An efficient gene-disruption method in Cryptococcus neoformans by double-joint PCR with NAT-split markers

Kim, Min Su; Kim, Seo Young; Yoon, Ja Kyung; Lee, Yin Won; Bahn, Yong-Sun

doi:10.1016/j.bbrc.2009.10.089

Cited by 71 publications

(80 citation statements)

References 24 publications

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“…For gene disruption, information on genomic DNA (exon and intron) structures for each gene was obtained from the serotype A C. neoformans genome database (http://www .broadinstitute.org/annotation/genome/cryptococcus_neoformans/MultiHome .html). The genes GRE2 (H99 gene identification number CNAG_02182.2 [hereinafter, only the five digits represented by X in CNAG_XXXXX.2 are given for H99 gene identification numbers]), PKP1 (00047), HSP12 (03143), and HSP122 (01446) in the C. neoformans serotype A H99 strain were deleted by overlap PCR or double-joint PCR with split markers and biolistic transformation as described previously (10,17,37). Primers for the generation of the 5Ј and 3Ј flanking regions of each gene and the dominant selectable nourseothricin resistance marker (NAT, encoding nourseothricin acetyltransferase) are described in Table S1 in the supplemental material.…”

Section: Methodsmentioning

confidence: 99%

Comparative Transcriptome Analysis Reveals Novel Roles of the Ras and Cyclic AMP Signaling Pathways in Environmental Stress Response and Antifungal Drug Sensitivity in Cryptococcus neoformans

et al. 2010

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The cyclic AMP (cAMP) pathway plays a central role in the growth, differentiation, and virulence of pathogenic fungi, including Cryptococcus neoformans. Three upstream signaling regulators of adenylyl cyclase (Cac1), Ras, Aca1, and Gpa1, have been demonstrated to control the cAMP pathway in C. neoformans, but their functional relationship remains elusive. We performed a genome-wide transcriptome analysis with a DNA microarray using the ras1⌬, gpa1⌬, cac1⌬, aca1⌬, and pka1⌬ pka2⌬ mutants. The aca1⌬, gpa1⌬, cac1⌬, and pka1⌬ pka2⌬ mutants displayed similar transcriptome patterns, whereas the ras1⌬ mutant exhibited transcriptome patterns distinct from those of the wild type and the cAMP mutants. Interestingly, a number of environmental stress response genes are modulated differentially in the ras1⌬ and cAMP mutants. In fact, the Ras signaling pathway was found to be involved in osmotic and genotoxic stress responses and the maintenance of cell wall integrity via the Cdc24-dependent signaling pathway. Notably, the Ras and cAMP mutants exhibited hypersensitivity to a polyene drug, amphotericin B, without showing effects on ergosterol biosynthesis, which suggested a novel method of antifungal combination therapy. Among the cAMP-dependent gene products that we characterized, two small heat shock proteins, Hsp12 and Hsp122, were found to be involved in the polyene antifungal drug susceptibility of C. neoformans.

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Section: Methodsmentioning

confidence: 99%

Comparative Transcriptome Analysis Reveals Novel Roles of the Ras and Cyclic AMP Signaling Pathways in Environmental Stress Response and Antifungal Drug Sensitivity in Cryptococcus neoformans

et al. 2010

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“…SCH9 deletion increases C. neoformans thermotolerance. Hence, the sch9D mutant survives well up to 41°in contrast to the wild-type (WT) strain (Wang et al 2004;Kim et al 2009). Sch9 also negatively regulates the production of the polysaccharide capsule, which is also a key virulence factor for C. neoformans (Wang et al 2004).…”

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confidence: 99%

“…Sch9 also negatively regulates the production of the polysaccharide capsule, which is also a key virulence factor for C. neoformans (Wang et al 2004). Nevertheless, the virulence of the sch9D mutant is attenuated, possibly due to a greater susceptibility to oxidative stress (Wang et al 2004;Kim et al 2009). SCH9 expression is induced by oxidative stress ).…”

