Transcription Factors Tec1 and Tec2 Play Key Roles in the Hyphal Growth and Virulence of Mucor lusitanicus Through Increased Mitochondrial Oxidative Metabolism.

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 (Δgpb1) 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 Δgpb1. 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.

The fungal NADPH oxidase is an essential element for the molecular dialog between Trichoderma and Arabidopsis.

Members of the fungal genus Trichoderma stimulate growth and reinforce plant immunity. Nevertheless, how fungal signaling elements mediate the establishment of a successful Trichoderma-plant interaction is largely unknown. In this work, we analyzed growth, root architecture and defense in an Arabidopsis-Trichoderma co-cultivation system, including the wild-type (WT) strain of the fungus and mutants affected in NADPH oxidase. Global gene expression profiles were assessed in both the plant and the fungus during the establishment of the interaction. Trichoderma atroviride WT improved root branching and growth of seedling as previously reported. This effect diminished in co-cultivation with the ∆nox1, ∆nox2 and ∆noxR null mutants. The data gathered of the Arabidopsis interaction with the ∆noxR strain showed that the seedlings had a heightened immune response linked to jasmonic acid in roots and shoots. In the fungus, we observed repression of genes involved in complex carbohydrate degradation in the presence of the plant before contact. However, in the absence of NoxR, such repression was lost, apparently due to a poor ability to adequately utilize simple carbon sources such as sucrose, a typical plant exudate. Our results unveiled the critical role played by the Trichoderma NoxR in the establishment of a fine-tuned communication between the plant and the fungus even before physical contact. In this dialog, the fungus appears to respond to the plant by adjusting its metabolism, while in the plant, fungal perception determines a delicate growth-defense balance.

Arf-like proteins (Arl1 and Arl2) are involved in mitochondrial homeostasis in Mucor circinelloides.

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.

Virulence Conferred by PumA Toxin from the Plasmid-Encoded PumAB Toxin-Antitoxin System is Regulated by Quorum System.

Toxin-antitoxin (TA) systems are small genetic elements composed of a toxin gene and its cognate antitoxin that are important for plasmid stabilization (plasmid-encoded) and bacterial virulence (chromosome-encoded). These systems are also related to biofilm and persister cell formations. Pseudomonas aeruginosa is an antibiotic-resistant human pathogen that produces virulence factors modulated by quorum sensing (QS) and can form biofilms. The type II PumAB TA system of pUM505, isolated from a clinical strain of P. aeruginosa, confers plasmid stability. Additionally, the PumA toxin increases P. aeruginosa virulence and is neutralized by the PumB antitoxin. In this study, we determined whether virulence conferred by PumA toxin is regulated by QS. The pumA gene was transferred to P. aeruginosa lasI/rhlI, a mutant strain in the LasI and RhlI QS systems, to analyze the effect on virulence of the transformants. pumA transfer did not increase bacterial virulence in lettuce and Caenorhabditis elegans, suggesting that the virulence conferred by PumA requires QS modulation. pumA mRNA levels drastically decreased in the P. aeruginosa lasI/rhlI (pUC_pumA) strain, suggesting positive regulation of pumA gene expression by QS. Supplementation of the growth medium of P. aeruginosa lasI/rhlI (pUC_pumA) with C4-AHL and 3-oxo-C12-AHL autoinducers increased pumA mRNA levels and restored bacterial virulence, suggesting that both autoinducers complemented the mutations and positively regulated the toxic effects of PumA. This strengthened the hypothesis that QS regulates bacterial virulence conferred by the PumA toxin. Thus, this report establishes an important function of QS in the virulence conferred by plasmid-encoded TA systems in bacterial pathogens.

The plant beneficial rhizobacterium Achromobacter sp. 5B1 influences root development through auxin signaling and redistribution.

Roots provide physical and nutritional support to plant organs that are above ground and play critical roles for adaptation via intricate movements and growth patterns. Through screening the effects of bacterial isolates from roots of halophyte Mesquite (Prosopis sp.) on Arabidopsis thaliana, we identified Achromobacter sp. 5B1 as a probiotic bacterium that influences plant functional traits. Detailed genetic and architectural analyses in Arabidopsis grown in vitro and in soil, cell division measurements, auxin transport and response gene expression and brefeldin A treatments demonstrated that root colonization with Achromobacter sp. 5B1 changes the growth and branching patterns of roots, which were related to auxin perception and redistribution. Expression analysis of auxin transport and signaling revealed a redistribution of auxin within the primary root tip of wild-type seedlings by Achromobacter sp. 5B1 that is disrupted by brefeldin A and correlates with repression of auxin transporters PIN1 and PIN7 in root provasculature, and PIN2 in the epidermis and cortex of the root tip, whereas expression of PIN3 was enhanced in the columella. In seedlings harboring AUX1, EIR1, AXR1, ARF7ARF19, TIR1AFB2AFB3 single, double or triple loss-of-function mutations, or in a dominant (gain-of-function) mutant of SLR1, the bacterium caused primary roots to form supercoils that are devoid of lateral roots. The changes in growth and root architecture elicited by the bacterium helped Arabidopsis seedlings to resist salt stress better. Thus, Achromobacter sp. 5B1 fine tunes both root movements and the auxin response, which may be important for plant growth and environmental adaptation.

Protective effect of supplementation with biotin against high-fructose-induced metabolic syndrome in rats.

Several reports have demonstrated that pharmacological concentrations of biotin reduce hyperglycemia, hypertriglyceridemia, and hypertension. We hypothesized that biotin could exert a protective effect on some illness-associated metabolic syndrome. To test this hypothesis, male Wistar rats were fed a diet containing 30% fructose in drinking water and classified into four groups: C, the control group; B, the group receiving biotin (intraperitoneal injection, 2 mg/kg); F, the group receiving fructose (30% w/v); and FB, the group receiving fructose-biotin. The administration of biotin began after the rats had been on a high-fructose diet for 12 weeks and continued for 4 weeks. Our results showed that food and fluid intake were diminished in the F and FB groups. However, the final body weights were similar between the groups. A significant increase in hepatic triglyceride and cholesterol content, plasma cholesterol, triglycerides, transaminases, low-density lipoprotein cholesterol (LDL-c), systolic blood pressure, and vasocontraction, as well as a decrease in high-density lipoprotein cholesterol (HDL-c) were observed in the F group. Glucose tolerance and insulin tolerance were also impaired in the F group. The administration of biotin ameliorated all these changes. Hepatic oxidative stress as well as macrovesicular fatty changes in hepatocytes caused by a high-fructose diet were also improved by biotin. Our findings demonstrate that biotin has a protective role against metabolic syndrome by improving insulin resistance associated with normal hepatic and serum levels of triglyceride and cholesterol, blood pressure, and the prevention of steatosis and hepatic oxidative damage. Therefore, biotin could be used as a therapeutic strategy in the pharmacological treatment of metabolic syndrome.