-
Journal of Fungi (Basel, Switzerland) Jan 2023Mucormycetes, a heterogeneous group of fungi, induce a life-threatening disease called mucormycosis. Immune deficiencies represent a major risk factor; hence, we wanted...
BACKGROUND
Mucormycetes, a heterogeneous group of fungi, induce a life-threatening disease called mucormycosis. Immune deficiencies represent a major risk factor; hence, we wanted to illuminate the role of complement and platelets in the defense against mucormycetes.
METHODS
(), (), (), (), (), and () spores were opsonized with human and mouse serum, and C1q, C3c, and terminal complement complex (C5b-9) deposition was measured. Additionally, thrombocytopenic, C3-deficient, or C6-deficient mice were intravenously infected with selected isolates. Survival and immunological parameters were monitored, and fungal burden was determined and compared to that of immunocompetent and neutropenic mice.
RESULTS
In vitro experiments showed significant differences in complement deposition between mucormycetes. isolates bound up to threefold more human C5b-9 than other mucormycetes. , , and bound high levels of murine C3c, whereas human C3c deposition was reduced on compared to and . Murine C3c deposition negatively correlated with virulence. Complement deficiencies and neutropenia, but not thrombocytopenia, were shown to be a risk factor for a lethal outcome.
CONCLUSION
Complement deposition varies between mucormycetes. Additionally, we demonstrated that complement and neutrophilic granulocytes, but not platelets, play an important role in a murine model of disseminated mucormycosis.
PubMed: 36836277
DOI: 10.3390/jof9020162 -
Infection and Drug Resistance 2022Metagenomic next-generation sequencing (mNGS) is a fast, sensitive and accurate diagnostic method for pathogens detection. However, reports on the application of mNGS in...
BACKGROUND
Metagenomic next-generation sequencing (mNGS) is a fast, sensitive and accurate diagnostic method for pathogens detection. However, reports on the application of mNGS in mucormycosis remain scarce.
METHODS
From January 2019 to December 2021, we recruited 13 patients with hematological malignancies who were suspected of mucormycosis and completed mNGS in D20. Then we retrospectively analyze the clinical data, diagnosis, therapeutic process, and outcomes. In order to evaluate the diagnostic value of mNGS in hematological malignancies patients with suspected mucormycosis.
RESULTS
All patients had high risk factors of Invasive Fungal Disease, including hematopoietic stem cell transplantation, immunosuppression, glucocorticoids, etc. The clinical presentations were pulmonary (n=9), rhino-orbito-cerebral (n=4). But the manifestations were nonspecific. All enrolled patients completed mNGS. And most (8/13, 61.54%) of the samples were from blood. Fungi can be detected in all specimens, including Rhizopus (n=7), Rhizomucor (n=4) and Mucor (n=2). In addition, 7/13 (53.85%) specimens were detected bacteria at the same time and virus were detected in 5/13 (38.46%). Histopathological examination was completed in 5 patients, 3 of which were completely consistent with the results of mNGS. After treatment, 6 patients were cured, while the other 7 patients died.
CONCLUSION
mNGS may be a complementary method for early diagnosis, especially for patients who are not suitable for histopathology examination or unable to obtain culture specimen. mNGS can also help detect bacteria and viruses simultaneously, allowing for appropriate and timely antibiotic administration and thus improving patient outcomes.
PubMed: 36570711
DOI: 10.2147/IDR.S393201 -
Frontiers in Microbiology 2021Daqu is an important saccharifying and fermenting agent. It provides various microorganisms and enzymes for the fermentation of Baijiu and plays a vital role in the...
