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.
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.
Mucor circinelloides
is a dimorphic fungus used to study cell differentiation that has emerged as a model to characterize mucormycosis. In this work, we identified four ADP-ribosylation factor ...(Arf)-encoding genes (
arf1–arf4)
and study their role in the morphogenesis and virulence. Arfs are key regulators of the vesicular trafficking process and are associated with both growth and virulence in fungi. Arf1 and Arf2 share 96% identity and Arf3 and Arf4 share 89% identity, which suggests that the genes arose through gene-duplication events in
M. circinelloides
. Transcription analysis revealed that certain
arf
genes are affected by dimorphism of
M. circinelloides
, such as the
arf2
transcript, which was accumulated during yeast development. Therefore, we created knockout mutants of four
arf
genes to evaluate their function in dimorphism and virulence. We found that both
arf1
and
arf2
are required for sporulation, but these genes also perform distinct functions;
arf2
participates in yeast development, whereas
arf1
is involved in aerobic growth. Conversely,
arf3
and
arf4
play only minor roles during aerobic growth. Moreover, we observed that all single
arf
-mutant strains are more virulent than the wild-type strain in mouse and nematode models, with the
arf3
mutant being most virulent. Lastly,
arf1
/
arf2
and
arf3
/
arf4
double mutations produced heterokaryon strains that did not reach the homokaryotic state, indicating that these genes participate in essential and redundant functions. Overall, this work reveals that Arfs proteins regulate important cellular processes in
M. circinelloides
such as morphogenesis and virulence, laying the foundation to characterize the molecular networks underlying this regulation.
The fungus
undergoes yeast-mold dimorphism, a developmental process associated with its capability as a human opportunistic pathogen. Dimorphism is strongly influenced by carbon metabolism, and hence ...the type of metabolism likely affects fungus virulence. We investigated the role of ethanol metabolism in
virulence. A mutant in the
gene (M5 strain) exhibited higher virulence than the wild-type (R7B) and the complemented (M5/pEUKA-
) strains, which were nonvirulent when tested in a mouse infection model. Cell-free culture supernatant (SS) from the M5 mutant showed increased toxic effect on nematodes compared to that from R7B and M5/pEUKA-
strains. The concentration of acetaldehyde excreted by strain M5 in the SS was higher than that from R7B, which correlated with the acute toxic effect on nematodes. Remarkably, strain M5 showed higher resistance to H
O
, resistance to phagocytosis, and invasiveness in mouse tissues and induced an enhanced systemic inflammatory response compared with R7B. The mice infected with strain M5 under disulfiram treatment exhibited only half the life expectancy of those infected with M5 alone, suggesting that acetaldehyde produced by
contributes to the toxic effect in mice. These results demonstrate that the failure in fermentative metabolism, in the step of the production of ethanol in
, contributes to its virulence, inducing a more severe tissue burden and inflammatory response in mice as a consequence of acetaldehyde overproduction.
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
.
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.
Fusarium oxysporum
f. sp.
lycopersici
is an important plant pathogen that has been used to understand the virulence mechanisms that soil inhabiting fungi exhibit during the infection process. In
F. ...oxysporum
many of the virulence factors are secreted, and the secretion process requires the formation of vesicles. Arf family members, represented by Arf (ADP- Ribosylation Factor), Arl (Arf-like), and Sar (Secretion-associated and Ras-related) proteins, are involved in the vesicle creation process. In this study we identified the Arf family members in
F. oxysporum
f. sp.
lycopersici
, which includes seven putative proteins: Arf1, Arf3, Arl1 through Arl3, Arl8B, and Sar1. Quantification of the mRNA levels of each
arf
encoding gene revealed that the highest expression corresponds to
arf1
in all tested conditions. The phylogenetic analysis revealed that no other Arf1 paralogue, such as Arf2 from yeast, is present in
F. oxysporum
f. sp.
lycopersici
. The essential function suggested of Arf1 in
F. oxysporum
f. sp.
lycopersici
was corroborated experimentally when, after several attempts, it was impossible to obtain a knockout mutant in
arf1
. Moreover,
arl3
mRNA levels increased significantly when plant tissue was added as a sole carbon source, suggesting that the product of these genes could play pivotal roles during plant infection, the corresponding mutant ∆
arl3
was less virulent compared to the wild-type strain. These results describe the role of
arl3
as a critical regulator of the virulence in
F. oxysporum
f. sp.
lycopersici
and stablish a framework for the
arf
family members to be studied in deeper details in this phytopathogen.
