PKC1 Is Essential for Protection against both Oxidative and Nitrosative Stresses, Cell Integrity, and Normal Manifestation of Virulence Factors in the Pathogenic Fungus Cryptococcus neoformans
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ABSTRACT Cell wall integrity is crucial for fungal growth, survival, and pathogenesis. Responses to environmental stresses are mediated by the highly conserved Pkc1 protein and its downstream components. In this study, we demonstrate that both oxidative and nitrosative stresses activate the PKC1 cell integrity pathway in wild-type cells, as measured by phosphorylation of Mpk1, the terminal protein in the PKC1 phosphorylation cascade. Furthermore, deletion of PKC1 shows that this gene is essential for defense against both oxidative and nitrosative stresses; however, other genes involved directly in the PKC1 pathway are dispensable for protection against these stresses. This suggests that Pkc1 may have multiple and alternative functions other than activating the mitogen-activated protein kinase cascade from a “top-down” approach. Deletion of PKC1 also causes osmotic instability, temperature sensitivity, severe sensitivity to cell wall-inhibiting agents, and alterations in capsule and melanin. Furthermore, the vital cell wall components chitin and its deacetylated form chitosan appear to be mislocalized in a pkc1Δ strain, although this mutant contains wild-type levels of both of these polymers. These data indicate that loss of Pkc1 has pleiotropic effects because it is central to many functions either dependent on or independent of PKC1 pathway activation. Notably, this is the first time that Pkc1 has been implicated in protection against nitrosative stress in any organism.Cryptococcus neoformans感染症の剖検例で, C. neoformansが膵ランゲルハンス島に好んで定着し, 内分泌細胞を侵襲することを知つた. このことは糖尿病の発症ないし増悪につながると想定されたので, この点を臨床的, 実験的に追求した. 1. 臨床例: 症例1.49歳, 男. 一次性糖尿病にクリプトコックス症が合併し, 多数のランゲルハンス島内にC. neoformansを認め, 内分泌細胞の壊死をみた. ラ島内のC. neoformansの直径は外のそれに比し, 大きかつた. 症例2.65歳, 女. 発熱で受診. 経時的に, 耐糖能障害増強し, インスリン分泌能は低下した. これはランゲルハンス島や肝にC. neoformansによる病変が生じたためと推察された. 症例3.76歳, 男. 糖尿病の有無は不明だが, ランゲルハンス島内に数個のC. neoformansがみられた. ランゲルハンス島外のC. neoformansと比べ, 同程度の大きさであつた. 2. 実験的: C. neoformansをrat尾静脈内に投与し, 15日後に糖静注負荷試験を行つた. 層殺後, ランゲルハンス島内ないしその辺縁C. neoformansが確認されたratでは, 著明な耐糖能障害とインスリン分泌能低下を示し, 二次性糖尿病を発症させ得た. 以上より, 臨床例においても, Cryptococcus症により二次性糖尿病が発症する可能性があると考えられた.
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ABSTRACT Analysis of 40 New York City Cryptococcus neoformans isolates revealed that 39 were typeable, of which 85 and 12.5% were Cryptococcus neoformans var. grubii (serotype A) and Cryptococcus neoformans var. neoformans (serotype D), respectively. The prevalence of serotype D isolates in New York City appears to be significantly higher than indicated by previous studies of North American isolates.
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Cryptococcus neoformans var. neoformans presently includes isolates which have been determined by the immunologic reactivity of their capsular polysaccharides to be serotype A and those which have been determined to be serotype D. However, recent analyses of the URA5 sequences and DNA fingerprinting patterns suggest significant genetic differences between the two serotypes. Therefore, we propose to recognize these genotypic distinctions, as well as previously reported phenotypic differences, by restricting C. neoformans var. neoformans to isolates which are serotype D and describing a new variety, C. neoformans var. grubii, for serotype A isolates.
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Ceramide synthase
Sphingolipid
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Immunomagnetic separation (IMS) was utilized for the selective isolation of Cryptococcus neoformans from environmental sources, such as soils and pigeon droppings. Magnetic beads coated with anti-cryptococcal IgG (serotypes A and B) were used to isolate the fungus. In a modeled spiking experiment using C. neoformans serotype A strain and anti-serotype A antibody, the recovery rate of the cells was more than 47%. Specificity experiments using C. neoformans and Candida albicans showed that the beads, when coated with specific antibody for C. neoformans, were highly effective for the separation of C. neoformans strains from C. albicans (more than 97%). The IMS of serotype B cells with purified anti-serotype B antibody indicated a high specificity. When this IMS technique was applied to soils and pigeon droppings, C. neoformans cells were selectively isolated from 3 out of 8 samples, and C. neoformans DNAs were identified by PCR. Therefore C. neoformans cells were thus selectively isolated and the efficiency of the technique further confirmed.
