Dataset Information

Generation of A Mucor circinelloides Reporter Strain-A Promising New Tool to Study Antifungal Drug Efficacy and Mucormycosis.

ABSTRACT: Invasive fungal infections caused by Mucorales (mucormycosis) have increased worldwide. These life-threatening infections affect mainly, but not exclusively, immunocompromised patients, and are characterized by rapid progression, severe tissue damage and an unacceptably high rate of mortality. Still, little is known about this disease and its successful therapy. New tools to understand mucormycosis and a screening method for novel antimycotics are required. Bioluminescent imaging is a powerful tool for in vitro and in vivo approaches. Hence, the objective of this work was to generate and functionally analyze bioluminescent reporter strains of Mucor circinelloides, one mucormycosis-causing pathogen. Reporter strains were constructed by targeted integration of the firefly luciferase gene under control of the M. circinelloides promoter Pzrt1. The luciferase gene was sufficiently expressed, and light emission was detected under several conditions. Phenotypic characteristics, virulence potential and antifungal susceptibility were indifferent to the wild-type strains. Light intensity was dependent on growth conditions and biomass, being suitable to determine antifungal efficacy in vitro. This work describes for the first time the generation of reporter strains in a basal fungus that will allow real-time, non-invasive infection monitoring in insect and murine models, and the testing of antifungal efficacy by means other than survival.

SUBMITTER: Binder U

PROVIDER: S-EPMC6315630 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Generation of A Mucor circinelloides Reporter Strain-A Promising New Tool to Study Antifungal Drug Efficacy and Mucormycosis.

Binder Ulrike U Navarro-Mendoza Maria Isabel MI Naschberger Verena V Bauer Ingo I Nicolas Francisco E FE Pallua Johannes D JD Lass-Flörl Cornelia C Garre Victoriano V

Genes 20181207 12

Invasive fungal infections caused by Mucorales (mucormycosis) have increased worldwide. These life-threatening infections affect mainly, but not exclusively, immunocompromised patients, and are characterized by rapid progression, severe tissue damage and an unacceptably high rate of mortality. Still, little is known about this disease and its successful therapy. New tools to understand mucormycosis and a screening method for novel antimycotics are required. Bioluminescent imaging is a powerful t ...[more]

PMID: 30544643

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Project description:Mucorales are ubiquitous environmental molds responsible for mucormycosis in diabetic, immunocompromised, and severely burned patients. Small outbreaks of invasive wound mucormycosis (IWM) have already been reported in burn units without extensive microbiological investigations. We faced an outbreak of IWM in our center and investigated the clinical isolates with whole-genome sequencing (WGS) analysis. We analyzed M. circinelloides isolates from patients in our burn unit (BU1, Hôpital Saint-Louis, Paris, France) together with nonoutbreak isolates from Burn Unit 2 (BU2, Paris area) and from France over a 2-year period (2013 to 2015). A total of 21 isolates, including 14 isolates from six BU1 patients, were analyzed by whole-genome sequencing (WGS). Phylogenetic classification based on de novo assembly and assembly free approaches showed that the clinical isolates clustered in four highly divergent clades. Clade 1 contained at least one of the strains from the six epidemiologically linked BU1 patients. The clinical isolates were specific to each patient. Two patients were infected with more than two strains from different clades, suggesting that an environmental reservoir of clonally unrelated isolates was the source of contamination. Only two patients from BU1 shared one strain, which could correspond to direct transmission or contamination with the same environmental source. In conclusion, WGS of several isolates per patients coupled with precise epidemiological data revealed a complex situation combining potential cross-transmission between patients and multiple contaminations with a heterogeneous pool of strains from a cryptic environmental reservoir.IMPORTANCE Invasive wound mucormycosis (IWM) is a severe infection due to environmental molds belonging to the order Mucorales. Severely burned patients are particularly at risk for IWM. Here, we used whole-genome sequencing (WGS) analysis to resolve an outbreak of IWM due to Mucor circinelloides that occurred in our hospital (BU1). We sequenced 21 clinical isolates, including 14 from BU1 and 7 unrelated isolates, and compared them to the reference genome (1006PhL). This analysis revealed that the outbreak was mainly due to multiple strains that seemed patient specific, suggesting that the patients were more likely infected from a pool of diverse strains from the environment rather than from direct transmission among them. This study revealed the complexity of a Mucorales outbreak in the settings of IWM in burn patients, which has been highlighted based on WGS combined with careful sampling.

