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12 pages, 1218 KiB

Open AccessArticle

Uncovering a Novel cyp51A Mutation and Antifungal Resistance in Aspergillus fumigatus through Culture Collection Screening

by Laís Pontes, Teppei Arai, Caio Augusto Gualtieri Beraquet, Ana Luisa Perini Leme Giordano, Franqueline Reichert-Lima, Edson Aparecido da Luz, Camila Fernanda de Sá, Larissa Ortolan Levy, Cibele Aparecida Tararam, Akira Watanabe, Maria Luiza Moretti and Angélica Zaninelli Schreiber

J. Fungi 2024, 10(2), 122; https://doi.org/10.3390/jof10020122 - 1 Feb 2024

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Abstract

Background: Aspergillus fumigatus is an important concern for immunocompromised individuals, often resulting in severe infections. With the emergence of resistance to azoles, which has been the therapeutic choice for Aspergillus infections, monitoring the resistance of these microorganisms becomes important, including the search for [...] Read more.

Background: Aspergillus fumigatus is an important concern for immunocompromised individuals, often resulting in severe infections. With the emergence of resistance to azoles, which has been the therapeutic choice for Aspergillus infections, monitoring the resistance of these microorganisms becomes important, including the search for mutations in the cyp51A gene, which is the gene responsible for the mechanism of action of azoles. We conducted a retrospective analysis covering 478 A. fumigatus isolates. Methods: This comprehensive dataset comprised 415 clinical isolates and 63 isolates from hospital environmental sources. For clinical isolates, they were evaluated in two different periods, from 1998 to 2004 and 2014 to 2021; for environmental strains, one strain was isolated in 1998, and 62 isolates were evaluated in 2015. Our primary objectives were to assess the epidemiological antifungal susceptibility profile; trace the evolution of resistance to azoles, Amphotericin B (AMB), and echinocandins; and monitor cyp51A mutations in resistant strains. We utilized the broth microdilution assay for susceptibility testing, coupled with cyp51A gene sequencing and microsatellite genotyping to evaluate genetic variability among resistant strains. Results: Our findings reveal a progressive increase in Minimum Inhibitory Concentrations (MICs) for azoles and AMB over time. Notably, a discernible trend in cyp51A gene mutations emerged in clinical isolates starting in 2014. Moreover, our study marks a significant discovery as we detected, for the first time, an A. fumigatus isolate carrying the recently identified TR46/F495I mutation within a sample obtained from a hospital environment. The observed cyp51A mutations underscore the ongoing necessity for surveillance, particularly as MICs for various antifungal classes continue to rise. Conclusions: By conducting resistance surveillance within our institution’s culture collection, we successfully identified a novel TR46/F495I mutation in an isolate retrieved from the hospital environment which had been preserved since 1998. Moreover, clinical isolates were found to exhibit TR34/L98H/S297T/F495I mutations. In addition, we observed an increase in MIC patterns for Amphotericin B and azoles, signaling a change in the resistance pattern, emphasizing the urgent need for the development of new antifungal drugs. Our study highlights the importance of continued monitoring and research in understanding the evolving challenges in managing A. fumigatus infections. Full article

(This article belongs to the Special Issue Young Investigators of Human Pathogenic Fungi)

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17 pages, 2425 KiB

Open AccessArticle

Zinc Starvation Induces Cell Wall Remodeling and Activates the Antioxidant Defense System in Fonsecaea pedrosoi

by Tayná Aparecida de Oliveira Santos, Lucas Weba Soares, Lucas Nojosa Oliveira, Dayane Moraes, Millena Silva Mendes, Célia Maria de Almeida Soares, Alexandre Melo Bailão and Mirelle Garcia Silva Bailão

J. Fungi 2024, 10(2), 118; https://doi.org/10.3390/jof10020118 - 31 Jan 2024

Viewed by 1046

Abstract

The survival of pathogenic fungi in the host after invasion depends on their ability to obtain nutrients, which include the transition metal zinc. This essential micronutrient is required to maintain the structure and function of various proteins and, therefore, plays a critical role [...] Read more.

