Outbreaks of Mucorales and the Species Involved.

The order Mucorales is an ancient group of fungi classified in the subphylum Mucoromycotina. Mucorales are mainly fast-growing saprotrophs that belong to the first colonizers of diverse organic materials and represent a permanent part of the human environment. Several species are able to cause human infections (mucormycoses) predominantly in patients with impaired immune system, diabetes, or deep trauma. In this review, we compiled 32 reports on community- and hospital-acquired outbreaks caused by Mucorales. The most common source of mucoralean outbreaks was contaminated medical devices that are responsible for 40.7% of the outbreaks followed by contaminated air (31.3%), traumatic inoculation of soil or foreign bodies (9.4%), and the contact (6.2%) or the ingestion (6.2%) of contaminated plant material. The most prevalent species were Rhizopus arrhizus and R. microsporus causing 57% of the outbreaks. The genus Rhizomucor was dominating in outbreaks related to contaminated air while outbreaks of Lichtheimia species and Mucor circinelloides were transmitted by direct contact. Outbreaks with the involvement of several species are reported. Subtyping of strains revealed clonality in two outbreaks and no close relation in two other outbreaks. Based on the existing data, outbreaks of Mucorales can be caused by heterogeneous sources consisting of different strains or different species. Person-to-person transmission cannot be excluded because Mucorales can sporulate on wounds. For a better understanding and prevention of outbreaks, we need to increase our knowledge on the physiology, ecology, and population structure of outbreak causing species and more subtyping data.

Evaluation of Aspergillus and Mucorales specific T-cells and peripheral blood mononuclear cell cytokine signatures as biomarkers of environmental mold exposure.

Mold specific T-cells have been described as a supportive biomarker to monitor invasive mycoses and mold exposure. This study comparatively evaluated frequencies and cytokine profiles of Aspergillus fumigatus and Mucorales reactive T-cells depending on environmental mold exposure. Peripheral blood mononuclear cells (PBMCs) obtained from 35 healthy donors were stimulated with mycelial lysates of A. fumigatus and three human pathogenic Mucorales species. CD154+ specific T-cells were quantified by flow cytometry. In a second cohort of 20 additional donors, flow cytometry was complemented by 13-plex cytokine assays. Mold exposure of the subjects was determined using a previously established questionnaire. Highly exposed subjects exhibited significantly greater CD154+A. fumigatus and Mucorales specific naïve and memory T-helper cell frequencies. Significant correlation (r = 0.48 - 0.79) was found between A. fumigatus and Mucorales specific T-cell numbers. Logistic regression analyses revealed that combined analysis of mold specific T-cell frequencies and selected cytokine markers (A. fumigatus: IL-5 and TNF-α, R. arrhizus: IL-17A and IL-13) significantly improves classification performance, resulting in 75-90 % predictive power using 10-fold cross-validation. In conclusion, mold specific T-cell frequencies and their cytokine signatures offer promising potential in the assessment of environmental mold exposure. The cytokines identified in this pilot study should be validated in the clinical setting, e. g. in patients with hypersensitivity pneumonitis.

Biochemical Characterization, Thermal Stability, and Partial Sequence of a Novel Exo-Polygalacturonase from the Thermophilic Fungus Rhizomucor pusillus A13.36 Obtained by Submerged Cultivation.

This work reports the production of an exo-polygalacturonase (exo-PG) by Rhizomucor pusillus A13.36 in submerged cultivation (SmC) in a shaker at 45°C for 96 h. A single pectinase was found and purified in order to analyze its thermal stability, by salt precipitation and hydrophobic interaction chromatography. The pectinase has an estimated Mw of approximately 43.5-47 kDa and optimum pH of 4.0 but is stable in pH ranging from 3.5 to 9.5 and has an optimum temperature of 61°C. It presents thermal stability between 30 and 60°C, has 70% activation in the presence of Ca2+, and was tested using citrus pectin with a degree of methyl esterification (DE) of 26%. Ea(d) for irreversible denaturation was 125.5 kJ/mol with positive variations of entropy and enthalpy for that and ΔG(d) values were around 50 kJ/mol. The hydrolysis of polygalacturonate was analyzed by capillary electrophoresis which displayed a pattern of sequential hydrolysis (exo). The partial identification of the primary sequence was done by MS MALDI-TOF and a comparison with data banks showed the highest identity of the sequenced fragments of exo-PG from R. pusillus with an exo-pectinase from Aspergillus fumigatus. Pectin hydrolysis showed a sigmoidal curve for the Michaelis-Menten plot.

