- Proteomic analysis of Paracoccidioides brasiliensis complex isolates: Correlation of the levels of differentially expressed proteins with in vivo virulence. [Journal Article]
- PlosPLoS One 2019; 14(7):e0218013
- CONCLUSIONS: Our findings may point to the mechanisms used by highly virulent P. brasiliensis isolates to withstand host immune reactions and to adapt to transient iron availability as strategies to survive and overcome stress conditions inside the host.
- Synthetic and minimalist vectors for Agrobacterium tumefaciens-mediated transformation of fungi. [Journal Article]
- GMGenet Mol Biol 2019 Jun 13
- We present a collection of minimalist binary vectors for transformation through ATMT applicable to several fungi species. pLUO plasmid binary vectors consist of a reporter module containing fluoresce…
We present a collection of minimalist binary vectors for transformation through ATMT applicable to several fungi species. pLUO plasmid binary vectors consist of a reporter module containing fluorescent proteins, mCherry or eGFP, flanked by a multiple cloning site and a transcription terminator site. They also present a synthetic gene allowing resistance to Hygromicin B flanked by alternate promoters, one for yeast and another for filamentous fungi. Left and right borders were added for Agrobacterium tumefaciens recognition, and a minimal broad-host range RK2 replication origin. Transformation was validated in the pathogenic fungus Paracoccidioides lutzii. Hence, we developed an efficient and reliable molecular tool for fungal transformation: minimalist, synthetic, modular, and available in four different versions, and these can still be readily modified using a few primers and few cloning steps.
- Drug Repurposing for Paracoccidioidomycosis Through a Computational Chemogenomics Framework. [Journal Article]
- FMFront Microbiol 2019; 10:1301
- Paracoccidioidomycosis (PCM) is the most prevalent endemic mycosis in Latin America. The disease is caused by fungi of the genus Paracoccidioides and mainly affects low-income rural workers after inh…
Paracoccidioidomycosis (PCM) is the most prevalent endemic mycosis in Latin America. The disease is caused by fungi of the genus Paracoccidioides and mainly affects low-income rural workers after inhalation of fungal conidia suspended in the air. The current arsenal of chemotherapeutic agents requires long-term administration protocols. In addition, chemotherapy is related to a significantly increased frequency of disease relapse, high toxicity, and incomplete elimination of the fungus. Due to the limitations of current anti-PCM drugs, we developed a computational drug repurposing-chemogenomics approach to identify approved drugs or drug candidates in clinical trials with anti-PCM activity. In contrast to the one-drug-one-target paradigm, our chemogenomics approach attempts to predict interactions between drugs, and Paracoccidioides protein targets. To achieve this goal, we designed a workflow with the following steps: (a) compilation and preparation of Paracoccidioides spp. genome data; (b) identification of orthologous proteins among the isolates; (c) identification of homologous proteins in publicly available drug-target databases; (d) selection of Paracoccidioides essential targets using validated genes from Saccharomyces cerevisiae; (e) homology modeling and molecular docking studies; and (f) experimental validation of selected candidates. We prioritized 14 compounds. Two antineoplastic drug candidates (vistusertib and BGT-226) predicted to be inhibitors of phosphatidylinositol 3-kinase TOR2 showed antifungal activity at low micromolar concentrations (<10 μM). Four antifungal azole drugs (bifonazole, luliconazole, butoconazole, and sertaconazole) showed antifungal activity at low nanomolar concentrations, validating our methodology. The results suggest our strategy for predicting new anti-PCM drugs is useful. Finally, we could recommend hit-to-lead optimization studies to improve potency and selectivity, as well as pharmaceutical formulations to improve oral bioavailability of the antifungal azoles identified.
