In Silico Prediction and Structure Based Multitargeted Molecular Docking Analysis of Selected Bioactive Compounds Against Mucormycosis

Background: During the second wave of the COVID-19 pandemic, an unusual increase in cases of mucormycosis was observed in India, owing to immunological dysregulation caused by the SARS-CoV-2 and the use of broad-spectrum antibiotics, particularly in patients with poorly controlled diabetes with ketoacidosisto have contributed to the rise and it has been declared an epidemic in several states of India.Because of the black colouring of dead and dying tissue caused by the fungus, it was dubbed "black fungus" by several Indian media outlets. In this study, attempts were taken to unmask novel therapeutic options to treat mucormycosis disease. Rhizopus species is the primary fungi responsible for 70% of mucormycosis cases.Results: We chose three important proteins from the Rhizopus delemar such as CotH3, Lanosterol 14 alpha-demethylase and Mucoricin which plays a crucial role in the virulence of Mucorales. Initially, we explored the physiochemical, structural and functional insights of proteins and later using AutoDock Vina, we applied computational protein-ligand binding modelling to perform a virtual screening around 300 selected compounds against these three proteins, including FDA-approved drugs, FDA-unapproved drugs, investigational-only drugs, and natural bioactive compounds. ADME parameters, Toxicity risk and biological activity of those compounds were approximated via in silico methods. Our computational studies identified six ligands as potential inhibitors against Rhizopus delemar, including 12,28-Oxamanzamine A, vialinin B and deoxytopsentin for CotH3; pramiconazole and saperconazole for Lanosterol 14 alpha-demethylase; and Hesperidin for Mucoricin. Interestingly, 12,28-Oxamanzamine A showed a maximum binding affinity with all three proteins(CotH3: -10.2 kcal/mol Lanosterol 14 alpha-demethylase: -10.9 kcal/mol Mucoricin:-8.6 kcal/mol).Conclusions: In summary, our investigation identified 12,28-Oxamanzamine A, vialinin B, deoxytopsentin, pramiconazole, saperconazole and hesperidin as potent bioactive compounds for treating mucormycosis that may be considered for further optimization techniques and in-vitro and in-vivo studies.

Species-specific differences in C-5 sterol desaturase function influence the outcome of azole antifungal exposure.

The azole antifungals inhibit sterol 14α-demethylase (S14DM), leading to depletion of cellular ergosterol and the synthesis of an aberrant sterol-diol that disrupts membrane function. In Candida albicans , sterol diol production is catalyzed by the C-5 sterol desaturase enzyme encoded by ERG3 . Accordingly, mutations that inactivate ERG3 enable the fungus to grow in the presence of the azoles. The purpose of this study was to compare the propensity of C-5 sterol desaturases from different fungal pathogens to produce the toxic diol upon S14DM inhibition and thus contribute to antifungal efficacy. The coding sequences of ERG3 homologs from C. albicans ( CaERG3 ), Candida glabrata ( CgERG3 ), Candida auris ( CaurERG3 ), Cryptococcus neoformans ( CnERG3 ), Aspergillus fumigatus ( AfERG3A-C ) and Rhizopus delemar ( RdERG3A/B ) were expressed in a C. albicans erg3Δ/Δ mutant to facilitate comparative analysis. All but one of the Erg3p-like proteins (AfErg3C) at least partially restored sterol C-5 desaturase activity, and to corresponding degrees rescued the stress and hyphal growth defects of the C. albicans erg3Δ/Δ mutant - confirming functional equivalence. Each C-5 desaturase enzyme conferred markedly different responses to fluconazole exposure in terms of the minimal inhibitory concentration (MIC) and residual growth observed at supra-MIC concentrations. Upon fluconazole-mediated inhibition of S14DM, the strains expressing each homolog also produced varying levels of 14α-methylergosta-8,24(28)-dien-3β,6α-diol. The RdErg3A and AfErg3A proteins are notable for low levels of sterol diol production and failing to confer appreciable azole sensitivity upon the C. albicans erg3Δ/Δ mutant. These findings suggest that species-specific properties of C5-sterol desaturase may be an important determinant of intrinsic azole sensitivity.

Evaluation of Sex Differences in Murine Diabetic Ketoacidosis and Neutropenic Models of Invasive Mucormycosis

There is increased concern that the quality, generalizability and reproducibility of biomedical research can be influenced by the sex of animals used. We studied the differences between male and female mice in response to invasive pulmonary mucormycosis including susceptibility to infection, host immune reaction and responses to antifungal therapy. We used diabetic ketoacidotic (DKA) or neutropenic mice infected with either Rhizopus delemar or Mucor circinelloides. The only difference detected was that when DKA mice were infected with M. circinelloides, female mice were more resistant to infection than male mice (median survival time of 5 vs. 2 days for female and male mice, respectively). However, a 100% lethality was detected among infected animals of both sexes. Treatment with either liposomal amphotericin B (L-AMB) or posaconazole (POSA) protected mice from infection and eliminated the difference seen between infected but untreated female and male mice. Treatment with L-AMB consistently outperformed POSA in prolonging survival and reducing tissue fungal burden of DKA and neutropenic mice infected with R. delemar or M. circinelloides, in both mouse sexes. While little difference was detected in cytokine levels among both sexes, mucormycosis infection in the DKA mouse model induced more inflammatory cytokines/chemokines involved in neutrophil (CXCL1) and macrophage (CXCL2) recruitment vs. uninfected mice. As expected, this inflammatory response was reduced in the neutropenic mouse model. Our studies show that there are few differences between female and male DKA or neutropenic mice infected with mucormycosis with no effect on the outcome of treatment or host immune response.

