Mucor circinelloides, a dimorphic opportunistic pathogen, expresses three heterotrimeric G‐protein beta subunits (Gpb1, Gpb2 and Gpb3). The Gpb1‐encoding gene is up‐regulated during mycelial growth ...compared with that in the spore or yeast stage. gpb1 deletion mutation analysis revealed its relevance for an adequate development during the dimorphic transition and for hyphal growth under low oxygen concentrations. Infection assays in mice indicated a phenotype with considerably reduced virulence and tissue invasiveness in the deletion mutants (Δgpb1) and decreased host inflammatory response. This finding could be attributed to the reduced filamentous growth in animal tissues compared with that of the wild‐type strain. Mutation in a regulatory subunit of cAMP‐dependent protein kinase A (PKA) subunit (PkaR1) resulted in similar phenotypes to Δgpb1. The defects exhibited by the Δgpb1 strain were genetically suppressed by pkaR1 overexpression, indicating that the PKA pathway is controlled by Gpb1 in M. circinelloides. Moreover, during growth under low oxygen levels, cAMP levels were much higher in the Δgpb1 than in the wild‐type strain, but similar to those in the ΔpkaR1 strain. These findings reveal that M. circinelloides possesses a signal transduction pathway through which the Gpb1 heterotrimeric G subunit and PkaR1 control mycelial growth in response to low oxygen levels.
Abstract
Mucormycosis is a fungal infection caused by Mucorales, with a high mortality rate. However, only a few virulence factors have been described in these organisms. This study showed that ...deletion of
rfs
, which encodes the enzyme for the biosynthesis of rhizoferrin, a siderophore, in
Mucor lusitanicus
, led to a lower virulence in diabetic mice and nematodes. Upregulation of
rfs
correlated with the increased toxicity of the cell-free supernatants of the culture broth (SS) obtained under growing conditions that favor oxidative metabolism, such as low glucose levels or the presence of H
2
O
2
in the culture, suggesting that oxidative metabolism enhances virulence through rhizoferrin production. Meanwhile, growing
M. lusitanicus
in the presence of potassium cyanide, N-acetylcysteine, a higher concentration of glucose, or exogenous cAMP, or the deletion of the gene encoding the regulatory subunit of PKA (
pkaR1
), correlated with a decrease in the toxicity of SS, downregulation of
rfs
, and reduction in rhizoferrin production. These observations indicate the involvement of the cAMP-PKA pathway in the regulation of rhizoferrin production and virulence in
M. lusitanicus
. Moreover,
rfs
upregulation was observed upon macrophage interaction or during infection with spores in mice, suggesting a pivotal role of
rfs
in
M. lusitanicus
infection.
Mezcal, a traditional beverage that originated in Mexico, is produced from species of the Agavaceae family. The esters associated with the yeasts utilized during fermentation are important for ...improving the organoleptic properties of the beverage. We improved the ester contents in a mezcal beverage by using the yeast Kluyveromyces marxianus, which was engineered with the ATF1 gene. ATF1 expression in the recombinant yeast significantly increased compared with that in the parental yeast, but its fermentative parameters were unchanged. Volatile-organic-compound-content analysis showed that esters had significantly increased in the mezcal produced with the engineered yeast. In a sensory-panel test, 48% of the panelists preferred the mezcal produced from the engineered yeast, 30% preferred the mezcal produced from the wild type, and 15 and 7% preferred the two mezcal types produced following the routine procedure. Correlation analysis showed that the fruitiness/sweetness description of the mezcal produced using the ATF1-engineered K. marxianus yeast correlated with the content of the esters, whose presence improved the organoleptic properties of the craft mezcal beverage.
Mucor circinelloides is one of the causal agents of mucormycosis, an emerging and high mortality rate fungal infection produced by asexual spores (sporangiospores) of fungi that belong to the order ...Mucorales. M. circinelloides has served as a model genetic system to understand the virulence mechanism of this infection. Although the G-protein signaling cascade plays crucial roles in virulence in many pathogenic fungi, its roles in Mucorales are yet to be elucidated. Previous study found that sporangiospore size and calcineurin are related to the virulence in Mucor, in which larger spores are more virulent in an animal mucormycosis model and loss of a calcineurin A catalytic subunit CnaA results in larger spore production and virulent phenotype. The M. circinelloides genome is known to harbor twelve gpa (gpa1 to gpa12) encoding G-protein alpha subunits and the transcripts of the gpa11 and gpa12 comprise nearly 72% of all twelve gpa genes transcript in spores. In this study we demonstrated that loss of function of Gpa11 and Gpa12 led to larger spore size associated with reduced activation of the calcineurin pathway. Interestingly, we found lower levels of the cnaA mRNAs in sporangiospores from the Δgpa12 and double Δgpa11/Δgpa12 mutant strains compared to wild-type and the ΔcnaA mutant had significantly lower gpa11 and gpa12 mRNA levels compared to wild-type. However, in contrast to the high virulence showed by the large spores of ΔcnaA, the spores from Δgpa11/Δgpa12 were avirulent and produced lower tissue invasion and cellular damage, suggesting that the gpa11 and gpa12 define a signal pathway with two branches. One of the branches controls spore size through regulation of calcineurin pathway, whereas virulences is controlled by an independent pathway. This virulence-related regulatory pathway could control the expression of genes involved in cellular responses important for virulence, since sporangiospores of Δgpa11/Δgpa12 were less resistant to oxidative stress and phagocytosis by macrophages than the ΔcnaA and wild-type strains. The characterization of this pathway could contribute to decipher the signals and mechanism used by Mucorales to produce mucormycosis.