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Pathogenic yeast Cryptococcus neoformans causes devastating cryptococcal meningoencephalitis. Our previous studies demonstrated that C. neoformans hyaluronic acid was required for invasion into human brain microvascular endothelial cells (HBMEC), which constitute the blood–brain barrier. In this report, we demonstrate that C. neoformans hyaluronic acid interacts with CD44 on HBMEC. Our results suggest that HBMEC CD44 is a primary receptor during C. neoformans infection, based on the following observations. First, anti-CD44 neutralizing antibody treatment was able to significantly reduce C. neoformans association with HBMEC. Second, C. neoformans association was considerably impaired using either CD44-knock-down HBMEC or C. neoformans hyaluronic acid-deficient strains. Third, overexpression of CD44 in HBMEC increased their association activity towards C. neoformans. Fourth, confocal microscopic images showed that CD44 was enriched at and around the C. neoformans association sites. Fifth, upon C. neoformans and HBMEC engagement, a subpopulation of CD44 and actin translocated to the host membrane rafts. Our results highlight the interactions between C. neoformans hyaluronic acid and host CD44 and the dynamic results of these interactions, which may represent events during the adhesion and entry of C. neoformans at HBMEC membrane rafts.
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Cryptococcus neoformansは、免疫不全患者を中心に発症するクリプトコッカス症の原因菌である。私達は、C. neoformansにエンドグリコセラミダーゼ（EGCase）の2つのホモログ（Endoglycoceramidase related Protein 1, 2; EGCrP1, 2） が存在することを見出した。この2つのホモログは、オリゴ糖鎖とセラミド間のβグリコシド結合を加水分解するEGCaseとは全く異なる基質特異性を示した。つまり、EGCrP1はグルコシルセラミド（GlcCer）に特異的な中性グルコセレブロシダーゼであり、EGCrP2はGlcCerのみならず種々のβ-グルコシドを加水分解するアグリコン特異性の広い酸性β-グルコシダーゼであった。これらの酵素遺伝子が欠損したC. neoformans変異株を作製、解析した結果、EGCrP1欠損株ではセラミドの合成過程で生成される未成熟型セラミドにグルコースが結合した、野生株にはない未成熟型GlcCerが蓄積した。一方、EGCrP2欠損株ではエルゴステリルβ-グルコシドが蓄積した。これらの結果は、EGCrP1はGlcCer合成系と共役してGlcCerの品質管理に、EGCrP2はエルゴステリルβ-グルコシド（EG）の分解に関与していることを示している。生理学的に重要なことは、EGCrP1欠損株では莢膜形成不全が、EGCrP2欠損株では液胞の肥大化と細胞分裂不全が観察されたことである。今回明らかにされたEGCrP1, 2が駆動する真菌特異的な糖脂質代謝経路は、新しい作用機序を備える抗深在性真菌薬の開発標的として有望と考えられる。
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A new criterion to divide the pattern of phosphorylation to produce ATP was suggested. According to whether it was related to redox reaction, the pattern of substrate-level phosphorylation could be divided into the oxidative substrate-level phosphorylation ( or the substrate-level phosphorylation caused by dehydrogenation of metabolites ) and the non-oxidative substrate-level phosphorylation ( or the substrate-level phosphorylation caused by dehydration of metabolites ). On the basis, a new noun, phosphorylation related to redox reaction, was suggested to be used to include classical oxidative phosphorylation (or respiratory chain phosphorylation ) and the oxidative substrate-level phosphorylation suggested above.
Phosphorylation cascade
Substrate-level phosphorylation
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Cryptococcus neoformans はエイズや悪性血液疾患などの易感染性宿主に重篤な脳髄膜炎を発症させるが, 発症メカニズムは明確になっていない. C. neoformans に対する感染防御の主体は Th1型の細胞性免疫であり, そのきっかけとなるパターン認識受容体による病原微生物の構成分子の認識が重要な役割を持っていると推測される. 筆者らはマウスの系で, クリプトコックスDNA が Toll 様受容体9依存的に免疫応答を誘導することを明らかにし, この誘導が既知のCpGモチーフを持つ細菌 DNA とは異なる様式で起こる可能性を示した. また, Cタイプレクチン受容体からのシグナルを伝えるアダプタータンパク質CARD9 を欠失したマウスでは免疫応答が低下し, C. neoformans に感受性になることから, C. neoformans に対する感染防御に何らかの Cタイプレクチンが関与することが示唆された. 本稿では C. neoformans 感染におけるパターン認識と感染防御反応について, 自験例を交えて概説する.
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