Evaluation of Sex Differences in Murine Diabetic Ketoacidosis and Neutropenic Models of Invasive Mucormycosis

There is increased concern that the quality, generalizability, and reproducibility of biomedical research can be influenced by the sex of animals used. We studied the differences between male and female mice in response to invasive pulmonary mucormycosis including susceptibility to infection, host immune reaction, and responses to antifungal therapy. We used diabetic ketoacidotic (DKA) or neutropenic mice infected with either Rhizopus delemar or Mucor circinelloides. The only difference detected was that when DKA mice were infected with M. circinelloides, female mice were more resistant to infection than male mice (median survival time of 5 vs. 2 days for female and male mice, respectively). However, a 100% lethality was detected among infected animals of both sexes. Treatment with either liposomal amphotericin B (L-AMB) or posaconazole (POSA) protected mice from infection and eliminated the difference seen between infected but untreated female and male mice. Treatment with L-AMB consistently outperformed POSA in prolonging survival and reducing tissue fungal burden of DKA and neutropenic mice infected with R. delemar or M. circinelloides, in both mouse sexes. Although little difference was detected in cytokine levels among both sexes, mucormycosis infection in the DKA mouse model induced more inflammatory cytokines/chemokines involved in neutrophil (CXCL1) and macrophage (CXCL2) recruitment vs. uninfected mice. As expected, this inflammatory response was reduced in the neutropenic mouse model. Our studies show that there are few differences between female and male DKA or neutropenic mice infected with mucormycosis with no effect on the outcome of treatment or host immune response.

Spórafelszíni fehérjéket kódoló gének jellemzése Mucor circinelloides-ben

A Mucorales rend fajai által okozott gombás fertőzéseket mucormikózisnak nevezzük. Ez a ritka, humán megbetegedés leggyakrabban olyan betegeknél fordul elő, akik immunszuppresszív kezelés alatt állnak vagy vérképzőszervi rosszindulatú daganat szövődményeként immunhiányos állapotban szenvednek. A mucormikózis etiológiai ágenseként leggyakrabban azonosított fajok a Rhizopus, Lichtheimia és Mucor nemzetségekhez tartoznak. A gombás fertőzésekre fogékony populáció növekedése miatt a mucormikózisos esetek száma is növekvő tendenciát mutat. A kezelés sikeressége nagymértékben függ a korai diagnózistól és a megfelelően megválasztott gombaellenes terápiától, valamint a háttérben meghúzódó, immunrendszert gyengítő állapot kezelésétől. Legtöbbször azonban már a diagnózis felállítása is kihívást jelentő feladat. A Mucorales rend tagjai rezisztenciát mutatnak a rutinszerűen alkalmazott gombaellenes szerek többségével szemben (pl. echinokandinok és azolok). A fent említett tények mindegyike sürgetővé teszi az azonosítást lehetővé tevő molekuláris módszerek fejlesztését, valamint új gombaellenes célpontok keresését. Ehhez elengedhetetlen a mucormikózis patomechanizmusának tisztázása, a gazda-patogén interakciók feltárása, valamint a potenciális virulencia faktorok és biomarkerek azonosítása. E célok eléréséhez szükségszerű egy olyan molekuláris és genetikai manipulációs módszer járomspórás gombákra történő adaptációjára, mely segítségével rutinszerűen állíthatók elő a génfunkciók vizsgálatára alkalmas mutáns törzsek. A legfrissebb kutatások rámutattak a CotH fehérje család fontosságára a járomspórás Rhizopus delemar patogenitása kapcsán. Jelen munka elsősorban e géncsalád átfogó elemzésére, mint például a virulenciában betöltött szerepének tisztázására összpontosult. A Mucor genomban azonosított feltételezett spórafelszíni fehérjéknek azonban csak egy része mutatott homológiát azokkal a Rhizopus fehérjékkel, melyek kapcsolatban állnak a gomba patogenitásával. A CotH fehérjék funkcionális elemzése során nyomon követtük a CRISPR-Cas9 rendszer által létrehozott genetikailag stabil mutánsok fenotípusos változásait, hogy megválaszolhassuk azt a kérdést, hogy a CotH fehérjék milyen szerepet játszanak a Mucor circinelloides gomba patogenezisében, illetve egyéb fiziológiai folyamataiban.