- Comparative Innate and Adaptive Immune Responses in Atlantic Bottlenose Dolphins (Tursiops truncatus) With Viral, Bacterial, and Fungal Infections. [Journal Article]
- FIFront Immunol 2019; 10:1125
- Free-ranging Atlantic bottlenose dolphins (n = 360) from two southeastern U.S. estuarine sites were given comprehensive health examinations between 2003 and 2015 as part of a multi-disciplinary resea…
Free-ranging Atlantic bottlenose dolphins (n = 360) from two southeastern U.S. estuarine sites were given comprehensive health examinations between 2003 and 2015 as part of a multi-disciplinary research project focused on individual and population health. The study sites (and sample sizes) included the Indian River Lagoon (IRL), Florida, USA (n = 246) and Charleston harbor and associated rivers (CHS), South Carolina, USA (n = 114). Results of a suite of clinicoimmunopathologic tests revealed that both populations have a high prevalence of infectious and neoplastic disease and a variety of abnormalities of their innate and adaptive immune systems. Subclinical infections with cetacean morbillivirus and Chlamydiaceae were detected serologically. Clinical evidence of orogenital papillomatosis was supported by the detection of a new strain of dolphin papillomavirus and herpesvirus by molecular pathology. Dolphins with cutaneous lobomycosis/lacaziasis were subsequently shown to be infected with a novel, uncultivated strain of Paracoccidioides brasiliensis, now established as the etiologic agent of this enigmatic disease in dolphins. In this review, innate and adaptive immunologic responses are compared between healthy dolphins and those with clinical and/or immunopathologic evidence of infection with these specific viral, bacterial, and fungal pathogens. A wide range of immunologic host responses was associated with each pathogen, reflecting the dynamic and complex interplay between the innate, humoral, and cell-mediated immune systems in the dolphin. Collectively, these studies document the comparative innate and adaptive immune responses to various types of infectious diseases in free-ranging Atlantic bottlenose dolphins. Evaluation of the type, pattern, and degree of immunologic response to these pathogens provides novel insight on disease immunopathogenesis in this species and as a comparative model. Importantly, the data suggest that in some cases infection may be associated with subclinical immunopathologic perturbations that could impact overall individual and population health.
- Immunohistochemical Cross-Reactivity Between Arthrographis kalrae and Highly Pathogenic Coccidioides posadasii, Histoplasma capsulatum, and Paracoccidioides Fungal Species. [Journal Article]
- MMycopathologia 2019; 184(3):393-402
- Recently, we have reported serological cross-reactivity between paracoccidioidomycosis ceti and paracoccidioidomycosis, histoplasmosis, and coccidioidomycosis. However, data on the interaction of Art…
Recently, we have reported serological cross-reactivity between paracoccidioidomycosis ceti and paracoccidioidomycosis, histoplasmosis, and coccidioidomycosis. However, data on the interaction of Arthrographis kalrae with the above pathogenic fungal infections are lacking. A. kalrae is a widely occurring ascomycetous fungus; causes superficial and deep mycoses; shows thermally dependent dimorphism; and has a genomic profile related to the above-mentioned fungal species. Our study aims to investigate cross-reactivity using eight murine sera, obtained from experimental infection with two A. kalrae isolates. The murine sera were incubated with fungal cells of A. kalrae, Coccidioides posadasii, Histoplasma capsulatum, Paracoccidioides sp., and P. brasiliensis. Thirty murine sera, obtained from experimental infection with six isolates of H. capsulatum, sera from three cases of dolphin paracoccidioidomycosis ceti, two human sera from patients with paracoccidioidomycosis, and a serum sample from a healthy person with a history of coccidioidomycosis, were also incubated with A. kalrae fungal cells and the respective fungal cells that caused the infection as positive controls. Sera derived from the mice infected with A. kalrae reacted strongly when incubated with the Paracoccidioides sp., P. brasiliensis, and C. posadasii, but no positive reaction was observed against the fungal cells of H. capsulatum. The murine sera infected with three out of six isolates of H. capsulatum, and all cetacean and human serum samples reacted positively with the fungal cells of A. kalrae. The present study demonstrated serological cross-reactions among A. kalrae infection, coccidioidomycosis, paracoccidioidomycosis, paracoccidioidomycosis ceti, and histoplasmosis.
