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13 pages, 2322 KiB

Open AccessArticle

Simplified All-In-One CRISPR-Cas9 Construction for Efficient Genome Editing in Cryptococcus Species

by Ping Zhang, Yu Wang, Chenxi Li, Xiaoyu Ma, Lan Ma and Xudong Zhu

J. Fungi 2021, 7(7), 505; https://doi.org/10.3390/jof7070505 - 24 Jun 2021

Cited by 5 | Viewed by 3177

Abstract

Cryptococcus neoformans and Cryptococcus deneoformans are opportunistic fungal pathogens found worldwide that are utilized to reveal mechanisms of fungal pathogenesis. However, their low homologous recombination frequency has greatly encumbered genetic studies. In preliminary work, we described a ‘suicide’ CRISPR-Cas9 system for use in [...] Read more.

Cryptococcus neoformans and Cryptococcus deneoformans are opportunistic fungal pathogens found worldwide that are utilized to reveal mechanisms of fungal pathogenesis. However, their low homologous recombination frequency has greatly encumbered genetic studies. In preliminary work, we described a ‘suicide’ CRISPR-Cas9 system for use in the efficient gene editing of C. deneoformans, but this has not yet been used in the C. neoformans strain. The procedures involved in constructing vectors are time-consuming, whether they involve restriction enzyme-based cloning of donor DNA or the introduction of a target sequence into the gRNA expression cassette via overlap PCR, as are sophisticated, thus impeding their widespread application. Here, we report the optimized and simplified construction method for all-in-one CRISPR-Cas9 vectors that can be used in C. neoformans and C. deneoformans strains respectively, named pNK003 (Genbank: MW938321) and pRH003 (Genbank: KX977486). Taking several gene manipulations as examples, we also demonstrate the accuracy and efficiency of the new simplified all-in-one CRISPR-Cas9 genome editing tools in both Serotype A and Serotype D strains, as well as their ability to eliminate Cas9 and gDNA cassettes after gene editing. We anticipate that the availability of new vectors that can simplify and streamline the technical steps for all-in-one CRISPR-Cas9 construction could accelerate genetic studies of the Cryptococcus species. Full article

(This article belongs to the Special Issue Molecular and Genetic Tools for Studying Cryptococcus spp.)

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12 pages, 1652 KiB

Open AccessArticle

Influence of Galectin-3 on the Innate Immune Response during Experimental Cryptococcosis

by Caroline Patini Rezende, Patricia Kellen Martins Oliveira Brito, Thiago Aparecido Da Silva, Andre Moreira Pessoni, Leandra Naira Zambelli Ramalho and Fausto Almeida

J. Fungi 2021, 7(6), 492; https://doi.org/10.3390/jof7060492 - 20 Jun 2021

Viewed by 2059

Abstract

Cryptococcus neoformans, the causative agent of cryptococcosis, is the primary fungal pathogen that affects the immunocompromised individuals. Galectin-3 (Gal-3) is an animal lectin involved in both innate and adaptive immune responses. The present study aimed to evaluate the influence of Gal-3 on [...] Read more.

Cryptococcus neoformans, the causative agent of cryptococcosis, is the primary fungal pathogen that affects the immunocompromised individuals. Galectin-3 (Gal-3) is an animal lectin involved in both innate and adaptive immune responses. The present study aimed to evaluate the influence of Gal-3 on the C. neoformans infection. We performed histopathological and gene profile analysis of the innate antifungal immunity markers in the lungs, spleen, and brain of the wild-type (WT) and Gal-3 knockout (KO) mice during cryptococcosis. These findings suggest that Gal-3 absence does not cause significant histopathological alterations in the analyzed tissues. The expression profile of the genes related to innate antifungal immunity showed that the presence of cryptococcosis in the WT and Gal-3 KO animals, compared to their respective controls, promoted the upregulation of the pattern recognition receptor (PRR) responsive to mannose/chitin (mrc1) and a gene involved in inflammation (ccr5), as well as the downregulation of the genes related to signal transduction (card9, fos, ikbkb, jun) and PRRs (cd209a, colec12, nptx1). The absence of Gal-3, in fungal infection, a positively modulated gene involved in phagocytosis (sftpd) and negatively genes involved in signal transduction (syk and myd88), proinflammatory cytokines il-1β and il-12b and cd209a receptor. Therefore, our results suggest that Gal-3 may play an essential role in the development of antifungal immune responses against cryptococcosis. Full article

(This article belongs to the Special Issue Molecular and Genetic Tools for Studying Cryptococcus spp.)