•Two Arf-like proteins are encoded in the genome of M. circinelloides.•Arl1 and Arl2 share 55% sequence identity with different expression profiles.•Arl1 is essential for M. circinelloides ...growth.•Arl1 and Arl2 dysfunction led to mislocalization of vesicles in the hyphae.•Arl1 is important for sporulation, virulence, and resistance to antifungal agents.
Mucor circinelloides is an etiologic agent of mucormycosis, a fungal infection produced by Mucorales often associated with mortality due to unavailability of antifungal drugs. Arl proteins belong to the Arf family and are involved in vesicle trafficking and tubulin assembly. This study identified two Arl (Arf-like)-encoding genes, arl1 and arl2, in M. circinelloides and explored their function in morphogenesis, virulence, and antifungal susceptibility. Although Arl1 and Arl2 proteins shared 55% amino acid sequence identity, arl1 and arl2 genes showed distinct transcriptional expression patterns. arl1 was expressed at higher levels than arl2 and induced in mycelia, suggesting a role in morphological transitions. Disruption of the arl1 and arl2 genes led to heterokaryon (Δarl1(+)(−)) and homokaryon (Δarl2) genotypes, respectively. The incapacity to generate homokaryon mutants for arl1 suggested that it is essential for growth of M. circinelloides. Deletion of each gene reduced the expression of the other, suggesting the existence of a positive cross-regulation between them. Thus, deletion of arl2 resulted in a ~60% reduction of arl1 expression, whereas the Δarl1(+)(−) showed ∼90% reduction of arl1 expression. Mutation of arl2 showed no phenotype or a mild phenotype between Δarl1(+)(−) and wild-type (WT), suggesting that all observed phenotypes in both mutant strains corresponded to arl1 low expression. The Δarl1(+)(−) produced a small amount of spores that showed increased sensitivity to dodecyl-sulfate and azoles, suggesting a defect in the cell wall that was further supported by decrease in saccharide content. These defects in the cell wall were possibly originated by abnormal vesicle trafficking since FM4-64 staining of both mutants Δarl1(+)(−) and Δarl2 revealed less well-localized endosomes compared to the WT. Moreover, aberrant vesicle trafficking may be responsible for the secretion of specific virulence-related proteins since cell-free medium from Δarl1(+)(−) were found to increase killing of Caenorhabditis elegans compared to WT.
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.
Dimorphic species of
, which are cosmopolitan fungi belonging to subphylum Mucoromycotina, are metabolically versatile. Some species of
are sources of biotechnological products, such as biodiesel ...from
and expression of heterologous proteins from
. Furthermore,
has been described as a model for understanding mucormycosis infections. However, little is known regarding the relationship between
and other soil inhabitants. In this study, we investigated the potential use of
as a biocontrol agent against fungal phytopathogens, namely
f. sp.
, and
, which destroy economically important crops. Results showed that aerobic cell-free supernatants of the culture broth (SS) from
inhibited the growth of the fungal phytopathogens in culture, soil, and tomato fruits. The SS obtained from a strain of
carrying the deletion of
gene, which encodes an enzyme involved in the synthesis of siderophore rhizoferrin, had a decreased inhibitory effect against the growth of the phytopathogens. Contrarily, this inhibitory effect was more evident with the SS from an
-overexpressing strain compared to the wild-type. This study provides a framework for the potential biotechnological use of the molecules secreted from
in the biocontrol of fungal phytopathogens.
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