Mucormycosis is a lethal and difficult-to-treat fungal infection caused by fungi of the order Mucorales.
Mucor lusitanicus,
a member of Mucorales, is commonly used as a model to understand disease ...pathogenesis. However, transcriptional control of hyphal growth and virulence in Mucorales is poorly understood. This study aimed to investigate the role of Tec proteins, which belong to the TEA/ATTS transcription factor family, in the hyphal development and virulence of
M. lusitanicus
. Unlike in the genome of Ascomycetes and Basidiomycetes, which have a single Tec homologue, in the genome of Mucorales, two Tec homologues, Tec1 and Tec2, were found, except in that of
Phycomyces blakesleeanus
, with only one Tec homologue.
tec1
and
tec2
overexpression in
M. lusitanicus
increased mycelial growth, mitochondrial content and activity, expression of the rhizoferrin synthetase-encoding gene
rfs
, and virulence in nematodes and wax moth larvae but decreased cAMP levels and protein kinase A (PKA) activity. Furthermore,
tec1
- and
tec2
-overexpressing strains required adequate mitochondrial metabolism to promote the virulent phenotype. The heterotrimeric G beta subunit 1-encoding gene deletant strain (Δ
gpb1
) increased cAMP-PKA activity, downregulation of both
tec
genes, decreased both virulence and hyphal development, but
tec1
and
tec2
overexpression restored these defects. Overexpression of allele-mutated variants of Tec1(S332A) and Tec2(S168A) in the putative phosphorylation sites for PKA increased both virulence and hyphal growth of Δ
gpb1
. These findings suggest that Tec homologues promote mycelial development and virulence by enhancing mitochondrial metabolism and rhizoferrin accumulation, providing new information for the rational control of the virulent phenotype of
M. lusitanicus
.
is a dimorphic and entomopathogenic fungus with different ecological roles in nature. In pathogenic fungi, yeast-to-mycelial conversion, which is controlled by environmental factors, is required for ...virulence. Here, we studied the effects of different stimuli on the morphology of two
strains and compared the toxicities of culture filtrates. In addition, we explored the role of volatiles as quorum sensing-like signals during dimorphic transition. The killing assays in
(Nematoda: Rhabditidae) showed that strain AI2 isolated from a mycosed insect cadaver had higher toxicity than strain AS5 isolated from soil. Furthermore, AI2 showed earlier yeast-to-mycelial switching than AS5. However, an increase in inoculum size induced faster yeast-to-mycelium conversion in AS5 cells, suggesting a cell-density-dependent phenomenon. Gas chromatography-mass spectrometry (GC-MS) analyses showed that the fingerprint of the volatiles was strain-specific; however, during the morphological switching, an inverse relationship between the abundance of total terpenes and 3-methylbutanol was observed in both strains. Fungal exposure to 3-methylbutanol retarded the yeast-to-mycelium transition. Hence, this study provides evidence that volatile compounds are associated with critical events in the life cycle of
.
Mucor circinelloides is an opportunistic dimorphic pathogen, with the dimorphic process controlled in parts by fermentative and oxidative metabolisms, which lead to yeast or mycelial growth, ...respectively. Dimorphic transition is important for pathogenesis since the mycelium represents the virulent morphology. We previously reported that the deletion of arl1 or arl2 stimulate anaerobic germination in M. circinelloides, suggesting an augmented fermentative metabolism. In the present study, we demonstrate that the heterokaryon Δarl1(+)(−) and homokaryon Δarl2 strains contain low number of mitochondria, which possibly results in a dysfunctional oxidative metabolism, marked by a low oxygen consumption in glucose and poor growth in glycerol as the unique carbon source. This dysfunction is compensated for by an increase in the glycolysis and fermentation in aerobic conditions, demonstrating growth kinetics similar to that in the wild-type strain. Moreover, as a consequence a high fermentative activity, the Δarl1(+)(−) and Δarl2 strains possibly increased the yeast cell growth during low oxygen concentrations in presence of glucose.