Stress response in medically important Mucorales.

Mucorales are saprobes, ubiquitously distributed and able to infect a heterogeneous population of human hosts. The fungi require robust stress responses to survive in human host. We tested the growth of Mucorales in the presence of different abiotic stress. Eight pathogenic species of Mucorales, including Rhizopus arrhizus, Rhizopus microsporus, Rhizomucor pusillus, Apophysomyces elegans, Licthemia corymbifera, Cunninghamella bertholletiae, Syncephalastrum racemosum and Mucor racemosus, were exposed to different stress inducers: osmotic (sodium chloride and d-sorbitol), oxidative (hydrogen peroxide and menadione), pH, cell wall and metal ions (Cu, Zn, Fe and Mg). Wide variation in stress responses was noted: R. arrhizus showed maximum resistance to both osmotic and oxidative stresses, whereas R. pusillus and M. indicus were relatively sensitive. Rhizopus arrhizus and R. microsporus showed maximum resistance to alkaline pH, whereas C. bertholletiae, L. corymbifera, M. racemosus and A. elegans were resistant to acidic pH. Maximum tolerance was noted in R. microsporus to Cu, R. microsporus and R. arrhizus to Fe and C. bertholletiae to Zn. In contrast, L. corymbifera, A. elegans and M. indicus were sensitive to Cu, Zn and Fe respectively. In conclusion, R. arrhizus showed high stress tolerance in comparison to other species of Mucorales, and this could be the possible reason for high pathogenic potential of this fungi.

Heat shock response of thermophilic fungi: membrane lipids and soluble carbohydrates under elevated temperatures.

The heat shock (HS) response is an adaptation of organisms to elevated temperature. It includes substantial changes in the composition of cellular membranes, proteins and soluble carbohydrates. To protect the cellular macromolecules, thermophilic organisms have evolved mechanisms of persistent thermotolerance. Many of those mechanisms are common for thermotolerance and the HS response. However, it remains unknown whether thermophilic species respond to HS by further elevating concentrations of protective components. We investigated the composition of the soluble cytosol carbohydrates and membrane lipids of the thermophilic fungi Rhizomucor tauricus and Myceliophthora thermophilaat optimum temperature conditions (41-43 °Ð¡), and under HS (51-53 °Ð¡). At optimum temperatures, the membrane lipid composition was characterized by a high proportion of phosphatidic acids (PA) (20-35 % of the total), which were the main components of the membrane lipids, together with phosphatidylcholines (PC), phosphatidylethanolamines (PE) and sterols (St). In response to HS, the proportion of PA and St increased, and the amount of PC and PE decreased. No decrease in the degree of fatty acid desaturation in the major phospholipids under HS was detected. The mycelium of all fungi at optimum temperatures contained high levels of trehalose (8-10 %, w/w; 60-95 % of the total carbohydrates), which is a hallmark of thermophilia. In contrast to mesophilic fungi, heat exposure decreased the trehalose level and the fungi did not acquire thermotolerance to lethal HS, indicating that trehalose plays a key role in this process. This pattern of changes appears to be conserved in the studied filamentous thermophilic fungi.

Biochemical characterization of a first fungal esterase from Rhizomucor miehei showing high efficiency of ester synthesis.

