the ergosterol biosynthesis pathway). Molecular modeling studies show a relatively high binding affinity for the CgHMGR enzyme by two secondary metabolites: isogingerenone B (diaryl heptanoid) and notoginsenoside J (polycyclic triterpene). These secondary metabolites were able to inhibit ergosterol synthesis and affect yeast viability in vitro. They also caused alterations in the ultrastructure of the yeast cytoplasmic membrane, as evidenced by transmission electron microscopy. The putative target of isogingerenone B and notoginsenoside J is distinct from that of azole drugs (the most common clinical antifungals). The target for the latter is the lanosterol 14 alpha-demethylase enzyme (Erg11). IMPORTANCE Multidrug resistance has emerged among yeasts of the genus Candida, posing a severe threat to global health. The problem has been exacerbated by the pandemic associated with COVID-19, during which resistant strains of Candida auris and Candida glabrata have been isolated from patients infected with the SARS-CoV-2 virus. To confront this challenge, the World Health Organization has invoked scientists to search for new antifungals with alternative molecular targets. This study identified 66 metabolites produced by the bacteria Streptomyces albidoflavus Q, 6 of which had promising properties for potential antifungal activity. The metabolites were tested in vitro as inhibitors of ergosterol synthesis and C. glabrata growth, with positive results. They were also found to damage the cytoplasmic membrane of the fungus. The corresponding molecular structures and their probable therapeutic target were established. The target is apparently distinct from that of azole drugs.

Plant growth-promoting and heavy metal-resistant Priestia and Bacillus strains associated with pioneer plants from mine tailings.

Open mine tailings dams are extreme artificial environments containing sizeable potentially toxic elements (PTEs), including heavy metals (HMs), transition metals, and metalloids. Furthermore, these tailings have nutritional deficiencies, including assimilable phosphorus sources, organic carbon, and combined nitrogen, preventing plant colonization. Bacteria, that colonize these environments, have mechanisms to tolerate the selective pressures of PTEs. In this work, several Priestia megaterium (formerly Bacillus megaterium), Bacillus mojavensis, and Bacillus subtilis strains were isolated from bulk tailings, anthills, rhizosphere, and endosphere of pioneer plants from abandoned mine tailings in Zacatecas, Mexico. Bacillus spp. tolerated moderate HMs concentrations, produced siderophores and indole-3-acetic acid (IAA), solubilized phosphates, and reduced acetylene in the presence of HMs. The strains harbored different PIB-type ATPase genes encoding for efflux pumps and Cation Diffusion Facilitator (CDF) genes. Moreover, nifH and nifD nitrogenase genes were detected in P. megaterium and B. mojavensis genomic DNA. They showed similarity with sequences of the beta-Proteobacteria species, which may represent likely horizontal transfer events. These Bacillus species precede the colonization of mine tailings by plants. Their phenotypic and genotypic features could be essential in the natural recovery of the sites by reducing the oxidative stress of HMs, fixing nitrogen, solubilizing phosphate, and accumulating organic carbon. These traits of the strains reflect the adaptations of Bacillus species to the mine tailings environment and could contribute to the success of phytoremediation efforts.

Promotion of the growth and yield of Zea mays by synthetic microbial communities from Jala maize.

Plant growth-promoting bacteria (PGPB) are a source of nutrient supply, stimulate plant growth, and even act in the biocontrol of phytopathogens. However, these phenotypic traits have rarely been explored in culturable bacteria from native maize landraces. In this study, synthetic microbial communities (SynCom) were assembled with a set of PGPB isolated from the Jala maize landrace, some of them with additional abilities for the biocontrol of phytopathogenic fungi and the stimulation of plant-induced systemic resistance (ISR). Three SynCom were designed considering the phenotypic traits of bacterial strains, including Achromobacter xylosoxidans Z2K8, Burkholderia sp. Z1AL11, Klebsiella variicola R3J3HD7, Kosakonia pseudosacchari Z2WD1, Pantoea ananatis E2HD8, Pantoea sp. E2AD2, Phytobacter diazotrophicus Z2WL1, Pseudomonas protegens E1BL2, and P. protegens E2HL9. Plant growth promotion in gnotobiotic and greenhouse seedlings assays was performed with Conejo landrace; meanwhile, open field tests were carried out on hybrid CPL9105W maize. In all experimental models, a significant promotion of plant growth was observed. In gnotobiotic assays, the roots and shoot length of the maize seedlings increased 4.2 and 3.0 times, respectively, compared to the untreated control. Similarly, the sizes and weights of the roots and shoots of the plants increased significantly in the greenhouse assays. In the open field assay performed with hybrid CPL9105W maize, the yield increased from 11 tons/ha for the control to 16 tons/ha inoculated with SynCom 3. In addition, the incidence of rust fungal infections decreased significantly from 12.5% in the control to 8% in the treatment with SynCom 3. All SynCom designs promoted the growth of maize in all assays. However, SynCom 3 formulated with A. xylosoxidans Z2K8, Burkholderia sp. Z1AL11, K. variicola R3J3HD7, P. ananatis E2HD8, P. diazotrophicus Z2WL1, and P. protegens E1BL2 displayed the best results for promoting plant growth, their yield, and the inhibition of fungal rust. This study demonstrated the biotechnological eco-friendly plant growth-promoting potential of SynCom assemblies with culturable bacteria from native maize landraces for more sustainable and economic agriculture.

