Cryptococcus neoformans CAP10 Gene Regulates the Immune Response in Mice

Lin, Li; Chen, Shanshan; Zhang, Junhui; Li, Xianghui; Wu, Juan; Lin, Ning

doi:10.1016/j.mycmed.2021.101160

Cited by 4 publications

(8 citation statements)

References 25 publications

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“…RNAi in human cells also indicated that the size and nucleotide sequence of the loop had obvious influence to the interference, which was in accord with the results of this study [38]. In addition, other studies also used the shRNA with the same long loops to successfully conduct interference withCAP10 gene of C. neoformans [22,39]. And we further explored the phenotype of the LAC1 knockdown strains and clarified at which passage the strains would present a visible attenuation of interfering effect.…”

Section: Discussionsupporting

confidence: 84%

“…The dsRNA from vectors has no requirement for in vitro synthesis of dsRNA [19] and is widely used in many fungal species [20]. The intracellular transcription of dsRNA is usually done in two ways: the reverse complementary RNA strands pair to form dsRNA after being transcribed by two promoters in the opposite direction, and a single RNA strand consisting of reverse RNA oligonucleotides matches itself to form a small hairpin RNA (shRNA) after being driven by a single promoter [18,19,21,22]. Both forming ways of dsRNA were previously used for the exploration of genic function (e.g., CAP10 , CAP59 , ADE2 ) of C. neoformans [18,19,22,23].…”

Section: Introductionmentioning

confidence: 99%

“…The intracellular transcription of dsRNA is usually done in two ways: the reverse complementary RNA strands pair to form dsRNA after being transcribed by two promoters in the opposite direction, and a single RNA strand consisting of reverse RNA oligonucleotides matches itself to form a small hairpin RNA (shRNA) after being driven by a single promoter [18,19,21,22]. Both forming ways of dsRNA were previously used for the exploration of genic function (e.g., CAP10 , CAP59 , ADE2 ) of C. neoformans [18,19,22,23]. The dsRNA from the former way is relatively inefficient in RNAi compared to hairpin RNA, perhaps due to the low efficiency of dsRNA formation [20].…”

Section: Introductionmentioning

confidence: 99%

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Simplified and effective RNA interference and CRISPR-Cas9 systems for Cryptococcus neoformans

Chen

Peng

et al. 2023

Preprint

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The 3,4-dihydroxyphenylalanine (DOPA) melanin is one of important virulence factors for Cryptococcus neoformans, which may trigger immune responses in the host. It is worth exploring the genetic function of C. neoformans, by which we may derive more antifungal strategies. Therefore, we established two systems that were constructed quickly and easily for the knock-down/knock-out of LAC1 gene: RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9. The RNAi system used pSilencer 4.1-CMV neo plasmid and short hairpin RNA to realize the effective transcriptional suppression. The CRISPR-Cas9 system used the PNK003 vectors to obtain a stable albino mutant strain. The results of phenotype, qRT-PCR, Transmission Electron Microscope (TEM) and spectrophotometry were used to assess the ability of melanin production. As a result, the RNAi system displayed attenuation of transcriptional suppression when the transformants continuously passed on new plates. However, the transcriptional suppression of long loop in short hairpin RNA were more powerful and lasted longer. The CRISPR-Cas9 system constructed an albino strain completely without the ability to produce melanin. Considering the weakening of transcriptional suppression, we recommend using a long loop for the RNAi system and 1st or 2nd passage of knockdown strains for the subsequent studies. Besides, the different capacities of melanin production might be useful for exploring the linear relation between melanin and immunoreaction of the host. In addition, we recommend applying the PNK003 vectors to other serotypes of C. neoformans for quick screening of possible trait-regulating genes because of its easy construction and valid knockout effect.

