Two Novel Dimorphism-Related Virulence Factors of Zymoseptoria tritici Identified Using Agrobacterium-Mediated Insertional Mutagenesis

Diseases caused by dimorphic phytopathogenic and systemic dimorphic fungi have markedly increased in prevalence in the last decades, and understanding the morphogenic transition to the virulent state might yield novel means of controlling dimorphic fungi. The dimorphic fungus Z. tritici causes significant economic impact on wheat production, and yet the regulation of the dimorphic switch, a key first step in successful plant colonization, is still largely unexplored in this fungus. The fungus is amenable to suppression by fungicides at this switch point, and the identification of the factors controlling the dimorphic switch provides a potential source of novel targets to control Septoria tritici blotch (STB). Inhibition of the dimorphic switch can potentially prevent penetration and avoid any damage to the host plant. The aim of the current work was to unveil genetic determinants of the dimorphic transition in Z. tritici by using a forward genetics strategy. Using this approach, we unveiled two novel factors involved in the switch to the pathogenic state and used reverse genetics and complementation to confirm the role of the novel virulence factors and further gained insight into the role of these genes, using transcriptome analysis via RNA-Seq. The transcriptomes generated potentially contain key determinants of the dimorphic transition.

Farnesol secretion as a possible driving force for maintaining Candida albicans as a diploid

Candida albicans is a pathogenic dimorphic fungus which is invariably found as a diploid in patients. C. albicans secretes the sesquiterpene farnesol both as a quorum sensing molecule which blocks the yeast to hypha conversion and as a virulence factor for pathogenicity. 20-25 μM farnesol kills other competing yeasts and fungi, often by triggering apoptosis, and yet wild type diploid C. albicans tolerates 300-500 μM farnesol. The recent availability of 10 haploid strains of C. albicans (5 mating type aand 5 mating type α) allowed us to compare their production of and sensitivity to farnesol. On average, the heterozygous diploid strains of C. albicans were 2.4 times more resistant to 20-40 μM farnesol than MTLa haploid cells and 4.6 times more resistant than MTLα haploid cells. Furthermore, the MTLa haploids produce approximately 10 times more farnesol than do the MTLα haploids. Prior work concluded that haploid strains exhibited such low fitness that C. albicans was thought to be an obligate diploid. We now suggest that increased farnesol secretion by the MTLa haploids and increased farnesol sensitivity of the MTLα haploids is a mechanism for maintaining the dominant heterozygous diploid status of C. albicans. This idea is based on the observation that the a-factor peptide pheromone is farnesylated but the α-factor pheromone is not farnesylated. Our working hypothesis is that farnesol is secreted in part via Ste6 and imported in part via Ste3, the proteins which export and import the farnesylated a-pheromone. We also examined whether farnesol was excreted in extracellular vesicles.

Brain paracoccidioidomycosis in an immunosuppressed patient with systemic lupus erythematosus

Background: Brain paracoccidioidomycosis (PCM) or neuroparacoccidioidomycosis (NPCM) is a fungal infection of the central nervous system (CNS) caused by Paracoccidioides brasiliensis, a dimorphic fungus. The CNS involvement is through bloodstream dissemination. The association between NPCM and systemic lupus erythematous (SLE) is rare. However, SLE patients are under risk of opportunistic infections given their immunosuppression status. Case Description: The aim of this case report is to present a 37-year-old female with diagnosis of SLE who presented with progressive and persistent headache in the past 4 months accompanied by the right arm weakness with general and neurologic examination unremarkable. The computerized tomography of the head showed left extra-axial parietooccipital focal hypoattenuation with adjacent bone erosion. The brain magnetic resonance imaging reported left parietooccipital subdural collection associated with focal leptomeningeal thickening with restriction to diffusion and peripheral contrast enhancement. The patient underwent a left craniotomy and dura mater biopsy showed noncaseous granulomatosis with multinucleated giant cells with rounded birefringent structures positive for silver stain, consistent with PCM. Management with itraconazole 200 mg daily was started with a total of 12 months of treatment, with patient presenting resolution of headache and right arm weakness. Conclusion: The diagnosis of NPCM is challenging and a high degree of suspicious should be considered in patients with persistent headache and immunosuppression.

Sporotrichosis in a U.S. Army Basic Trainee

ABSTRACT Lymphocutaneous sporotrichosis is a mycotic disease caused by traumatic inoculation of the dimorphic fungus Sporothrix schenckii, found ubiquitously in decaying vegetation. The potential for outbreaks, difficulty with timely diagnosis, as well as the prolonged treatment course and recovery from surgical intervention can have a substantial impact on the medical readiness of service members. Treatment with the antifungal therapy of choice, itraconazole, requires close patient monitoring for the duration of treatment for clinical response, drug tolerance, as well as therapeutic drug levels. We present a case of a U.S. Army enlistee with sporotrichosis contracted during basic training, highlighting the impact of delayed diagnosis and prolonged treatment course and recovery on medical readiness.