Invasive Fungal Infection Caused by Magnusiomyces capitatus in an Immunocompromised Pediatric Patient with Acute Lymphoblastic Leukemia in Mexico City: A Case Report.

Magnusiomyces capitatus (also denominated "Geotrichum capitatum" and "the teleomorph stage of Saprochaete capitata") mainly affects immunocompromised patients with hematological malignancies in rare cases of invasive fungal infections (IFIs). Few cases have been reported for pediatric patients with acute lymphoblastic leukemia (ALL), in part because conventional diagnostic methods do not consistently detect M. capitatus in infections. The current contribution describes a systemic infection in a 15-year-old female diagnosed with ALL. She arrived at the Children's Hospital of Mexico City with a fever and neutropenia and developed symptoms of septic shock 4 days later. M. capitatus ENCB-HI-834, the causal agent, was isolated from the patient's blood, urine, bile, and peritoneal fluid samples. It was identified with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and a phylogenetic reconstruction using internal transcribed spacer (ITS) and 28S ribosomal sequences. The phylogenetic sequence of M. capitatus ENCB-HI-834 clustered with other M. capitatus-type strains with a 100% identity. In vitro antifungal testing, conducted with the Sensititre YeastOne susceptibility system, found the following minimum inhibitory concentration (MIC) values (µg/mL): posaconazole 0.25, amphotericin B 1.0, fluconazole > 8.0, itraconazole 0.25, ketoconazole 0.5, 5-flucytosine ≤ 0.06, voriconazole 0.25, and caspofungin > 16.0. No clinical breakpoints have been defined for M. capitatus. This is the first clinical case reported in Mexico of an IFI caused by M. capitatus in a pediatric patient with ALL. It emphasizes the importance of close monitoring for a timely and accurate diagnosis of neutropenia-related IFIs to determine the proper treatment with antibiotics, antifungals, and chemotherapy for instance including children with ALL.

Fermentation performance of a Mexican native Clavispora lusitaniae strain for xylitol and ethanol production from xylose, glucose and cellobiose.

Lignocellulose hydrolysates are rich in fermentable sugars such as xylose, cellobiose and glucose, with high potential in the biotechnology industry to obtain bioproducts of higher economic value. Thus, it is important to search for and study new yeast strains that co-consume these sugars to achieve better yields and productivity in the processes. The yeast Clavispora lusitaniae CDBB-L-2031, a native strain isolated from mezcal must, was studied under various culture conditions to potentially produce ethanol and xylitol due to its ability to assimilate xylose, cellobiose and glucose. This yeast produced ethanol under microaerobic conditions with yields of 0.451 gethanol/gglucose and 0.344 gethanol/gcellobiose, when grown on 1% glucose or cellobiose, respectively. In mixtures (0.5% each) of glucose:xylose and glucose:xylose:cellobiose the yields were 0.367 gethanol/gGX and 0. 380 gethanol/gGXC, respectively. Likewise, in identical conditions, C. lusitaniae produced xylitol from xylose with a yield of 0.421 gxylitol/gxylose. In 5% glucose or xylose, this yeast had better ethanol and xylitol titers and yields, respectively. However, glucose negatively affected xylitol production in the mixture of both sugars (3% each), producing only ethanol. Xylose reductase (XR) and xylitol dehydrogenase (XDH) activities were evaluated in cultures growing on xylose or glucose, obtaining the highest values in cultures on xylose at 8 h (25.9 and 6.22 mU/mg, respectively). While in glucose cultures, XR and XDH activities were detected once this substrate was consumed (4.06 and 3.32 mU/mg, respectively). Finally, the XYL1 and XYL2 genes encoding xylose reductase and xylitol dehydrogenase, respectively, were up-regulated by xylose, whereas glucose down-regulated their expression.