Daqu is an important saccharifying and fermenting agent. It provides various microorganisms and enzymes for the fermentation of Baijiu and plays a vital role in the formation of Baijiu flavor. However, it is difficult to obtain information on microbial growth and metabolism in time for Daqu production. Therefore, the "Qu Xiang" obtained by smelling is an important index in the traditional production process to evaluate the microbial fermentation in the process of Daqu-making, "Qu Xiang" mainly represents the volatile flavor compounds in Daqu. The microbial diversity and volatile metabolites on 0, 6, 16, and 29 days of the fermentation process were measured using high-throughput sequencing and gas chromatography-mass spectrometry. Significant differences were found in the composition of the microbial community. , , , and were the main bacterial genera. , , and are the main fungal genera. A total of 32 differential volatile metabolites were detected in samples at four time points using differential metabolic analysis. The correspondence of prevailing microorganisms with differential metabolites distinguished by Spearman correlation and two-way orthogonal partial least square analysis show that Saccharopolyspora exhibited a significant connection for the 12 differential metabolites. A significant positive correlation was observed between and 13 different metabolites. These findings further understanding of the metabolism of microorganisms in Daqu fermentation and also help to control the microorganisms in the Daqu-making process, to obtain more stable Baijiu products.
PubMed: 34630343
DOI: 10.3389/fmicb.2021.688981 -
Applied and Environmental Microbiology Mar 2023Cavities are created by hydrophobic interactions between residue side chain atoms during the folding of enzymes. Redesigning cavities can improve the thermostability and...
Cavities are created by hydrophobic interactions between residue side chain atoms during the folding of enzymes. Redesigning cavities can improve the thermostability and catalytic activity of the enzyme; however, the synergistic effect of cavities remains unclear. In this study, Rhizomucor miehei lipase (RML) was used as a model to explore volume fluctuation and spatial distribution changes of the internal cavities, which could reveal the roles of internal cavities in the thermostability and catalytic activity. We present an inside out cavity engineering (CE) strategy based on computational techniques to explore how changes in the volumes and spatial distribution of cavities affect the thermostability and catalytic activity of the enzyme. We obtained 12 single-point mutants, among which the melting temperatures () of 8 mutants showed an increase of more than 2°C. Sixteen multipoint mutations were further designed by spatial distribution rearrangement of internal cavities. The of the most stable triple variant, with mutations including T21V (a change of T to V at position 21), S27A, and T198L (T21V/S27A/T198L), was elevated by 11.0°C, together with a 28.7-fold increase in the half-life at 65°C and a specific activity increase of 9.9-fold (up to 5,828 U mg), one of the highest lipase activities reported. The possible mechanism of decreased volumes and spatial rearrangement of the internal cavities improved the stability of the enzyme, optimizing the outer substrate tunnel to improve the catalytic efficiency. Overall, the inside out computational redesign of cavities method could help to deeply understand the effect of cavities on enzymatic stability and activity, which would be beneficial for protein engineering efforts to optimize natural enzymes. In the present study, R. miehei lipase, which is widely used in various industries, provides an opportunity to explore the effects of internal cavities on the thermostability and catalytic activity of enzymes. Here, we execute high hydrostatic pressure molecular dynamics (HP-MD) simulations to screen the critical internal cavity and reshape the internal cavities through site-directed mutation. We show that as the global internal cavity volume decreases, cavity rearrangement can improve the stability of the protein while optimizing the substrate channel to improve the catalytic efficiency. Our results provide significant insights into understanding the mechanism of action of the internal cavity. Our strategy is expected to be applied to other enzymes to promote increases in thermostability and catalytic activity.
Topics: Lipase; Enzyme Stability; Temperature; Enzymes, Immobilized; Rhizomucor
PubMed: 36912632
DOI: 10.1128/aem.02172-22 -
International Journal of Molecular... Jun 2022One of the indispensable applications of lipases in modification of oils and fats is the possibility to tailor the fatty acid content of triacylglycerols (TAGs), to meet...