- Unravelling the interactions of the environmental host Acanthamoeba castellanii with fungi through the recognition by mannose-binding proteins. [Journal Article]
- CMCell Microbiol 2019 Jun 07; :e13066
- Free-living amoebae (FLAs) are major reservoirs for a variety of bacteria, viruses, and fungi. The most studied mycophagic FLA, Acanthamoeba castellanii (Ac), is a potential environmental host for en…
Free-living amoebae (FLAs) are major reservoirs for a variety of bacteria, viruses, and fungi. The most studied mycophagic FLA, Acanthamoeba castellanii (Ac), is a potential environmental host for endemic fungal pathogens such as Cryptococcus spp., Histoplasma capsulatum, Blastomyces dermatitides, and Sporothrix schenckii. However, the mechanisms involved in this interaction are poorly understood. The aim of this work was to characterize the molecular instances that enable Ac to interact with and ingest fungal pathogens, a process that could lead to selection and maintenance of possible virulence factors. The interaction of Ac with a variety of fungal pathogens was analysed in a multifactorial evaluation that included the role of multiplicity of infection over time. Fungal binding to Ac surface by living image consisted of a quick process, and fungal initial extrusion (vomocytosis) was detected from 15 to 80 min depending on the organism. When these fungi were cocultured with the amoeba, only Candida albicans and Cryptococcus neoformans were able to grow, whereas Paracoccidioides brasiliensis and Sporothrix brasiliensis displayed unchanged viability. Yeasts of H. capsulatum and Saccharomyces cerevisiae were rapidly killed by Ac; however, some cells remained viable after 48 hr. To evaluate changes in fungal virulence upon cocultivation with Ac, recovered yeasts were used to infect Galleria mellonella, and in all instances, they killed the larvae faster than control yeasts. Surface biotinylated extracts of Ac exhibited intense fungal binding by FACS and fluorescence microscopy. Binding was also intense to mannose, and mass spectrometry identified Ac proteins with affinity to fungal surfaces including two putative transmembrane mannose-binding proteins (MBP, L8WXW7 and MBP1, Q6J288). Consistent with interactions with such mannose-binding proteins, Ac-fungi interactions were inhibited by mannose. These MBPs may be involved in fungal recognition by amoeba and promotes interactions that allow the emergence and maintenance of fungal virulence for animals.
- Clinical and epidemiological features of paracoccidioidomycosis due to Paracoccidioides lutzii. [Journal Article]
- PNPLoS Negl Trop Dis 2019; 13(6):e0007437
- CONCLUSIONS: Compared with the classical clinical-epidemiological profile of PCM caused by P. brasiliensis, the results of this descriptive study did not show significant clinical or epidemiological differences that could be attributed to the species P. lutzii. Future studies may confirm or refute the existence of clinical differences between the two fungal species.
- Antifungal activity of two oxadiazole compounds for the paracoccidioidomycosis treatment. [Journal Article]
- PNPLoS Negl Trop Dis 2019; 13(6):e0007441
- Paracoccidioidomycosis (PCM) is a neglected disease present in Latin America with difficulty in treatment and occurrence of serious sequelae. Thus, the development of alternative therapies is imperat…
Paracoccidioidomycosis (PCM) is a neglected disease present in Latin America with difficulty in treatment and occurrence of serious sequelae. Thus, the development of alternative therapies is imperative. In the current work, two oxadiazole compounds (LMM5 and LMM11) presented fungicidal activity against Paracoccidioides spp. The minimum inhibitory and fungicidal concentration values ranged from 1 to 32 μg/mL, and a synergic effect was observed for both compounds when combined with Amphotericin B. LMM5 and LMM11 were able to reduce CFU counts (≥2 log10) on the 5th and 7th days of time-kill curve, respectively. The fungicide effect was confirmed by fluorescence microscopy (FUN-1/FUN-2). The hippocratic screening and biochemical analysis were performed in Balb/c male mice that received a high dose of each compound, and the compounds showed no in vivo toxicity. The treatment of experimental PCM with the new oxadiazoles led to significant reduction in CFU (≥1 log10). Histopathological analysis of the groups treated exhibited control of inflammation, as well as preserved lung areas. These findings suggest that LMM5 and LMM11 are promising hits structures, opening the door for implementing new PCM therapies.
- Novel 4-methoxynaphthalene-N-acylhydrazones as potential for paracoccidioidomycosis and tuberculosis co-infection. [Journal Article]
- FMFuture Microbiol 2019; 14:587-598
- CONCLUSIONS: We highlight the compound 4k, as a potential agent for the treatment of patients co-infected with paracoccidioidomycosis and tuberculosis.
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- Biliary Paracoccidioidomycosis: An Unusual Infection Simulating Malignant Neoplasm. [Journal Article]
- AHAnn Hepatol 2019 Jan - Feb; 18(1):258-262
- Paracoccidioidomycosis is a systemic granulomatous disease caused by the dimorphic fungus Paracoccidioides brasiliensis and is restricted to Latin America. It normally affects lungs, skin and lymph n…
Paracoccidioidomycosis is a systemic granulomatous disease caused by the dimorphic fungus Paracoccidioides brasiliensis and is restricted to Latin America. It normally affects lungs, skin and lymph nodes. Abdominal organs are usually not involved. In rare cases paracoccidioidomycosis may simulate neoplasm. Herein we describe our experience with four cases of paracoccidioidomycosis mimicking cholangiocarcinoma. To the best of our knowledge, this is the largest case series on this subject produced in English. Paracoccidioidomycosis must be considered as a differential diagnosis of cholangiocarcinoma, especially in individuals who come from endemic areas.