Antifungal Activity of Fibrate-Based Compounds and Substituted Pyrroles That Inhibit the Enzyme 3-Hydroxy-methyl-glutaryl-CoA Reductase of Candida glabrata (CgHMGR), Thus Decreasing Yeast Viability and Ergosterol Synthesis.

Due to the emergence of multidrug-resistant strains of yeasts belonging to the Candida genus, there is an urgent need to discover antifungal agents directed at alternative molecular targets. The aim of the current study was to evaluate the capacity of three different series of synthetic compounds to inhibit the Candida glabrata enzyme denominated 3-hydroxy-methyl-glutaryl-CoA reductase and thus affect ergosterol synthesis and yeast viability. Compounds 1c (α-asarone-related) and 5b (with a pyrrolic core) were selected as the best antifungal candidates among over 20 synthetic compounds studied. Both inhibited the growth of fluconazole-resistant and fluconazole-susceptible C. glabrata strains. A yeast growth rescue experiment based on the addition of exogenous ergosterol showed that the compounds act by inhibiting the mevalonate synthesis pathway. A greater recovery of yeast growth occurred for the C. glabrata 43 fluconazole-resistant (versus fluconazole-susceptible) strain and after treatment with 1c (versus 5b). Given that the compounds decreased the concentration of ergosterol in the yeast strains, they probably target ergosterol synthesis. According to the docking analysis, the inhibitory effect of 1c and 5b could possibly be mediated by their interaction with the amino acid residues of the catalytic site of the enzyme. Since 1c displayed higher binding energy than α-asarone and 5b, it is the best candidate for further research, which should include structural modifications to increase its specificity and potency. The derivatives could then be examined with in vivo animal models using a therapeutic dose. IMPORTANCE Within the context of the COVID-19 pandemic, there is currently an epidemiological alert in health care services due to outbreaks of Candida auris, Candida glabrata, and other fungal species multiresistant to conventional antifungals. Therefore, it is important to propose alternative molecular targets, as well as new antifungals. The three series of synthetic compounds herein designed and synthesized are inhibitors of ergosterol synthesis in yeasts. Of the more than 20 compounds studied, two were selected as the best antifungal candidates. These compounds were able to inhibit the growth and synthesis of ergosterol in C. glabrata strains, whether susceptible or resistant to fluconazole. The rational design of antifungal compounds derived from clinical drugs (statins, fibrates, etc.) has many advantages. Future studies are needed to modify the structure of the two present test compounds to obtain safer and less toxic antifungals. Moreover, it is important to carry out a more in-depth mechanistic approach.

Intracellular Aminopeptidase Activity Determination from the Fungus Sporisorium reilianum: Purification and Biochemical Characterization of psrAPEi Enzyme.

The aims of this study were to, first, determine the intracellular aminopeptidase activity (APEi) and second, purify and biochemically characterize one intracellular aminopeptidase enzyme from the phytopathogen fungus Sporisorium reilianum (psrAPEi), the causal agent of head smut in corn. The fungus produced APEi activity in all media cultures evaluated. The psrAPEi was purified by a procedure that involved ammonium sulfate fractionation and four chromatographic steps using an FPLC system (Fast Protein Liquid Chromatography). Results showed an estimated molecular mass of 52.2 kDa. Enzymatic activity was optimal at pH 7.0 and 35 °C and was inhibited by EDTA-Na2, 1,10-phenanthroline, bestatin, and PMSF. This aminopeptidase showed a preference for leucine, arginine, and lysine at the N-position. The Km and Vmax values were 3.72 µM and 188.0 µmol/min, respectively, for L-lysyl-4-nitroanilide. This is the first study to report on intracellular aminopeptidase activity in S. reilianum and the purification and characterization of an intracellular metallo-serine-aminopeptidase (psrAPEi).

Molecular Recognition of Citroflavonoids Naringin and Naringenin at the Active Site of the HMG-CoA Reductase and DNA Topoisomerase Type II Enzymes of Candida spp. and Ustilago maydis.