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Section: Discussionsupporting

confidence: 84%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Simplified and effective RNA interference and CRISPR-Cas9 systems for Cryptococcus neoformans

Chen

Peng

et al. 2023

Preprint

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“…Cryptococcus spp. virulence is associated with the polysaccharide capsule, which increases pathogenicity, modulates the immune response, and protects against oxidative stress [ 114 , 115 , 116 ]. The presence of melanin in the cell wall protects against antifungal drug attacks at elevated temperatures and contributes to the modulation of the immune response [ 117 , 118 ].…”

Section: Fungal Infectionsmentioning

confidence: 99%

Nanotechnology-Based Approaches for Voriconazole Delivery Applied to Invasive Fungal Infections

et al. 2023

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Invasive fungal infections increase mortality and morbidity rates worldwide. The treatment of these infections is still limited due to the low bioavailability and toxicity, requiring therapeutic monitoring, especially in the most severe cases. Voriconazole is an azole widely used to treat invasive aspergillosis, other hyaline molds, many dematiaceous molds, Candida spp., including those resistant to fluconazole, and for infections caused by endemic mycoses, in addition to those that occur in the central nervous system. However, despite its broad activity, using voriconazole has limitations related to its non-linear pharmacokinetics, leading to supratherapeutic doses and increased toxicity according to individual polymorphisms during its metabolism. In this sense, nanotechnology-based drug delivery systems have successfully improved the physicochemical and biological aspects of different classes of drugs, including antifungals. In this review, we highlighted recent work that has applied nanotechnology to deliver voriconazole. These systems allowed increased permeation and deposition of voriconazole in target tissues from a controlled and sustained release in different routes of administration such as ocular, pulmonary, oral, topical, and parenteral. Thus, nanotechnology application aiming to delivery voriconazole becomes a more effective and safer therapeutic alternative in the treatment of fungal infections.

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“…The dsRNA from vectors has no requirement for in vitro synthesis of dsRNA [23] and is widely used in many fungal species [24]. The intracellular transcription of dsRNA is usually done in two ways: the reverse complementary RNA strands are paired to form dsRNA after being transcribed by two promoters in the opposite direction, and a single RNA strand consisting of reverse RNA oligonucleotides matches itself to form a small hairpin RNA (shRNA) after being driven by a single promoter [22,23,25,26]. Both forming ways of dsRNA were previously used for the exploration of genic function (e.g., CAP10, CAP59, ADE2) of C. neoformans [22,23,26,27].…”

Section: Introductionmentioning

confidence: 99%

Simplified and effective RNA interference and CRISPR‐Cas9 systems for Cryptococcus neoformans

Chen

Peng

et al. 2023

J Basic Microbiol

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The 3,4‐dihydroxyphenylalanine (DOPA) melanin is one of the important virulence factors for Cryptococcus neoformans, which may trigger immune responses in the host. While the production of DOPA melanin is catalyzed by laccase that is predominantly encoded by LAC1 gene. Therefore, regulating the genetic expression of C. neoformans is conducive to exploring the impact of interested molecules on the host. In this work, we established two systems that were constructed quickly and easily for the knock‐down/knock‐out of LAC1 gene: RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats CRISPR‐Cas9. The RNAi system was constructed by pSilencer 4.1‐CMV neo plasmid and short hairpin RNA to achieve effective transcriptional suppression. The CRISPR‐Cas9 system was used the PNK003 vectors to obtain a stable albino mutant strain. The results of phenotype, quantitative real‐time polymerase chain reaction, transmission electron microscope, and spectrophotometry were used to assess the ability of melanin production. As a result, the RNAi system displayed attenuation of transcriptional suppression when the transformants continuously passed on new plates. However, the transcriptional suppression of long loop in short hairpin RNA was more powerful and lasted longer. An albino strain produced by CRISPR‐Cas9 was completely unable to synthesize melanin. In conclusion, strains with different capacities of melanin production were obtained by RNAi and CRISPR‐Cas9 systems, which might be useful for exploring the linear relation between melanin and immunoreaction of the host. In addition, the two systems in this article might be convenient to quickly screen the possible trait‐regulating genes of other serotypes of C. neoformans.

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Cryptococcus neoformans CAP10 Gene Regulates the Immune Response in Mice

Cited by 4 publications

References 25 publications

Simplified and effective RNA interference and CRISPR-Cas9 systems for Cryptococcus neoformans

Simplified and effective RNA interference and CRISPR-Cas9 systems for Cryptococcus neoformans

Nanotechnology-Based Approaches for Voriconazole Delivery Applied to Invasive Fungal Infections

Simplified and effective RNA interference and CRISPR‐Cas9 systems for Cryptococcus neoformans

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