One of the indispensable applications of lipases in modification of oils and fats is the possibility to tailor the fatty acid content of triacylglycerols (TAGs), to meet specific requirements from various applications in food, nutrition, and cosmetic industries. Oleic acid (C18:1) and stearic acid (C18:0) are two common long fatty acids in the side chain of triglycerides in plant fats and oils that have similar chemical composition and structures, except for an unsaturated bond between C9 and C10 in oleic acid. Two lipases from (RML) and (ROL) show activity in reactions involving oleate and stearate, and share high sequence and structural identity. In this research, the preference for one of these two similar fatty acid side chains was investigated for the two lipases and was related to the respective enzyme structure. From transesterification reactions with 1:1 (molar ratio) mixed ethyl stearate (ES) and ethyl oleate (EO), both RML and ROL showed a higher activity towards EO than ES, but RML showed around 10% higher preference for ES compared with ROL. In silico results showed that stearate has a less stable interaction with the substrate binding crevice in both RML and ROL and higher tendency to freely move out of the substrate binding region, compared with oleate whose structure is more rigid due to the existence of the double bond. However, Trp88 from RML which is an Ala at the identical position in ROL shows a significant stabilization effect in the substrate interaction in RML, especially with stearate as a ligand.
Topics: Fungal Proteins; Lipase; Molecular Docking Simulation; Oleic Acids; Rhizomucor; Rhizopus oryzae; Sequence Analysis, Protein; Stearates; Structure-Activity Relationship; Substrate Specificity
PubMed: 35806072
DOI: 10.3390/ijms23137072 -
Medicina Oral, Patologia Oral Y Cirugia... Dec 2008Mucormycosis is a rare oportunistic infection typically described in diabetic patients with a ketoacidotic status, as well as neutropenic patients. The infection is... (Review)
Review
Mucormycosis is a rare oportunistic infection typically described in diabetic patients with a ketoacidotic status, as well as neutropenic patients. The infection is caused by a group of saprophytic fungi of the class Phycomicetes, being the most frequent ones the Rhizomucor, Rhizopus and Mucor. Its hystological findings include vascular trombosis and tissue necrosis, predominantly in the rino-orbito-cerebral area. Even though the frequency of presentation is very low, given its rapid evolution and severe consequences which include a high mortality rate, it is very important to be aware of the main features of the disease and treat it promptly. Although the diagnosis is based on the high clinical suspect, the computed tomography (CT) and the magnetic resonance image (MRI) plays an important role in determining the extension. The patients should receive treatment in a reference hospital so that a multidisciplinary approach is ensured. In this sense, we present a case of rhino-orbito-cerebralmucormycosis in a diabetic patient, recently treated in our Department. A comprehensive review of the literature has been performed to update the physiopathology and diagnosis. Finally, we describe the different treatment options focusing in the surgical approach, as well as the medical treatment with amphotericine and posaconzole.
Topics: Aged; Brain Diseases; Diabetes Complications; Humans; Male; Mucormycosis; Nose Diseases; Orbital Diseases
PubMed: 19047969
DOI: No ID Found -
Frontiers in Microbiology 2021The fungi present during pile-fermentation of Sichuan dark tea play a pivotal role in the development of its aroma and physical characteristics. Samples of tea leaves...
The fungi present during pile-fermentation of Sichuan dark tea play a pivotal role in the development of its aroma and physical characteristics. Samples of tea leaves were collected on days 0 (YC-raw material), 8 (W1-first turn), 16 (W2-second turn), 24 (W3-third turn), and 32 (W4-out of pile) during pile-fermentation. High-throughput sequencing revealed seven phyla, 22 classes, 41 orders, 85 families, 128 genera, and 184 species of fungi. During fermentation, the fungal diversity index declined from the W1 to W3 stages and then increased exponentially at the W4 stage. A bar plot and heatmap revealed that , , , , , , and were abundant during piling, of which was the most abundant. Cluster analysis revealed that the W4 stage of fermentation is critical for fungal growth, diversity, and the community structure in Sichuan dark tea. This study revealed the role of fungi during pile-fermentation in the development of the essence and physical characteristics of Sichuan dark tea. This study comes under one of the Sustainable Development Goals of United Nations Organization (UNO) to "Establish Good Health and Well-Being."
PubMed: 34421866
DOI: 10.3389/fmicb.2021.706714 -
A Rapid and Specific Real-Time PCR Assay for the Detection of Clinically Relevant Mucorales Species.International Journal of Molecular... Dec 2022Infections triggered by filamentous fungi placed in the order Mucorales, phylum Zygomycota, can cause serious harm to immunocompromised patients. Since there is lack of...