The survival of pathogenic fungi in the host after invasion depends on their ability to obtain nutrients, which include the transition metal zinc. This essential micronutrient is required to maintain the structure and function of various proteins and, therefore, plays a critical role in various biological processes. The host’s nutritional immunity limits the availability of zinc to pathogenic fungi mainly by the action of calprotectin, a component of neutrophil extracellular traps. Here we investigated the adaptive responses of Fonsecaea pedrosoi to zinc-limiting conditions. This black fungus is the main etiological agent of chromoblastomycosis, a chronic neglected tropical disease that affects subcutaneous tissues. Following exposure to a zinc-limited environment, F. pedrosoi induces a high-affinity zinc uptake machinery, composed of zinc transporters and the zincophore Pra1. A proteomic approach was used to define proteins regulated by zinc deprivation. Cell wall remodeling, changes in neutral lipids homeostasis, and activation of the antioxidant system were the main strategies for survival in the hostile environment. Furthermore, the downregulation of enzymes required for sulfate assimilation was evident. Together, the adaptive responses allow fungal growth and development and reveals molecules that may be related to fungal persistence in the host. Full article

(This article belongs to the Special Issue Young Investigators of Human Pathogenic Fungi)

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18 pages, 5121 KiB

Open AccessArticle

Molecular Study of Pneumocystis jirovecii in Respiratory Samples of HIV Patients in Chile

by Isabel Iturrieta-González, Carolina Chahin, Johanna Cabrera, Carla Concha, Pamela Olivares-Ferretti, Javier Briones, Fernando Vega, Luis Bustos-Medina and Flery Fonseca-Salamanca

J. Fungi 2024, 10(2), 117; https://doi.org/10.3390/jof10020117 - 31 Jan 2024

Viewed by 1157

Abstract

Pneumocystis is an opportunistic fungus that causes potentially fatal pneumonia (PCP) in immunocompromised patients. The objective of this study was to determine the prevalence of P. jirovecii in HIV patients through phenotypic and molecular study, to investigate the genetic polymorphisms of P. jirovecii [...] Read more.

Pneumocystis is an opportunistic fungus that causes potentially fatal pneumonia (PCP) in immunocompromised patients. The objective of this study was to determine the prevalence of P. jirovecii in HIV patients through phenotypic and molecular study, to investigate the genetic polymorphisms of P. jirovecii at the mitochondrial gene mtLSU and at the nuclear dihydropteroate synthase gene (DHPS), and by analysis of molecular docking to study the effect of DHPS mutations on the enzymatic affinity for sulfamethoxazole. A PCP prevalence of 28.3% was detected, with mtLSU rRNA genotypes 3 (33.3%) and 2 (26.6%) being the most common. A prevalence of 6.7% (1/15) mutations in the DHPS gene was detected, specifically at codon 55 of the amino acid sequence of dihydropteroate synthase. Molecular docking analysis showed that the combination of mutations at 55 and 98 codons is required to significantly reduce the affinity of the enzyme for sulfamethoxazole. We observed a low rate of mutations in the DHPS gene, and molecular docking analysis showed that at least two mutations in the DHPS gene are required to significantly reduce the affinity of dihydropteroate synthase for sulfamethoxazole. Full article

(This article belongs to the Special Issue Young Investigators of Human Pathogenic Fungi)

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22 pages, 6008 KiB

Open AccessArticle

Proteomics of Paracoccidioides lutzii: Overview of Changes Triggered by Nitrogen Catabolite Repression

by Vanessa Rafaela Milhomem Cruz-Leite, André Luís Elias Moreira, Lana O’Hara Souza Silva, Moises Morais Inácio, Juliana Alves Parente-Rocha, Orville Hernandez Ruiz, Simone Schneider Weber, Célia Maria de Almeida Soares and Clayton Luiz Borges

J. Fungi 2023, 9(11), 1102; https://doi.org/10.3390/jof9111102 - 12 Nov 2023

Viewed by 1154

Abstract

Members of the Paracoccidioides complex are the causative agents of Paracoccidioidomycosis (PCM), a human systemic mycosis endemic in Latin America. Upon initial contact with the host, the pathogen needs to uptake micronutrients. Nitrogen is an essential source for biosynthetic pathways. Adaptation to nutritional [...] Read more.