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24 pages, 1894 KiB

Open AccessArticle

What Are the Best Parents for Hybrid Progeny? An Investigation into the Human Pathogenic Fungus Cryptococcus

by Man You and Jianping Xu

J. Fungi 2021, 7(4), 299; https://doi.org/10.3390/jof7040299 - 15 Apr 2021

Cited by 5 | Viewed by 2056

Abstract

Hybridization between more divergent organisms is likely to generate progeny with more novel genetic interactions and genetic variations. However, the relationship between parental genetic divergence and progeny phenotypic variation remains largely unknown. Here, using strains of the human pathogenic Cryptococcus, we investigated [...] Read more.

Hybridization between more divergent organisms is likely to generate progeny with more novel genetic interactions and genetic variations. However, the relationship between parental genetic divergence and progeny phenotypic variation remains largely unknown. Here, using strains of the human pathogenic Cryptococcus, we investigated the patterns of such a relationship. Twenty-two strains with up to 15% sequence divergence were mated. Progeny were genotyped at 16 loci. Parental strains and their progeny were phenotyped for growth ability at two temperatures, melanin production at seven conditions, and susceptibility to the antifungal drug fluconazole. We observed three patterns of relationships between parents and progeny for each phenotypic trait, including (i) similar to one of the parents, (ii) intermediate between the parents, and (iii) outside the parental phenotypic range. We found that as genetic distance increases between parental strains, progeny showed increased fluconazole resistance and growth at 37 °C but decreased melanin production under various oxidative and nitrosative stresses. Our findings demonstrate that, depending on the traits, both evolutionarily more similar strains and more divergent strains may be better parents to generate progeny with hybrid vigor. Together, the results indicate the enormous potential of Cryptococcus hybrids in their evolution and adaptation to diverse conditions. Full article

(This article belongs to the Special Issue Molecular and Genetic Tools for Studying Cryptococcus spp.)

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Review

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14 pages, 593 KiB

Open AccessReview

Methods for Manipulating Cryptococcus Spores

by Anna B. Frerichs, Mingwei Huang, Sébastien C. Ortiz and Christina M. Hull

J. Fungi 2022, 8(1), 4; https://doi.org/10.3390/jof8010004 - 22 Dec 2021

Cited by 5 | Viewed by 6693

Abstract

Spores are essential for the long-term survival of many diverse organisms, due to their roles in reproduction and stress resistance. In the environmental human fungal pathogen, Cryptococcus, basidiospores are robust cells with the ability to cause disease in animal models of infection. [...] Read more.

Spores are essential for the long-term survival of many diverse organisms, due to their roles in reproduction and stress resistance. In the environmental human fungal pathogen, Cryptococcus, basidiospores are robust cells with the ability to cause disease in animal models of infection. Here we describe methods for producing and purifying Cryptococcus basidiospores in quantities sufficient for large-scale analyses. The production of high numbers of pure spores has facilitated the development of new assays, including quantitative germination assays, and enabled transcriptomic, proteomic, and virulence studies, leading to discoveries of behaviors and properties unique to spores and spore-mediated disease. Full article

(This article belongs to the Special Issue Molecular and Genetic Tools for Studying Cryptococcus spp.)

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14 pages, 1443 KiB

Open AccessReview

Optimizing Transformation Frequency of Cryptococcus neoformans and Cryptococcus gattii Using Agrobacterium tumefaciens

by Jianmin Fu, Nohelli E. Brockman and Brian L. Wickes

J. Fungi 2021, 7(7), 520; https://doi.org/10.3390/jof7070520 - 29 Jun 2021

Cited by 1 | Viewed by 1979

Abstract

The transformation of Cryptococcus spp. by Agrobacterium tumefaciens has proven to be a useful genetic tool. A number of factors affect transformation frequency. These factors include acetosyringone concentration, bacterial cell to yeast cell ratio, cell wall damage, and agar concentration. Agar concentration was [...] Read more.

The transformation of Cryptococcus spp. by Agrobacterium tumefaciens has proven to be a useful genetic tool. A number of factors affect transformation frequency. These factors include acetosyringone concentration, bacterial cell to yeast cell ratio, cell wall damage, and agar concentration. Agar concentration was found to have a significant effect on the transformant number as transformants increased with agar concentration across all four serotypes. When infection time points were tested, higher agar concentrations were found to result in an earlier transfer of the Ti-plasmid to the yeast cell, with the earliest transformant appearing two h after A. tumefaciens contact with yeast cells. These results demonstrate that A. tumefaciens transformation efficiency can be affected by a variety of factors and continued investigation of these factors can lead to improvements in specific A. tumefaciens/fungus transformation systems. Full article

(This article belongs to the Special Issue Molecular and Genetic Tools for Studying Cryptococcus spp.)

