室內空氣污染物特性、所致健康影響及其控制研究─室內生物性氣膠之採樣分析技術開發(2/2)

Abstract

嗜肺性退伍軍人菌(Legionella pneumophila)為一重要之室內生物氣膠，主要 透過空氣傳播感染人體，而傳統微生物培養之評估法有其限制存在，因此本研究 欲透過對L. pneumophila 蛋白質體分析，嘗試瞭解L. pneumohphila 在不同生長階 段下存在之特殊蛋白及其生理意義。 研究發現退伍軍人菌在營養環境下培養24 小時處於指數生長期，可培養菌 數對數值(log CFU/ml)由3.94 ± 0.10 增加至5.53 ± 0.24 ，培養至第48 及72 小 時處於停滯生長期，可培養菌數對數值分別為8.28 ± 0.13 及8.43 ± 0.14 ，其繁 殖增生週期為3 小時。當退伍軍人菌處於無營養提供之環境時，其在培養基上繁 殖增生的能力則會隨著時間的增加逐漸喪失，起始可培養之菌數濃度對數值8.16 ± 0.11 ，第20 天時已下降至2.51 ± 0.47 ，第35 天則完全喪失其在培養基上繁殖增生能力。蛋白質體分析發現不同生長階段之蛋白質數目表現不同，由指數生長期進 入停滯生長期隨著營養來源侷限影響，蛋白質總數有逐漸增加之趨勢，24 小時、 48 小時及72 小時分別為 282 點、336 點及357 點，另外，當退伍軍人菌處於無 營養提供之壓力環境下，隨著時間的增加蛋白質數目表現則呈現逐漸下降的趨 勢，由起始336 點，經過1 天即下降至290 點，至無培養能力階段第4 天及第 35 天則分別只剩258 點及253 點。 質量足夠進入質譜分析之蛋白質點共完成99 點蛋白質身份鑑定，其中發現 一退伍軍人菌之特殊重要蛋白Mip(macrophage infectivity potentiator)，為退伍軍 人菌得以進入宿主體內之毒力因子，且它不僅存在於營養階段，當退伍軍人菌處 於無營養的不良環境時，其表現量更是隨著時間而明顯增加，即使在無培養能力 之VBNC 階段，Mip 蛋白質量亦持續增加，不僅證明了VBNC 狀態的存在，亦 顯示了無法培養的退伍軍人菌具有明顯的侵犯宿主的能力。

Legionella pneumophila is an important indoor bioaerosol. The transmission root of L. pneumophila is inhalation of contaminated aerosols. The traditional detection method—culture is encountered several limitations. The aim of this study is to explore the protein profile and related physiological functions at different growth stages using proteomic technique. The log culturable concentration (Log CFU/ml) of L. pneumophila was rasied from 3.94 ± 0.10 to 5.53 ± 0.24 at exponential phase, and continuously raised to 8.28 ± 0.13(48th hr) and 8.43 ± 0.14(72nd hr) at the stationary phase. The Log CFU/ml of L. pneumophila in non-nutrient condition was declined from 8.16 ± 0.11 to 2.51 ± 0.47 at 20th day, and loss its culturability at 35th day. In the proteomics analyze, the expressive protein numbers were gradually increased in nutrient condition: 282 protein spots at exponential phase, followed by early stationary phase (336 spots) and stationary phase (357 spots). However, the protein number was decreased in non-nutrient condition. It stared with 336 spots, declined to 290 spots at day 1st, and reduced to 258 spots and 253 spots at non-culturable day 4th and 35th. Among 99 identified proteins, an important protein—Mip (macrophage infectivity potentiator) which is related to infection was not only found in nutrient condition but also in non-nutrient condition. With the prolong starvation period, the expression of Mip protein was mounting. It represented the possibility of infection for non-culturable L. pneumophila.