Members of the Paracoccidioides complex are the causative agents of Paracoccidioidomycosis (PCM), a human systemic mycosis endemic in Latin America. Upon initial contact with the host, the pathogen needs to uptake micronutrients. Nitrogen is an essential source for biosynthetic pathways. Adaptation to nutritional stress is a key feature of fungi in host tissues. Fungi utilize nitrogen sources through Nitrogen Catabolite Repression (NCR). NCR ensures the scavenging, uptake and catabolism of alternative nitrogen sources, when preferential ones, such as glutamine or ammonium, are unavailable. The NanoUPLC-MS^E proteomic approach was used to investigate the NCR response of Paracoccidioides lutzii after growth on proline or glutamine as a nitrogen source. A total of 338 differentially expressed proteins were identified. P. lutzii demonstrated that gluconeogenesis, β-oxidation, glyoxylate cycle, adhesin-like proteins, stress response and cell wall remodeling were triggered in NCR-proline conditions. In addition, within macrophages, yeast cells trained under NCR-proline conditions showed an increased ability to survive. In general, this study allows a comprehensive understanding of the NCR response employed by the fungus to overcome nutritional starvation, which in the human host is represented by nutritional immunity. In turn, the pathogen requires rapid adaptation to the changing microenvironment induced by macrophages to achieve successful infection. Full article

(This article belongs to the Special Issue Young Investigators of Human Pathogenic Fungi)

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15 pages, 3037 KiB

Open AccessArticle

Paracoccidioides brasiliensis Induces α3 Integrin Lysosomal Degradation in Lung Epithelial Cells

by Bruna Rocha Almeida, Bianca Carla Silva Campitelli Barros, Debora Tereza Lucas Barros, Cristina Mary Orikaza and Erika Suzuki

J. Fungi 2023, 9(9), 912; https://doi.org/10.3390/jof9090912 - 8 Sep 2023

Viewed by 913

Abstract

Studies on the pathogen–host interaction are crucial for the understanding of the mechanisms involved in the establishment, maintenance, and spread of infection. In recent years, our research group has observed that the P. brasiliensis species interact with integrin family receptors and increase the [...] Read more.

Studies on the pathogen–host interaction are crucial for the understanding of the mechanisms involved in the establishment, maintenance, and spread of infection. In recent years, our research group has observed that the P. brasiliensis species interact with integrin family receptors and increase the expression of α3 integrin in lung epithelial cells within 5 h of infection. Interestingly, α3 integrin levels were reduced by approximately 99% after 24 h of infection with P. brasiliensis compared to non-infected cells. In this work, we show that, during infection with this fungus, α3 integrin is increased in the late endosomes of A549 lung epithelial cells. We also observed that the inhibitor of the lysosomal activity bafilomycin A1 was able to inhibit the decrease in α3 integrin levels. In addition, the silencing of the charged multivesicular body protein 3 (CHMP3) inhibited the reduction in α3 integrin levels induced by P. brasiliensis in A549 cells. Thus, together, these results indicate that this fungus induces the degradation of α3 integrin in A549 lung epithelial cells by hijacking the host cell endolysosomal pathway. Full article

(This article belongs to the Special Issue Young Investigators of Human Pathogenic Fungi)

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13 pages, 1839 KiB

Open AccessArticle

Proteomic Profiling of Paracoccidioides brasiliensis in Response to Phenacylideneoxindol Derivative: Unveiling Molecular Targets and Pathways

by Lívia do Carmo Silva, Olivia Basso Rocha, Igor Godinho Portis, Thaynara Gonzaga Santos, Kleber Santiago Freitas e Silva, Raimundo Francisco dos Santos Filho, Silvio Cunha, Antônio Alonso, Célia Maria de Almeida Soares and Maristela Pereira

J. Fungi 2023, 9(8), 854; https://doi.org/10.3390/jof9080854 - 16 Aug 2023

Viewed by 814

Abstract

Background: The treatment of paracoccidioidomycosis (PCM) is a challenge, and the discovery of new antifungal compounds is crucial. The phenacylideneoxindoles exhibited promising antifungal activity against Paracoccidioides spp., but their mode of action remains unknown. Methods: Through proteomic analysis, we investigated the effects of [...] Read more.