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21 pages, 1608 KiB

Open AccessReview

Cryptococcus neoformans and Cryptococcus gattii Species Complexes in Latin America: A Map of Molecular Types, Genotypic Diversity, and Antifungal Susceptibility as Reported by the Latin American Cryptococcal Study Group

by Carolina Firacative, Wieland Meyer and Elizabeth Castañeda

J. Fungi 2021, 7(4), 282; https://doi.org/10.3390/jof7040282 - 9 Apr 2021

Cited by 23 | Viewed by 4522

Abstract

Cryptococcosis, a potentially fatal mycosis, is caused by members of the Cryptococcus neoformans and Cryptococcus gattii species complexes. In Latin America, cryptococcal meningitis is still an important health threat with a significant clinical burden. Analysis of publicly available molecular data from 5686 clinical, [...] Read more.

Cryptococcosis, a potentially fatal mycosis, is caused by members of the Cryptococcus neoformans and Cryptococcus gattii species complexes. In Latin America, cryptococcal meningitis is still an important health threat with a significant clinical burden. Analysis of publicly available molecular data from 5686 clinical, environmental, and veterinary cryptococcal isolates from member countries of the Latin American Cryptococcal Study Group showed that, as worldwide, C. neoformans molecular type VNI is the most common cause of cryptococcosis (76.01%) in HIV-infected people, followed by C. gattii molecular type VGII (12.37%), affecting mostly otherwise healthy hosts. These two molecular types also predominate in the environment (68.60% for VNI and 20.70% for VGII). Among the scarce number of veterinary cases, VGII is the predominant molecular type (73.68%). Multilocus sequence typing analysis showed that, in Latin America, the C. neoformans population is less diverse than the C. gattii population (D of 0.7104 vs. 0.9755). Analysis of antifungal susceptibility data showed the presence of non-wild-type VNI, VGI, VGII, and VGIII isolates in the region. Overall, the data presented herein summarize the progress that has been made towards the molecular epidemiology of cryptococcal isolates in Latin America, contributing to the characterization of the genetic diversity and antifungal susceptibility of these globally spreading pathogenic yeasts. Full article

(This article belongs to the Special Issue Molecular and Genetic Tools for Studying Cryptococcus spp.)

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14 pages, 2555 KiB

Open AccessReview

Tools for Assessing Translation in Cryptococcus neoformans

by Corey M. Knowles, Kelcy M. McIntyre and John C. Panepinto

J. Fungi 2021, 7(3), 159; https://doi.org/10.3390/jof7030159 - 24 Feb 2021

Cited by 4 | Viewed by 2561

Abstract

Cryptococcus neoformans is a ubiquitous environmental fungus capable of establishing an infection in a human host. Rapid changes in environments and exposure to the host immune system results in a significant amount of cellular stress, which is effectively combated at the level of [...] Read more.

Cryptococcus neoformans is a ubiquitous environmental fungus capable of establishing an infection in a human host. Rapid changes in environments and exposure to the host immune system results in a significant amount of cellular stress, which is effectively combated at the level of translatome reprogramming. Repression of translation following stress allows for the specific reallocation of limited resources. Understanding the mechanisms involved in regulating translation in C. neoformans during host infection is critical in the development of new antifungal drugs. In this review, we discuss the main tools available for assessing changes in translation state and translational output during cellular stress. Full article

(This article belongs to the Special Issue Molecular and Genetic Tools for Studying Cryptococcus spp.)

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10 pages, 264 KiB

Open AccessReview

Genetic Transformation in Cryptococcus Species

by Ping Wang

J. Fungi 2021, 7(1), 56; https://doi.org/10.3390/jof7010056 - 15 Jan 2021

Cited by 6 | Viewed by 3065

Abstract

Genetic transformation plays an imperative role in our understanding of the biology in unicellular yeasts and filamentous fungi, such as Saccharomyces cerevisiae, Aspergillus nidulans, Cryphonectria parasitica, and Magnaporthe oryzae. It also helps to understand the virulence and drug resistance mechanisms of [...] Read more.

Genetic transformation plays an imperative role in our understanding of the biology in unicellular yeasts and filamentous fungi, such as Saccharomyces cerevisiae, Aspergillus nidulans, Cryphonectria parasitica, and Magnaporthe oryzae. It also helps to understand the virulence and drug resistance mechanisms of the pathogenic fungus Cryptococcus that causes cryptococcosis in health and immunocompromised individuals. Since the first attempt at DNA transformation in this fungus by Edman in 1992, various methods and techniques have been developed to introduce DNA into this organism and improve the efficiency of homology-mediated gene disruption. There have been many excellent summaries or reviews covering the subject. Here we highlight some of the significant achievements and additional refinements in the genetic transformation of Cryptococcus species. Full article

(This article belongs to the Special Issue Molecular and Genetic Tools for Studying Cryptococcus spp.)

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