Mucoricin is a Ricin-Like Toxin that is Critical for the Pathogenesis of Mucormycosis

Fungi of the order Mucorales cause mucormycosis, a lethal infection with an incompletely understood pathogenesis. We now demonstrate that Mucorales fungi produce a toxin that plays a central role in virulence. Polyclonal antibodies against this toxin inhibit its ability to damage human cells in vitro, and prevent hypovolemic shock, organ necrosis, and death in mice with mucormycosis. RNAi inhibition of the toxin in Rhizopus delemar, compromises the ability of the fungus to damage host cells and attenuates virulence in mice. This 17 kDa toxin has structural and functional features of the plant toxin, ricin, including the ability to inhibit protein synthesis by its N-glycosylase activity, the existence of a motif that mediates vascular leak, and a lectin sequence. Antibodies against the toxin inhibit R. delemar- or toxin-mediated vascular permeability in vitro and cross-react with ricin. A monoclonal anti-ricin B chain antibody binds to the toxin and also inhibits its ability to cause vascular permeability. Therefore, we propose the name “mucoricin” for this toxin. Not only is mucoricin important in the pathogenesis of mucormycosis but our data suggest that a ricin- like toxin is produced by organisms beyond the plant and bacterial kingdoms. Importantly, mucoricin should be a promising therapeutic target.

Genotypic Characterization of Rhizopus Spp. Tempeh and Usar: Traditional Inoculum of Tempeh in Indonesia

Soybeans tempeh (tempeh) is processed by fermentation using Rhizopus spp. Tempeh is an important source of protein in Indonesia. The traditional inoculum in fermentation locally is known as Usar which is made from the leaves of Hibiscus tiliaceus. However, Rhizopus information from Usar is still limited. Therefore, this study aims to identify and investigate the genetic diversity of Rhizopus species from Usar and tempeh based on the Internal Transcribed Spacer (ITS) sequence and the Random Amplified Polymorphic DNA (RAPD) markers. Twenty-three Rhizopus strains were isolated from Usar and ten Rhizopus strains were isolated from tempeh. Based on ITS sequences, the isolates were similar to R Rhizopus microsporus (30 isolates) and Rhizopus delemar (3 isolates) with 98-99% similarity. The genetics of R. microsporus and R. delemar are varied and different from the genetics of R. microsporus from tempeh. The growth temperature of R. microsporus varies from 33 to 48°C and R. delemar can grow to a maximum at 33°C. The role of R. microsporus and R. delemar from Usar in determining the quality of tempeh is still limited. Therefore, it needs to be investigated further.

Kualitas Tempe Menggunakan Rhizopus delemar TB 26 dan R. delemar TB 37 yang Diisolasi dari Inokulum Tradisional Tempe "daun waru"

Mikroorganisme utama dalam pembuatan tempe ialah Rhizopus. Penelitian ini bertujuan untuk mendapatkan informasi tentang potensi Rhizopus delemar TB 26 dan R. delemar TB 37 yang berasal dari "daun waru" dalam menentukan kualitas tempe. Tempe dibuat menggunakan R. delemar TB 26 (Tempe TB 26), R. delemar TB 37 (Tempe TB 37), dan inokulum komersial tempe (Tempe K). Uji organoleptik, aktivitas antioksidan, dan analisis proksimat telah dilakukan untuk menentukan kualitas tempe. Hasil penelitian menunjukkan bahwa tekstur, warna dan komposisi kimia Tempe TB 26, Tempe TB 37, dan Tempe K memenuhi syarat mutu tempe yang ditetapkan di Indonesia yang tertera pada SNI 3144:2015. Secara organoleptik, citarasa Tempe TB 26 dan Tempe TB 37 lebih disukai panelis dibandingkan dengan Tempe K. Dengan demikian, R. delemar TB 26 dan R. delemar TB 37 yang masing masing digunakan membuat Tempe TB 26 dan Tempe TB 37 berpotensi dikembangkan sebagai inokulum tempe. Kesimpulannya, penelitian ini telah berhasil untuk menganalisis kualitas Tempe TB 26 dan Tempe TB 37 yang dibandingkan dengan tempe komersial.Quality of Tempeh using Rhizopus delemar TB 26 and R. delemar TB 37 Isolated from Traditional Inoculum of Tempeh "daun waru"AbstractRhizopus is the main microorganism in tempeh fermentation. This study aims to obtain information about the potential of R. delemar TB 26 and R. delemar TB 37 isolated from traditional inoculums of tempeh "waru leaves" in determining the quality of tempeh. Tempeh was made using R. delemar TB 26 (Tempe TB 26), R. delemar TB 37 (Tempe TB 37), and commercial inoculum of tempeh (Tempe K). Organoleptic test, antioxidant activity, and proximate analysis were done to measure the quality of tempeh. The results showed that the texture, color and chemical composition of Tempe TB 26 and Tempe TB 37 fulfilled the tempeh quality requirements as stated in SNI 3144: 2015. Tempe TB 26 and Tempe TB 37 were more preferred by panelists compared to Tempe K. Therefore, R. delemar TB 26 and R. delemar TB 37, which were used to make Tempe TB 26 and Tempe TB 37, could potentially be developed as tempeh inoculum. As conclusion, Tempe TB 26 and Tempe TB 37 could be analyzed and the comparison to commercial tempeh was also successfully identified.