BACKGROUND: Esterases with excellent merits suitable for commercial use in ester production field are still insufficient. The aim of this research is to advance our understanding by seeking for more unusual esterases and revealing their characterizations for ester synthesis. METHODOLOGY/PRINCIPAL FINDINGS: A novel esterase-encoding gene from Rhizomucor miehei (RmEstA) was cloned and expressed in Escherichia coli. Sequence analysis revealed a 975-bp ORF encoding a 324-amino-acid polypeptide belonging to the hormone-sensitive lipase (HSL) family IV and showing highest similarity (44%) to the Paenibacillus mucilaginosus esterase/lipase. Recombinant RmEstA was purified to homogeneity: it was 34 kDa by SDS-PAGE and showed optimal pH and temperature of 6.5 and 45°C, respectively. The enzyme was stable to 50°C, under a broad pH range (5.0-10.6). RmEstA exhibited broad substrate specificity toward p-nitrophenol esters and short-acyl-chain triglycerols, with highest activities (1,480 U mg(-1) and 228 U mg(-1)) for p-nitrophenyl hexanoate and tributyrin, respectively. RmEstA efficiently synthesized butyl butyrate (92% conversion yield) when immobilized on AOT-based organogel. CONCLUSION: RmEstA has great potential for industrial applications. RmEstA is the first reported esterase from Rhizomucor miehei.

High-throughput screening of B factor saturation mutated Rhizomucor miehei lipase thermostability based on synthetic reaction.

Conventional lipase screening methods are mostly based on hydrolytic activity, which may not always be the best method to assess the enzyme activity, especially for evaluating synthetic activity. Here we developed a high throughput and visual method to screen clones with high synthetic activity and used it to assess lipases thermostability. All mutants' lipase synthetic activity were identified through esterification of caprylic acid and ethanol with methyl red as the pH indicator adding in the substrates on according to the color change halo around the colony on culture plates since synthetic reaction was often accompanied with a rise in pH. After two rounds operation with the pH indicator screening method, we obtained a double mutant Asn120Lys/Lys131Phe from the Rhizomucor miehei lipase saturation mutated library based on amino acid residue B factors. The mutant's initial synthetic activity was a little higher than wild type and its thermostability in synthetic reaction was enhanced, which remained 63.1% residual activity after being heated at 70°C for 5h comparing to 51.0% of wild type. The double mutant with the two residue replacements balanced well between stability and activity. Yeast surface display technology and the pH indicator method, combined with colony screening were shown to facilitate high-throughput screening for lipase synthetic activity.

Heterothallic mating in Mucor irregularis and first isolate of the species outside of Asia.

This study reports on the discovery of heterothallic mating in Mucor irregularis (formerly Rhizomucor variabilis var. variabilis) and it extends the range of this species from Asia to the United States. We report on a case of primary cutaneous mucormycosis, involving the forearms of a cotton farmer from North Carolina, in which the infection was cured using amphotericin B therapy. Intraspecific crosses between the North Carolina strain DUMC 150.04 and M. irregularis CBS 103.93, the ex-type strain of R. variabilis var. variabilis from China, resulted in the formation of abundant fertile zygospores. By way of contrast, interspecific crosses between the North Carolina isolate and the ex-neotype strain of M. hiemalis NRRL 3624 resulted in the formation of putative azygospores by M. irregularis DUMC 150.04.

Differentiation of Rhizomucor species on the basis of their different sensitivities to lovastatin.

The opportunistic pathogens Rhizomucor pusillus and Rhizomucor miehei may be agents of frequently fatal mycotic diseases. In the present study, the susceptibilities of 27 clinical and environmental isolates of R. miehei and R. pusillus to lovastatin under different culturing conditions were investigated. Most of the R. miehei strains grew at lovastatin concentrations as high as 64 to 128 microg/ml. In contrast, the inhibitory effect of lovastatin on all of the R. pusillus strains was evident at lovastatin concentrations as low as 1 to 2 microg/ml. A simple and reliable method for species-level differentiation, based on the significantly higher sensitivity of R. pusillus to lovastatin than that of R. miehei, was elaborated. According this, on malt extract agar containing 6 mug of lovastatin/ml, R. pusillus is not able to produce colonies, while R. miehei will form compact colonies.

