渡辺 哲

Aspergillus fumigatus has been shown to trigger the activation of nuclear factor-kappaB (NF-kappaB), but it remains unclear whether other transcription factors are also induced following infection by this organism. In this study, we demonstrated that A. fumigatus also triggers activator protein 1 (AP-1), a transcription factor that plays an important role during the production of cytokines and chemokines. Swollen conidia strongly induce the activation of AP-1, and more than 80% of these were stained positively with anti-beta-glucan antibodies by fluorescence microscopy. Hyphae were also stained with anti-beta-glucan antibodies, albeit significantly weaker compared with swollen conidia. Furthermore, our present findings also showed that A. fumigatus triggers the activation of AP-1 in a dectin-1 (receptor for beta-glucan)-dependent manner. These data thus suggest that AP-1 is triggered by beta-glucan recognition on the surface of A. fumigatus. We also showed that Syk tyrosine kinase is required for AP-1 induction in this pathway. We therefore speculate that the dectin-1/Syk/AP-1 signaling pathway plays an important role in the host defense response to fungal infection.

ハイブリッド型ヒスチジンキナーゼ(HHK) や応答レギュレータータンパク質 (RR) は二成分シグナル伝達系の構成タンパク質であり、薬剤耐性、浸透圧感受性、病原性、菌糸生育などを制御していることが知られている。病原性酵母Cryptococcus neoformansはゲノム中に7個のHHK遺伝子、2個のRR遺伝子をもつ。HHK遺伝子の一つCnNIK1遺伝子はフェニルピロール系抗真菌物質フルジオキソニルに対する感受性を制御している。今回、CnHHK2、CnHHK3、CnHHK5、CnHHK6、CnHHK7（以上HHK遺伝子）、CnSKN7、CnSSK1（以上RR遺伝子）の機能を調べるため遺伝子破壊を行ったところ、CnHHK4を除く全ての遺伝子破壊株を得ることができた。HHK遺伝子破壊株のうち、CnHHK2、CnHHK5、CnHHK6、CnHHK7 遺伝子破壊株は明確な表現型を示さなかったが、CnHHK3遺伝子破壊株は高温および高浸透圧条件下において生育遅延が認められ、本遺伝子は温度や浸透圧適応に重要な役割を果たしていることが示唆された。RR遺伝子に関しては、CnSKN7、CnSSK1遺伝子破壊株はいずれも高温、高浸透圧に対して感受性を示し、また、フルジオキソニルに対して耐性であった。C. neoformansにおいては、温度、浸透圧、フェニルピロール系薬剤に対して、CnNIK1、CnHHK3およびCnSKN7、CnSSK1が協調的に応答していることが示唆された。

2007年中にわが国において新たに上市された注射用抗菌薬はなかったが、2007年1月、キャンディン系抗真菌薬ミカファンギンが造血幹細胞移植患者におけるアスペルギルス症およびカンジダ症の予防に対する適応を取得した。国内ではタゾバクタム/ピペラシリンナトリウム(YP-18)の第III相試験、ドリペネム水和物、メロペネム三水和物の追加適応取得のための第III相試験が進行中、もしくは準備中である。海外ではグリコペプタイド系抗菌薬dalbavancin,telavancinが複雑性皮膚・軟部組織感染症を目的適応症として承認申請中である。キャンディン系抗真菌薬であるcaspofungin,anidulafunginは各々2001年、2006年に米国において承認済み、新規アゾール系抗真菌薬isavuconazoleの第III相試験が進行中である。(著者抄録)

The oumber of patients with invasive fungal infection (IFI) has dramatically increased since the beginning of the 1980s. Asjiergillus fumigatus, the most common species recovered from aspergillosis, is an important pathogen of IFL Recently, new antifungal agents have become available in Japan, but mortality from aspergillosis is still high. Early initiation of therapy seems to improve the survival rate. Study of virulence factors of the fungus may lead to the development of novel diagnostic tools or advancements in therapy. Many candidates of the fungal virulence factors have been studied including proteases and mycotoxins. We previously discussed the influence of fungal secondary metabolites such as gliotoxin and other low molecular components on the virulence, and showed that A. fumigatus produces potent cytotoxic substances other than gliotoxin. Studies are in progress to clarify the significance of the unknown substances.

SETTING: University medical school in Japan. OBJECTIVE: To clarify the influence of prior intradermal purified protein derivative (PPD) injection on QuantiFERON-TB Gold (QFT-G). DESIGN: Ninety-seven sixth-year university medical students aged 20-29 years concurrently underwent QFT-G and tuberculin skin test (TST). The first negative QFT-G and the first TST &lt 15 mm were followed by a second QFT-G one month later. RESULTS: Five of the 97 (5%) subjects tested positive for the first QFT-G. Thirty-three underwent a second QFT-G, five of whom (15%) turned positive, demonstrating the booster phenomenon of QFT-G. CONCLUSIONS: Prior intradermal PPD injection may boost QFT-G. Further studies of the diagnostic significance and immunological mechanisms of this phenomenon are needed. For clinical application, especially during contact screening, QFT-G should be evaluated while keeping in mind the possible influence of prior PPD intradermal injection. © 2007 The Union.