Genetic Modification of Mucor circinelloides for Canthaxanthin Production by Heterologous Expression of β-carotene Ketolase Gene

Canthaxanthin is a reddish-orange xanthophyll with strong antioxidant activity and higher bioavailability than carotenes, primarily used in food, cosmetics, aquaculture, and pharmaceutical industries. The spiking market for natural canthaxanthin promoted researchers toward genetic engineering of heterologous hosts for canthaxanthin production. Mucor circinelloides is a dimorphic fungus that produces β-carotene as the major carotenoid and is considered as a model organism for carotenogenic studies. In this study, canthaxanthin-producing M. circinelloides strain was developed by integrating the codon-optimized β-carotene ketolase gene (bkt) of the Haematococcus pluvialis into the genome of the fungus under the control of strong promoter zrt1. First, a basic plasmid was constructed to disrupt crgA gene, a negative regulator of carotene biosynthesis resulted in substantial β-carotene production, which served as the building block for canthaxanthin by further enzymatic reaction of the ketolase enzyme. The genetically engineered strain produced a significant amount (576 ± 28 μg/g) of canthaxanthin, which is the highest amount reported in Mucor to date. Moreover, the cell dry weight of the recombinant strain was also determined, producing up to more than 9.0 g/L, after 96 h. The mRNA expression level of bkt in the overexpressing strain was analyzed by RT-qPCR, which increased by 5.3-, 4.1-, and 3-folds at 24, 48, and 72 h, respectively, compared with the control strain. The canthaxanthin-producing M. circinelloides strain obtained in this study provided a basis for further improving the biotechnological production of canthaxanthin and suggested a useful approach for the construction of more valuable carotenoids, such as astaxanthin.

Drk1, a Dimorphism Histidine Kinase, Contributes to Morphology, Virulence, and Stress Adaptation in Paracoccidioides brasiliensis

P. brasiliensis is a thermally dimorphic fungus belonging to Paracoccidioides complex, causative of a systemic, endemic mycosis limited to Latin American countries. Signal transduction pathways related to important aspects as surviving, proliferation according to the biological niches are linked to the fungal pathogenicity in many species, but its elucidation in P. brasiliensis remains poorly explored. As Drk1, a hybrid histidine kinase, plays regulators functions in other dimorphic fungi species, mainly in dimorphism and virulence, here we investigated its importance in P. brasilensis. We, therefore generated the respective recombinant protein, anti-PbDrk1 polyclonal antibody and a silenced strain. The Drk1 protein shows a random distribution including cell wall location that change its pattern during osmotic stress condition; moreover the P. brasiliensis treatment with anti-PbDrk1 antibody, which does not modify the fungus’s viability, resulted in decreased virulence in G. mellonella model and reduced interaction with pneumocytes. Down-regulating PbDRK1 yielded phenotypic alterations such as yeast cells with more elongated morphology, virulence attenuation in G. mellonella infection model, lower amount of chitin content, increased resistance to osmotic and cell wall stresses, and also caspofungin, and finally increased sensitivity to itraconazole. These observations highlight the importance of PbDrk1 to P. brasiliensis virulence, stress adaptation, morphology, and cell wall organization, and therefore it an interesting target that could help develop new antifungals.

Pulmonary histoplasmosis: A case report

Histoplasmosis is a systemic fungal infection caused by dimorphic fungus Histoplasma capsulatum. It is widely distributed throughout the world. In present study, the case of a 50-year-old male patient diagnosed as pulmonary histoplasmosis. Initially, the patient was suspected as a case of alcoholic hepatitis but subsequently the diagnosis of pulmonary histoplasmosis was confirmed by lung biopsy. The symptoms were dissuaded following amphotericin B and itraconazole treatment.

Cat1 Regulate ROS Metabolism and Microsclerotia Formation in the Dimorphic Fungus Nomuraea Rileyi

Catalases are the most important enzymes in the metabolism of reactive oxygen species (ROS), as they convert hydrogen peroxide (H2O2) into water and molecular oxygen. They are also involved in virulence, and in oxidative, heat, hyperosmotic stress and UV-B radiation responses in some entomopathogenic fungi. In this study, the Cat1 gene from Nomuraea rileyi was cloned and its function was studied by gene deletion. The NrCat1 deletion mutant (ΔNrCat1) was generated using the split-marker method. No significant differences in colony growth or dimorphic switching of ΔNrCat1 were observed under regular culture conditions, whereas oxidative stress inhibited colony growth and the yeast-hyphal transition. In contrast, there was no significant difference in tolerance to hyperosmotic stress between ΔNrCat1 and wild type (WT) strains. In the ΔNrCat1 strain, microsclerotia (MS) formation time of the ΔNrCat1 was delayed, and MS size was less uniform than in the WT. MS yield was decreased by 76% in the ΔNrCat1 strain compared to the WT strain. Furthermore, virulence was attenuated in the ΔNrCat1 strain. Gene expression analysis showed that NrCat2, NrCat4, and NrAox are up-regulated to compensate for NrCat1 deletion. Thus, the NrCat1 gene in N. rileyi appears to be involved in essential functions, including H2O2 metabolism, MS formation, and virulence.

Disseminated sporotrichosis in a person with human immunodeficiency virus disease

Introduction. Disseminated sporotrichosis is an incapacitating infection caused by the dimorphic fungus Sporothrix schenckii. Because this condition may mimic the presentation of tuberculosis, syphilis and other bacterial infections, the diagnosis may be missed or delayed. Case Presentation. We describe a case of disseminated sporotrichosis in a patient with poorly controlled human immunodeficiency virus infection. The patient was initially treated for bacterial skin infections. The differential diagnosis also included tuberculosis and syphilis. Only after appropriate specimens had been sent for microbiological and histopathological investigations was the diagnosis of disseminated sporotrichosis made and appropriate treatment started. The patient showed a good clinical response to itraconazole. Conclusion. This report highlights the importance of having a high index of suspicion of endemic mycoses when managing immunocompromised patients. The report also demonstrates that a delay in the diagnosis of sporotrichosis increases morbidity and results in unnecessary and inappropriate treatment with associated costs and adverse effects.