Background: The treatment of paracoccidioidomycosis (PCM) is a challenge, and the discovery of new antifungal compounds is crucial. The phenacylideneoxindoles exhibited promising antifungal activity against Paracoccidioides spp., but their mode of action remains unknown. Methods: Through proteomic analysis, we investigated the effects of (E)-3-(2-oxo-2-phenylethylidene)indolin-2-one on P. brasiliensis. In addition, we investigated the metabolic alterations of P. brasiliensis in response to the compound. Furthermore, the effects of the compound on the membrane, ethanol production, and reactive oxygen species (ROS) production were verified. Results: We identified differentially regulated proteins that revealed significant metabolic reorganization, including an increase in ethanol production, suggesting the activation of alcoholic fermentation and alterations in the rigidity of fungal cell membrane with an increase of the ergosterol content and formation of ROS. Conclusions: These findings enhance our understanding of the mode of action and response of P. brasiliensis to the investigated promising antifungal compound, emphasizing its potential as a candidate for the treatment of PCM. Full article

(This article belongs to the Special Issue Young Investigators of Human Pathogenic Fungi)

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14 pages, 1286 KiB

Open AccessArticle

Candida palmioleophila: A New Emerging Threat in Brazil?

by Gisela Lara da Costa, Melyssa Negri, Rodrigo Prado Rodrigues de Miranda, Danielly Corrêa-Moreira, Tatiana Castro Abreu Pinto, Livia de Souza Ramos, Deisiany Gomes Ferreira, Bruna Salomão, Tulio Machado Fumian, Camille Ferreira Mannarino, Tatiana Prado, Marise Pereira Miagostovich, André Luis Souza dos Santos and Manoel Marques Evangelista Oliveira

J. Fungi 2023, 9(7), 770; https://doi.org/10.3390/jof9070770 - 21 Jul 2023

Cited by 1 | Viewed by 1462

Abstract

Human activity directly or indirectly causes climate change, promoting changes in the composition of the atmosphere. This change is beyond the variation of the natural climate. In this manner, climate change could create an environmental pressure which is enough to trigger new fungal [...] Read more.

Human activity directly or indirectly causes climate change, promoting changes in the composition of the atmosphere. This change is beyond the variation of the natural climate. In this manner, climate change could create an environmental pressure which is enough to trigger new fungal diseases. In addition to climate alterations, the onset of the COVID-19 pandemic has also been associated with the emergence of fungal pathogens. Fungi showed that an inability to grow at high temperatures limits the capacity of fungi to infect mammals. However, fungi can develop thermotolerance, gradually adapting to rising temperatures due to climate change, and generating a greater number of disease-causing organisms. In the present study, we reported the detection and identification of Candida palmioleophila isolates recovered from raw sewage samples in Niteroi city, Rio de Janeiro State, Brazil, during a monitoring program for measuring SARS-CoV-2 presence and concentration. Using polyphasic taxonomy to identify the species and evaluating some virulence aspects of this species, such as biofilm formation and extracellular enzyme production, our data highlight this species as a possible emerging pathogen in Brazil, especially in the pandemic context. Full article

(This article belongs to the Special Issue Young Investigators of Human Pathogenic Fungi)

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21 pages, 4732 KiB

Open AccessArticle

Quantifying Isoprenoids in the Ergosterol Biosynthesis by Gas Chromatography–Mass Spectrometry

by Maximilian Liebl, Ludwig Huber, Hesham Elsaman, Petra Merschak, Johannes Wagener, Fabio Gsaller and Christoph Müller

J. Fungi 2023, 9(7), 768; https://doi.org/10.3390/jof9070768 - 20 Jul 2023

Viewed by 1299

Abstract

The ergosterol pathway is a promising target for the development of new antifungals since its enzymes are essential for fungal cell growth. Appropriate screening assays are therefore needed that allow the identification of potential inhibitors. We developed a whole-cell screening method, which can [...] Read more.