Pathogens of the Aspergillus species are frequently seen in deep-seated mycoses. We previously demonstrated that the culture filtrate of Aspergillus fumigatus (CF) has immunosuppressive effects on polymorphonuclear leukocytes (PMNs), which act as the main phagocytes to hyphae of Aspergillus fumigatus (A. fumigatus). But little is known about the gene expression profiles involved in it. Therefore we investigated the changes in gene expression in human PMNs treated with CF or gliotoxin at two time points, using microarray analysis. CF and gliotoxin changed the expression of 548 and 381 genes, respectively. Only 51 genes showed the same expression patterns with the two stimulants, and CF-induced changes in gene expression occurred comparatively earlier than those induced by gliotoxin. Among 31 genes encoding apoptosis, which were up- or down-regulated in this assay, only 3 genes were similarly changed by both kinds of stimulation. Apoptosis was detected and quantified using two apoptosis assays. CF and gliotoxin changed the expessions of only 3 out of 19 regulated genes related to inflammatory mediators and receptors similarly. The up-regulation of the gene encoding annexin 1 (ANXA1), which is known to be involved in extravasation and apoptosis of neutrophils, may play a role in the immunosuppressive effect of A. fumigatus. The difference in expression changes between CF and gliotoxin is presumed to be caused by the interaction among the components of CF and therefore the interaction is an area of interest for further investigation.

Histidine kinase（HK）は多くの真菌でフェニルピロール系抗真菌剤に対する感受性や浸透圧調節に関わる MAPK シグナル伝達経路を制御することが知られているタンパク質リン酸化酵素である。今回，ヒト病原真菌 Cryptococcus neoformans の HK をコードすると推定される遺伝子 CnNIK1 をクローニングし，構造解析を行った。塩基配列，アミノ酸配列を用いた相同検索の結果，アカパンカビ，イネいもち病菌をはじめとする真菌類の HK 遺伝子の活性部位を中心に高く保存されていた。B4500 由来の栄養要求性菌株 TAD1 を用いて，栄養要求性マーカー遺伝子と CnNIK1 遺伝子との置換による遺伝子破壊を行い，形質転換株TAD12を取得した。フェニルピロール系抗真菌剤フルジオキソニルに対する感受性について比較したところ，YPD 液体培地を用いた場合の野生型株に対する MIC は 3 μg/ml であったのに対して，TAD12 では MIC 値が 48 μg/ml よりも大きく，本菌の CnNIK1 遺伝子は他の真菌類と同様にフェニルピロール系抗真菌剤に対する感受性に関与していた。一方，高浸透圧条件下での生育は野生型株と差異が見られなかった。浸透圧調節を制御する MAPK である Hog1 のリン酸化状態を比較したところ，野生型株より低レベルなリン酸化が確認された。現在，動物モデルを用いた感染実験を行い，本遺伝子の病原性への関与を検証中である。

Gliotoxin, one of the mycotoxins produced by Aspergillus fumigatus, has various, potent bioactivities. However, it has not been considered to be a toxic (or virulence) factor because of its slow production. The aim of the present study was to investigate the effects of aeration on the cytotoxicity of A. fumigatus culture filtrate, and to determine the optimal condition for the rapid production of gliotoxin from this fungus. Fungal culture filtrates were made in three different containers under various conditions of aeration and O2 concentration. These filtrates were compared in terms of their cytotoxicity on murine macrophages and analyzed by gas chromatography. The culture filtrate showed high cytotoxicity when it was made under highly aerated conditions, but it was significantly less cytotoxic when prepared under non-aerated conditions. The cytotoxic activity became evident within 15 h of culture at 20% O2, when the fungus had already started producing gliotoxin. The culture filtrates also contained some other as yet unidentified substances that might also to some extent contribute to the cytotoxicity. In light of these results, the authors propose that a highly aerated condition is responsible for the rapid production of gliotoxin, and that gliotoxin might play an important role in the respiratory infection by A. fumigatus, with other toxic substances acting additively or synergistically.

近年の移植医療,抗悪性腫瘍療法の発達に伴い,深在性真菌症の発症頻度は増加し,かつその様相も変化してきている.Candida albicans感染例の減少により,カンジダ症全体の頻度は減少しているが,C.glabrata,C.krusei感染の相対的増加が見られる.アスペルギルス症はここ十年あまりの間に発症頻度は急増している.中心となる原因菌はA.fumigatusであるが,A.flavus,A.terreus,A.nigerなどによる感染も徐々に増加しつつある.更に,Aspergillus属菌以外のFusarium属菌や接合菌,Scedosporium属菌などによる糸状菌感染,輸入感染症の増加も深刻な問題である

真菌による髄膜炎,脳炎は稀ではあるが,近年の免疫低下宿主の増加に伴い,その頻度は高くなっているものと思われる.加えて海外渡航の普及により本来日本に見られなかった,いわゆる輸入真菌による感染の頻度も高くなっている.これらの中には必ずしも宿主免疫の低下を伴っていなくても発症し,髄膜炎,脳炎を引き起こすものもあるため注意が必要である.症状や画像所見などに特異的なものはないため,診断にあたっては患者の基礎疾患,免疫状態,病歴(特に海外渡航歴),身体所見を詳細に把握することが重要である.病理所見,血清中,髄液中の抗原抗体検査が極めて有用である菌種もあるが,確定診断のためには原則として菌の分離,培養が必要である