Project description:Mucor circinelloides is a zygomycete fungus and an emerging opportunistic pathogen in immunocompromised patients, especially transplant recipients and in some cases otherwise healthy individuals. We have discovered a novel example of size dimorphism linked to virulence. M. circinelloides is a heterothallic fungus: (+) sex allele encodes SexP and (-) sex allele SexM, both of which are HMG domain protein sex determinants. M. circinelloides f. lusitanicus (Mcl) (-) mating type isolates produce larger asexual sporangiospores that are more virulent in the wax moth host compared to (+) isolates that produce smaller less virulent sporangiospores. The larger sporangiospores germinate inside and lyse macrophages, whereas the smaller sporangiospores do not. sexM? mutants are sterile and still produce larger virulent sporangiospores, suggesting that either the sex locus is not involved in virulence/spore size or the sexP allele plays an inhibitory role. Phylogenetic analysis supports that at least three extant subspecies populate the M. circinelloides complex in nature: Mcl, M. circinelloides f. griseocyanus, and M. circinelloides f. circinelloides (Mcc). Mcc was found to be more prevalent among clinical Mucor isolates, and more virulent than Mcl in a diabetic murine model in contrast to the wax moth host. The M. circinelloides sex locus encodes an HMG domain protein (SexP for plus and SexM for minus mating types) flanked by genes encoding triose phosphate transporter (TPT) and RNA helicase homologs. The borders of the sex locus between the three subspecies differ: the Mcg sex locus includes the promoters of both the TPT and the RNA helicase genes, whereas the Mcl and Mcc sex locus includes only the TPT gene promoter. Mating between subspecies was restricted compared to mating within subspecies. These findings demonstrate that spore size dimorphism is linked to virulence of M. circinelloides species and that plasticity of the sex locus and adaptations in pathogenicity have occurred during speciation of the M. circinelloides complex.

Project description:Mucor circinelloides is a pathogenic fungus and etiologic agent of mucormycosis. In 2013, cases of gastrointestinal illness after yogurt consumption were reported to the US FDA, and the producer found that its products were contaminated with Mucor. A previous study found that the Mucor strain isolated from an open contaminated yogurt exhibited virulence in a murine systemic infection model and showed that this strain is capable of surviving passage through the gastrointestinal tract of mice. In this study, we isolated another Mucor strain from an unopened yogurt that is closely related but distinct from the first Mucor strain and subsequently examined if Mucor alters the gut microbiota in a murine host model. DNA extracted from a ten-day course of stool samples was used to analyze the microbiota in the gastrointestinal tracts of mice exposed via ingestion of Mucor spores. The bacterial 16S rRNA gene and fungal ITS1 sequences obtained were used to identify taxa of each kingdom. Linear regressions revealed that there are changes in bacterial and fungal abundance in the gastrointestinal tracts of mice which ingested Mucor. Furthermore, we found an increased abundance of the bacterial genus Bacteroides and a decreased abundance of the bacteria Akkermansia muciniphila in the gastrointestinal tracts of exposed mice. Measurements of abundances show shifts in relative levels of multiple bacterial and fungal taxa between mouse groups. These findings suggest that exposure of the gastrointestinal tract to Mucor can alter the microbiota and, more importantly, illustrate an interaction between the intestinal mycobiota and bacteriota. In addition, Mucor was able to induce increased permeability in epithelial cell monolayers in vitro, which might be indicative of unstable intestinal barriers. Understanding how the gut microbiota is shaped is important to understand the basis of potential methods of treatment for gastrointestinal illness. How the gut microbiota changes in response to exposure, even by pathogens not considered to be causative agents of food-borne illness, may be important to how commercial food producers prevent and respond to contamination of products aimed at the public. This study provides evidence that the fungal microbiota, though understudied, may play an important role in diseases of the human gut.

Dataset Information

Generation of A Mucor circinelloides Reporter Strain-A Promising New Tool to Study Antifungal Drug Efficacy and Mucormycosis.

Publications

Generation of A Mucor circinelloides Reporter Strain-A Promising New Tool to Study Antifungal Drug Efficacy and Mucormycosis.

Similar Datasets

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