The ergosterol pathway is a promising target for the development of new antifungals since its enzymes are essential for fungal cell growth. Appropriate screening assays are therefore needed that allow the identification of potential inhibitors. We developed a whole-cell screening method, which can be used to identify compounds interacting with the enzymes of isoprenoid biosynthesis, an important part of the ergosterol biosynthesis pathway. The method was validated according to the EMEA guideline on bioanalytical method validation. Aspergillus fumigatus hyphae and Saccharomyces cerevisiae cells were lysed mechanically in an aqueous buffer optimized for the enzymatic deconjugation of isoprenoid pyrophosphates. The residual alcohols were extracted, silylated and analyzed by GC-MS. The obtained isoprenoid pattern provides an indication of the inhibited enzyme, due to the accumulation of specific substrates. By analyzing terbinafine-treated A. fumigatus and mutant strains containing tunable gene copies of erg9 or erg1, respectively, the method was verified. Downregulation of erg9 resulted in a high accumulation of intracellular farnesol as well as elevated levels of geranylgeraniol and isoprenol. The decreased expression of erg1 as well as terbinafine treatment led to an increased squalene content. Additional analysis of growth medium revealed high farnesyl pyrophosphate levels extruded during erg9 downregulation. Full article

(This article belongs to the Special Issue Young Investigators of Human Pathogenic Fungi)

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11 pages, 3817 KiB

Open AccessArticle

Efficient Generation of Multiple Seamless Point Mutations Conferring Triazole Resistance in Aspergillus fumigatus

by Mariana Handelman and Nir Osherov

J. Fungi 2023, 9(6), 644; https://doi.org/10.3390/jof9060644 - 2 Jun 2023

Viewed by 1275

Abstract

Aspergillus fumigatus is a common human fungal pathogen that can cause a range of diseases. Triazoles are used to treat A. fumigatus infections, but resistance is increasing due to mutations in genes such as cyp51A, hmg1 and overexpression of efflux pumps. Verifying [...] Read more.

Aspergillus fumigatus is a common human fungal pathogen that can cause a range of diseases. Triazoles are used to treat A. fumigatus infections, but resistance is increasing due to mutations in genes such as cyp51A, hmg1 and overexpression of efflux pumps. Verifying the importance of these mutations is time-consuming, and although the use of CRISPR-Cas9 methods has shortened this process, it still relies on the construction of repair templates containing a selectable marker. Here, employing in vitro-assembled CRISPR-Cas9 along with a recyclable selectable marker, we devised a quick and easy way to effectively and seamlessly introduce mutations conferring triazole resistance in A. fumigatus. We used it to introduce, alone and in combination, triazole resistance-conferring mutations in cyp51A, cyp51B and hmg1. With the potential to seamlessly introduce genes imparting resistance to additional existing and novel antifungals, toxic metals, and environmental stressors, this technique can considerably improve the ability to introduce dominant mutations in A. fumigatus. Full article

(This article belongs to the Special Issue Young Investigators of Human Pathogenic Fungi)

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15 pages, 1697 KiB

Open AccessArticle

Extracellular Vesicles from Candida haemulonii var. vulnera Modulate Macrophage Oxidative Burst

by Bianca T. M. Oliveira, Thales M. H. Dourado, Patrick W. S. Santos, Tamires A. Bitencourt, Carlos R. Tirapelli, Arnaldo L. Colombo and Fausto Almeida

J. Fungi 2023, 9(5), 562; https://doi.org/10.3390/jof9050562 - 12 May 2023

Cited by 5 | Viewed by 1945

Abstract

Members of the Candida haemulonii species complex are multidrug-resistant emergent yeast pathogens able to cause superficial and invasive infections in risk populations. Fungal extracellular vesicles (EVs) play a critical role in the pathogenicity and virulence of several species and may perform essential functions [...] Read more.