Infections triggered by filamentous fungi placed in the order Mucorales, phylum Zygomycota, can cause serious harm to immunocompromised patients. Since there is lack of a standardized PCR (polymerase chain reaction) assay for early diagnosis of this fungal infection, this work was aimed to develop a new PCR assay able to detect the presence of Mucorales genera in clinical specimens. Here, we describe a novel diagnostic TaqMan MGB probe assay for precise and rapid detection of the most common clinical species of Mucorales. Zygomycete-specific oligonucleotides were designed to specifically amplify and bind highly conserved sequences of fungal 28S rRNA gene. Additionally, we succeeded in differentiating Mucorales species (i.e., , , , and ) in artificially infected serum samples, suggesting that the quantitative capability of this real-time PCR assay could potentially optimize the diagnosis of mucormycosis.
Topics: Humans; Mucorales; Real-Time Polymerase Chain Reaction; Mucormycosis; RNA, Ribosomal, 28S; Immunocompromised Host
PubMed: 36499395
DOI: 10.3390/ijms232315066 -
Biology Dec 2021l-asparaginase (EC 3.5.1.1) hydrolyzes l-asparagine to produce l-aspartate and ammonia and is widely found in microorganisms, plants, and some rodent sera....
l-asparaginase (EC 3.5.1.1) hydrolyzes l-asparagine to produce l-aspartate and ammonia and is widely found in microorganisms, plants, and some rodent sera. l-asparaginase used for industrial production should have good thermostability. We heterologously expressed l-asparaginase from , selected nine loci for site-directed mutagenesis by rational design, and obtained two mutants with significantly improved thermostability. The optimal temperature of mutants S302I and S302M was 50 °C. After incubating the mutant and wild-type enzymes at 45 °C for 35 h, the residual activity of the wild-type enzyme (WT) was only about 10%. In contrast, the residual activity of S302I and S302M was more than 50%. After combination mutagenesis, 168-pMA5-A344E/S302I was constructed using the food-safe host strain 168. Additionally, a 5' untranslated region (UTR) modification strategy was adopted to enhance the expression level of -derived l-asparaginase in . In a 5-L fermenter scale-up experiment, the enzyme activity of recombinant 168-pMA5-UTR-A344E/S302I reached 521.9 U·mL by fed-batch fermentation.
PubMed: 34943261
DOI: 10.3390/biology10121346 -
Journal of Fungi (Basel, Switzerland) Mar 2021β-Galactosidases of Mucoromycota are rarely studied, although this group of filamentous fungi is an excellent source of many industrial enzymes. In this study, 99...
β-Galactosidases of Mucoromycota are rarely studied, although this group of filamentous fungi is an excellent source of many industrial enzymes. In this study, 99 isolates from the genera , , , , and , were screened for their β-galactosidase activity using a chromogenic agar approach. Ten isolates from the best producers were selected, and the activity was further investigated in submerged (SmF) and solid-state (SSF) fermentation systems containing lactose and/or wheat bran substrates as enzyme production inducers. Wheat bran proved to be efficient for the enzyme production under both SmF and SSF conditions, giving maximum specific activity yields from 32 to 12,064 U/mg protein and from 783 to 22,720 U/mg protein, respectively. Oligosaccharide synthesis tests revealed the suitability of crude β-galactosidases from Szeged Microbiological Collection (SZMC) 11360 and SZMC 11025 to catalyze transgalactosylation reactions. In addition, the crude enzyme extracts had transfructosylation activity, resulting in the formation of fructo-oligosaccharide molecules in a sucrose-containing environment. The maximal oligosaccharide concentration varied between 0.0158 and 2.236 g/L depending on the crude enzyme and the initial material. Some oligosaccharide-enriched mixtures supported the growth of probiotics, indicating the potential of the studied enzyme extracts in future prebiotic synthesis processes.
PubMed: 33808917
DOI: 10.3390/jof7030229