To the best of our knowledge, this is the first study to demonstrate the control of members of Arf family on the mitochondrial population in a Mucor species.
•Arl1 and Arl2 control the mitochondrial content in M. circinelloides.•Δarl1(+)/(−) and Δarl2 decreased the mitochondrial cellular content.•Δarl1(+)/(−) and Δarl2 increased the glycolysis and fermentative metabolism.•Δarl1(+)/(−) and Δarl2 enhance the yeast growth under low oxygen level conditions.•Yeast growth of Δarl1(+)/(−) is more virulent compared to the WT.
Fungal alcohol dehydrogenases (ADHs) participate in growth under aerobic or anaerobic conditions, morphogenetic processes, and pathogenesis of diverse fungal genera. These processes are associated ...with metabolic operation routes related to alcohol, aldehyde, and acid production. The number of ADH enzymes, their metabolic roles, and their functions vary within fungal species. The most studied ADHs are associated with ethanol metabolism, either as fermentative enzymes involved in the production of this alcohol or as oxidative enzymes necessary for the use of ethanol as a carbon source; other enzymes participate in survival under microaerobic conditions. The fast generation of data using genome sequencing provides an excellent opportunity to determine a correlation between the number of ADHs and fungal lifestyle. Therefore, this review aims to summarize the latest knowledge about the importance of ADH enzymes in the physiology and metabolism of fungal cells, as well as their structure, regulation, evolutionary relationships, and biotechnological potential.
The genus Fusarium can be utilized to produce a great variety of secondary metabolites under specific culture conditions, including pigments of increasing biotechnological interest, such as ...bikaverin. Such pigments are important due to the biological properties they possess, including antitumor and antibiotic activities, among others. In Fusarium fujikuroi, bik1-bik6 have been identified as the genes that are responsible for the synthesis of bikaverin. Therefore, in this study, we screened for the presence of bik genes and examined changes in mRNA levels of the bik genes under the influence of NH
NO
(0.024, 0.048, 0.50, 1.0, and 4.60 g L
) and NH
Cl (0.50 and 1.0 g L
) as nitrogen sources for the phytopathogen Fusarium oxysporum f. sp. lycopersici. Our results indicated the presence of at least six bik (bik1-bik6) genes and showed increased mRNA levels for bik4, bik5, and bik6 in conditions where NH
NO
was used at pH 3.0. The characteristic coloration of bikaverin was obtained in 10 out of 16 culture conditions, except when the fungus was grown with higher concentrations of NH
NO
(1.0 and 4.60 g L
). The pigment was chloroform-extracted from the culture conditions of NH
NO
(0.024, 0.048, and 0.50 g L
) and NH
Cl (0.50 and 1.0 g L
) with 3 and 9 days of incubation. Analysis via visible spectroscopy and matrix-assisted laser desorption ionization-time of flight mass spectrometry were used for the identification of bikaverin.
Mucor circinelloides
is a dimorphic Zygomycete fungus that produces ethanol under aerobic conditions in the presence of glucose, which indicates that it is a Crabtree-positive fungus. To determine ...the physiological role of the alcohol dehydrogenase (ADH) activity elicited under these conditions, we obtained and characterized an allyl alcohol-resistant mutant that was defective in ADH activity, and examined the effect of
adh
mutation on physiological parameters related to carbon and energy metabolism. Compared to the Adh
+
strain R7B, the ADH-defective (Adh
-
) strain M5 was unable to grow under anaerobic conditions, exhibited a considerable reduction in ethanol production in aerobic cultures when incubated with glucose, had markedly reduced growth capacity in the presence of oxygen when ethanol was the sole carbon source, and exhibited very low levels of NAD
+
-dependent alcohol de-hydrogenase activity in the cytosolic fraction. Further characterization of the M5 strain showed that it contains a 10-bp deletion that interrupts the coding region of the
adhl
gene. Complementation with the wild-type allele
adh1
+
by transformation of M5 remedied all the defects caused by the
adh1
mutation. These findings indicate that in
M. circinelloides
, the product of the
adh1
gene mediates the Crabtree effect, and can act as either a fermentative or an oxidative enzyme, depending on the nutritional conditions, thereby participating in the association between fermentative and oxidative metabolism. It was found that the spores of
M. circinelloides
possess low mRNA levels of the ethanol assimilation genes (
adl2
and
acs2
), which could explain their inability to grow in the alcohol.
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