[Rennet production by Rhizomucor miehei NRRL 3169]. / Producción de renina por Rhizomucor miehei NRRL 3169.

The production of rennet was studied, using different strains of the fungus Rhizomucor miehei. The selection and preservation of strains, type of growth, media design and operation conditions were evaluated. The experiments were carried out in Erlenmeyer flasks in rotary shaker at 250 rpm and 2.5 eccentricity, and in mechanically stirred fermentors of the New Brunswick type, at 30 degrees C. In the studies concerning strain selection, the best strain was Rhizomucor miehei NRRL 3169. The major titles of enzyme were obtained in batch process at 168 h, with 884 SU/ml, whereas in mechanically stirred fermentors the best value was 1160 SU/ml. These values were far more superior to former ones published by various experts.

Physicochemical properties of a novel alpha-L-arabinofuranosidase from Rhizomucor pusillus HHT-1.

The zygomycete fungus Rhizomucor pusillus HHT-1, cultured on L(+)arabinose as a sole carbon source, produced extracellular alpha-L-arabinofuranosidase. The enzyme was purified by (NH4)2SO4 fractionation, gel filtration, and ion exchange chromatography. The molecular mass of this monomeric enzyme was 88 kDa. The native enzyme had a pI of 4.2 and displayed a pH optimum and stability of 4.0 and 7.0-10.0, respectively. The temperature optimum was 65 degrees C, and it was stable up to 70 degrees C. The Km and Vmax for p-nitrophenyl alpha-L-arabinofuranoside were 0.59 mM and 387 micromol x min(-1) x mg(-1) protein, respectively. Activity was not stimulated by metal cofactors. The N-terminal amino acid sequence did not show any similarity to other arabinofuranosidases. Higher hydrolytic activity was recorded with pnitrophenyl alpha-L-arabinofuranoside, arabinotriose, and sugar beet arabinan; lower hydrolytic activity was recorded with oat-spelt xylan and arabinogalactan, indicating specificity for the low molecular mass L(+)-arabinose containing oligosaccharides with furanoside configuration.

Characterization of an alg2 mutant of the zygomycete fungus Rhizomucor pusillus.

The zygomycete fungus Rhizomucor pusillus secretes an aspartic proteinase (MPP) that contains asparagine ( N )-linked oligosaccharides at two sites. Mutant strain 1116 defective in N -glycosylation secretes MPP with truncated oligo-saccharide chains. Lipid-linked oligosaccharides in mutant 1116 were labeled with [6-(3)H]glucosamine and [2-(3)H]mannose, prepared by cycles of solvent extraction, and analyzed by gel filtration chromatography on a Bio-Gel P-4 column after mild acid-hydrolysis. Mutant 1116 accumulated an intermediate, Man(1)GlcNAc(2)-dolichol pyrophosphate (PP-Dol), whereas wild-type strain F27 synthesized the fully assembled oligosaccharide precursor Glc(3)Man(9)GlcNAc(2)-PP-Dol. Consistent with this, alg2 encoding a mannosyltransferase in the lipid-linked oligosaccharide biosynthetic pathway in mutant 1116 had a 5 bp insertion that generated a stop codon in the middle of the coding sequence. Transformation of mutant 1116 with the intact alg2 gene on a pUC19-derived plasmid generated transformants that contained multicopies of alg2 at the alg2 locus. Glycosylation of the total proteins in the transformants was recovered to the same level as in strain F27, as determined with peroxidase-concanavalin A. These transformants produced MPP mainly with the same N -linked oligosaccharides as that produced by strain F27, but still with truncated oligosaccharides in small amounts. All of these data show that Alg2 is an alpha-1,3 or alpha-1,6 mannosyltransferase that elongates Man(1)GlcNAc(2)-PP-Dol to Man(2)GlcNAc(2)-PP-Dol. The slower growth of mutant 1116 was significantly recovered on introduction of alg2. The viability of the alg2 mutants of the zygomycete R.pusillus makes a contrast with the lethal effect of ALG2 mutations in the yeast Saccharomyces cerevisiae.