真菌が引き起こす致命的な感染症の治療法開発にゲノム研究が貢献(Genomic Research Aids in the Effort to Understand How Best to Treat Deadly Infections Caused by a Fungus)

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有害で逆説的な薬効に関与する遺伝子を同定。 UMass Amherst team identifies genes involved in a harmful, paradoxical drug effect

2022-10-06 マサチューセッツ大学アマースト校

研究チームは、毎年約10万人の重症免疫不全者を死に至らしめる致命的な真菌感染症、Aspergillus fumigatusの治療薬カスポファンギンの使用に光を当てる重要な遺伝子の発見した。
研究チームは、約半数がCPE(カスポファンギン・パラドキシカル効果)である67の臨床サンプルのゲノムを配列決定し、グループ間の遺伝的差異を見つけ出し、統計的手法であるGWA(ゲノムワイド関連解析)を用いて、これらの遺伝子変異が高濃度のカスポファンギンでの増殖パターンとどのように関連しているかを明らかにした。
その後、科学者達は、遺伝子工学技術であるCRISPRを使って、それらの候補遺伝子をゲノムから削除して、遺伝子欠失変異体を作り、そのうちの2つの遺伝子が逆説的効果に関与していると断定できるようにした。

<関連情報>

Aspergillus fumigatusにおけるCaspofunginの逆説的効果に関与する遺伝子のゲノムおよび分子レベルでの同定 Genomic and Molecular Identification of Genes Contributing to the Caspofungin Paradoxical Effect in Aspergillus fumigatus

Shu Zhao, Adela Martin-Vicente, Ana Cristina Colabardini, Lilian Pereira Silva, David C. Rinker, Jarrod R. Fortwendel, Gustavo Henrique Goldman, John G. Gibbons
Microbiology Spectrum  Published:12 September 2022
DOI: https://doi.org/10.1128/spectrum.00519-22

ABSTRACT

Aspergillus fumigatus is a deadly opportunistic fungal pathogen responsible for ~100,000 annual deaths. Azoles are the first line antifungal agent used against A. fumigatus, but azole resistance has rapidly evolved making treatment challenging. Caspofungin is an important second-line therapy against invasive pulmonary aspergillosis, a severe A. fumigatus infection. Caspofungin functions by inhibiting β-1,3-glucan synthesis, a primary and essential component of the fungal cell wall. A phenomenon termed the caspofungin paradoxical effect (CPE) has been observed in several fungal species where at higher concentrations of caspofungin, chitin replaces β-1,3-glucan, morphology returns to normal, and growth rate increases. CPE appears to occur in vivo, and it is therefore clinically important to better understand the genetic contributors to CPE. We applied genomewide association (GWA) analysis and molecular genetics to identify and validate candidate genes involved in CPE. We quantified CPE across 67 clinical isolates and conducted three independent GWA analyses to identify genetic variants associated with CPE. We identified 48 single nucleotide polymorphisms (SNPs) associated with CPE. We used a CRISPR/Cas9 approach to generate gene deletion mutants for seven genes harboring candidate SNPs. Two null mutants, ΔAfu3g13230 and ΔAfu4g07080 (dscP), resulted in reduced basal growth rate and a loss of CPE. We further characterized the dscP phosphatase-null mutant and observed a significant reduction in conidia production and extremely high sensitivity to caspofungin at both low and high concentrations. Collectively, our work reveals the contribution of Afu3g13230 and dscP in CPE and sheds new light on the complex genetic interactions governing this phenotype.

IMPORTANCE This is one of the first studies to apply genomewide association (GWA) analysis to identify genes involved in an Aspergillus fumigatus phenotype. A. fumigatus is an opportunistic fungal pathogen that causes hundreds of thousands of infections and ~100,000 deaths each year, and antifungal resistance has rapidly evolved in this species. A phenomenon called the caspofungin paradoxical effect (CPE) occurs in some isolates, where high concentrations of the drug lead to increased growth rate. There is clinical relevance in understanding the genetic basis of this phenotype, since caspofungin concentrations could lead to unintended adverse clinical outcomes in certain cases. Using GWA analysis, we identified several interesting candidate polymorphisms and genes and then generated gene deletion mutants to determine whether these genes were important for CPE. Two of these mutant strains (ΔAfu3g13230 and ΔAfu4g07080/ΔdscP) displayed a loss of the CPE. This study sheds light on the genes involved in clinically important phenotype CPE.

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