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confidence: 99%

Rewiring of Signaling Networks Modulating Thermotolerance in the Human Pathogen Cryptococcus neoformans

et al. 2017

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Thermotolerance is a crucial virulence attribute for human pathogens, including the fungus Cryptococcus neoformans that causes fatal meningitis in humans. Loss of the protein kinase Sch9 increases C. neoformans thermotolerance, but its regulatory mechanism has remained unknown. Here, we studied the Sch9-dependent and Sch9-independent signaling networks modulating C. neoformans thermotolerance by using genome-wide transcriptome analysis and reverse genetic approaches. During temperature upshift, genes encoding for molecular chaperones and heat shock proteins were upregulated, whereas those for translation, transcription, and sterol biosynthesis were highly suppressed. In this process, Sch9 regulated basal expression levels or induced/repressed expression levels of some temperature-responsive genes, including heat shock transcription factor (HSF1) and heat shock proteins (HSP104 and SSA1). Notably, we found that the HSF1 transcript abundance decreased but the Hsf1 protein became transiently phosphorylated during temperature upshift. Nevertheless, Hsf1 is essential for growth and its overexpression promoted C. neoformans thermotolerance. Transcriptome analysis using an HSF1 overexpressing strain revealed a dual role of Hsf1 in the oxidative stress response and thermotolerance. Chromatin immunoprecipitation demonstrated that Hsf1 binds to the step-type like heat shock element (HSE) of its target genes more efficiently than to the perfect-or gap-type HSE. This study provides insight into the thermotolerance of C. neoformans by elucidating the regulatory mechanisms of Sch9 and Hsf1 through the genome-scale identification of temperature-dependent genes. KEYWORDS Hsf1; Sch9; transcriptome analysis; high temperature; Cryptococcus neoformans R ESPONSE and adaptation to their host's physiological temperature, 37°, are key virulence attributes for most human fungal pathogens that infect from natural environments at ambient temperature. Cryptococcus neoformans is an example of such a pathogen. This basidiomycetous fungus exists in diverse environment niches and generates infectious yeast cells or spores through bisexual and unisexual differentiation, which are inhaled through the respiratory tract in the host and initially colonize the lungs (Idnurm et al. 2005;Lin and Heitman 2006). C. neoformans is subsequently disseminated into the bloodstream, breaches the central nervous system, and eventually elicits fatal meningoencephalitis, responsible for an estimated hundreds of thousands of deaths annually (Park et al. 2009). During this infectious process, the ability to sense and adapt to temperature upshift are crucial factors for the pathogen to establish the initial colonization of the lungs. Therefore, most mutants that are severely defective in thermotolerance tend to be highly attenuated in virulence. Due to such reasons, signaling cascades governing thermotolerance in fungi have been intensively investigated toward their exploitation as potential antifungal drug targets (Bahn et al. 2007;Bahn and Jung 2013). In...

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“…grubii serotype A genome database) was deleted with a NAT split marker (29). A gene-specific disruption cassette, which contains 350 bp of the 5Ј-and 3Ј-flanking regions of egcrp1, an 860-bp fragment of the promoter sequence and the ATG start codon of the C. neoformans actin gene (30), a 310-bp fragment of the terminator sequence and the stop codon of C. neoformans TRP1 (31), and a gene for the selectable marker NAT (32), was designed as shown in supplemental Fig.…”

Section: Methodsmentioning

confidence: 99%

“…Finally, the combined overlap PCR product was inserted into T-vector pMD20 to construct p⌬egcrp1 after adding adenine overhang using the Mighty TA cloning kit for PrimeSTAR (Takara Bio Inc.). A NAT split marker containing the 200-bp overlapping sequence was PCR-amplified with primers CN00623N-U and NSL-2 (29) for the 5Ј-region of NAT and primers NSR-2 (29) and CN00623-D for the 3Ј-region of NAT (supplemental Fig. S1A) using p⌬egcrp1 as a template.…”

Section: Methodsmentioning

confidence: 99%

Quality Control of Fungus-specific Glucosylceramide in Cryptococcus neoformans by Endoglycoceramidase-related Protein 1 (EGCrP1)

Ishibashi

Ikeda

Sakaguchi

et al. 2012

Journal of Biological Chemistry

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An efficient gene-disruption method in Cryptococcus neoformans by double-joint PCR with NAT-split markers

Cited by 71 publications

References 24 publications

Comparative Transcriptome Analysis Reveals Novel Roles of the Ras and Cyclic AMP Signaling Pathways in Environmental Stress Response and Antifungal Drug Sensitivity in Cryptococcus neoformans

Comparative Transcriptome Analysis Reveals Novel Roles of the Ras and Cyclic AMP Signaling Pathways in Environmental Stress Response and Antifungal Drug Sensitivity in Cryptococcus neoformans

Rewiring of Signaling Networks Modulating Thermotolerance in the Human Pathogen Cryptococcus neoformans

Quality Control of Fungus-specific Glucosylceramide in Cryptococcus neoformans by Endoglycoceramidase-related Protein 1 (EGCrP1)

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