Members of the Candida haemulonii species complex are multidrug-resistant emergent yeast pathogens able to cause superficial and invasive infections in risk populations. Fungal extracellular vesicles (EVs) play a critical role in the pathogenicity and virulence of several species and may perform essential functions during infections, such as carrying virulence factors that behave in two-way communications with the host, affecting survival and fungal resistance. Our study aimed to describe EV production from Candida haemulonii var. vulnera and evaluate whether murine macrophage RAW 264.7 cells respond to their stimuli by generating an oxidative response after 24 h. For this purpose, reactive oxygen species detection assays demonstrated that high concentrations of yeast and EVs (10¹⁰ particles/mL) of Candida haemulonii did not change macrophage viability. However, the macrophages recognized these EVs and triggered an oxidative response through the classical NOX-2 pathway, increasing O₂^•− and H₂O₂ levels. However, this stress did not cause lipid peroxidation in the RAW 264.7 cells and neither lead to the activation of the COX-2–PGE₂ pathway. Thus, our data suggest that low concentrations of C. haemulonii EVs are not recognized by the classical pathway of the oxidative burst generated by macrophages, which might be an advantage allowing the transport of virulence factors via EVs, not identified by the host immune system that could work as fine tube regulators during infections caused by C. haemulonii. In contrast, C. haemulonii var. vulnera and high EV concentrations activated microbicidal actions in macrophages. Therefore, we propose that EVs could participate in the virulence of the species and that these particles could be a source of antigens to be exploited as new therapeutic targets. Full article

(This article belongs to the Special Issue Young Investigators of Human Pathogenic Fungi)

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19 pages, 2261 KiB

Open AccessArticle

Expanding the Toolbox for Functional Genomics in Fonsecaea pedrosoi: The Use of Split-Marker and Biolistic Transformation for Inactivation of Tryptophan Synthase (trpB) Gene

by Luísa Dan Favilla, Tatiana Sobianski Herman, Camila da Silva Goersch, Rosangela Vieira de Andrade, Maria Sueli Soares Felipe, Anamélia Lorenzetti Bocca and Larissa Fernandes

J. Fungi 2023, 9(2), 224; https://doi.org/10.3390/jof9020224 - 8 Feb 2023

Cited by 1 | Viewed by 1772

Abstract

Chromoblastomycosis (CBM) is a disease caused by several dematiaceous fungi from different genera, and Fonsecaea is the most common which has been clinically isolated. Genetic transformation methods have recently been described; however, molecular tools for the functional study of genes have been scarcely [...] Read more.

Chromoblastomycosis (CBM) is a disease caused by several dematiaceous fungi from different genera, and Fonsecaea is the most common which has been clinically isolated. Genetic transformation methods have recently been described; however, molecular tools for the functional study of genes have been scarcely reported for those fungi. In this work, we demonstrated that gene deletion and generation of the null mutant by homologous recombination are achievable for Fonsecaea pedrosoi by the use of two approaches: use of double-joint PCR for cassette construction, followed by delivery of the split-marker by biolistic transformation. Through in silico analyses, we identified that F. pedrosoi presents the complete enzymatic apparatus required for tryptophan (trp) biosynthesis. The gene encoding a tryptophan synthase trpB —which converts chorismate to trp—was disrupted. The ΔtrpB auxotrophic mutant can grow with external trp supply, but germination, viability of conidia, and radial growth are defective compared to the wild-type and reconstituted strains. The use of 5-FAA for selection of trp^- phenotypes and for counter-selection of strains carrying the trp gene was also demonstrated. The molecular tools for the functional study of genes, allied to the genetic information from genomic databases, significantly boost our understanding of the biology and pathogenicity of CBM causative agents. Full article

(This article belongs to the Special Issue Young Investigators of Human Pathogenic Fungi)

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