Two agents from natural sources, citroflavonoids naringin and naringenin, can target enzymes in pathogenic yeasts responsible for hospital infections and crop failure. The aim of this study was to examine the molecular recognition site for naringin and naringenin on the HMGR and TOPOII enzymes of eleven Candida species and one phytopathogen, U. maydis, and evaluate yeast susceptibility to these flavonoids. The HMGR and TOPOII enzymes were analyzed in silico. The alignment of the two enzymes in the twelve pathogenic organisms with the corresponding enzyme of Homo sapiens revealed highly conserved amino acid sequences. Modeling studies of the enzymes indicated highly conserved structures. According to molecular docking simulations, both citroflavonoids recognized the amino acid residues of the active site of the enzymes. Binding energy values were higher for naringin (-10.75 and -9.38 kcal/mol, respectively) than simvastatin on HMGR (-9.9) and curcumin on TOPOII (-8.37). The appraisal of twenty-nine virtual mutations provided evidence of probable mechanisms of resistance (high binding energy) or susceptibility (low energy) to the drugs and emphasized the role of key residues. An in vitro susceptibility evaluation of the twelve yeasts demonstrated that the two flavonoids have similar or better MIC values than those reported for the reference compounds, obtaining the lowest with Candida dubliniensis (2.5 µg/ml) and U. maydis (5 µg/ml). Based on the present findings, naringin and naringenin could possibly be effective for treating diseases caused by pathogenic yeasts of the Candida species and U. maydis, presumably by inhibition of their HMGR and TOPOII enzymes. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12088-021-00980-0.

Vacuolar proteases and autophagy in phytopathogenic fungi: A review.

Autophagy (macroautophagy) is a survival and virulence mechanism of different eukaryotic pathogens. Autophagosomes sequester cytosolic material and organelles, then fuse with or enter into the vacuole or lysosome (the lytic compartment of most fungal/plant cells and many animal cells, respectively). Subsequent degradation of cargoes delivered to the vacuole via autophagy and endocytosis maintains cellular homeostasis and survival in conditions of stress, cellular differentiation, and development. PrA and PrB are vacuolar aspartyl and serine endoproteases, respectively, that participate in the autophagy of fungi and contribute to the pathogenicity of phytopathogens. Whereas the levels of vacuolar proteases are regulated by the expression of the genes encoding them (e.g., PEP4 for PrA and PRB1 for PrB), their activity is governed by endogenous inhibitors. The aim of the current contribution is to review the main characteristics, regulation, and role of vacuolar soluble endoproteases and Atg proteins in the process of autophagy and the pathogenesis of three fungal phytopathogens: Ustilago maydis, Magnaporthe oryzae, and Alternaria alternata. Aspartyl and serine proteases are known to participate in autophagy in these fungi by degrading autophagic bodies. However, the gene responsible for encoding the vacuolar serine protease of U. maydis has yet to be identified. Based on in silico analysis, this U. maydis gene is proposed to be orthologous to the Saccharomyces cerevisiae genes PRB1 and PBI2, known to encode the principal protease involved in the degradation of autophagic bodies and its inhibitor, respectively. In fungi that interact with plants, whether phytopathogenic or mycorrhizal, autophagy is a conserved cellular degradation process regulated through the TOR, PKA, and SNF1 pathways by ATG proteins and vacuolar proteases. Autophagy plays a preponderant role in the recycling of cell components as well as in the fungus-plant interaction.

Three-Component Synthesis of 2-Amino-3-cyano-4H-chromenes, In Silico Analysis of Their Pharmacological Profile, and In Vitro Anticancer and Antifungal Testing.

Chromenes are compounds that may be useful for inhibiting topoisomerase and cytochrome, enzymes involved in the growth of cancer and fungal cells, respectively. The aim of this study was to synthesize a series of some novel 2-amino-3-cyano-4-aryl-6,7-methylendioxy-4H-chromenes 4a-o and 2-amino-3-cyano-5,7-dimethoxy-4-aryl-4H-chromenes 6a-h by a three-component reaction, and test these derivatives for anticancer and antifungal activity. Compounds 4a and 4b were more active than cisplatin (9) and topotecan (7) in SK-LU-1 cells, and more active than 9 in PC-3 cells. An evaluation was also made of the series of compounds 4 and 6 as potential antifungal agents against six Candida strains, finding their MIC50 to be less than or equal to that of fluconazole (8). Molecular docking studies are herein reported, for the interaction of 4 and 6 with topoisomerase IB and the active site of CYP51 of Candida spp. Compounds 4a-o and 6a-h interacted in a similar way as 7 with key amino acids of the active site of topoisomerase IB and showed better binding energy than 8 at the active site of CYP51. Hence, 4a-o and 6a-h are good candidates for further research, having demonstrated their dual inhibition of enzymes that participate in the growth of cancer and fungal cells.