圖1. 感染宿主細胞的弓漿蟲寄生蟲。紫色是宿主細胞及寄生蟲的細胞核，白色顯示寄生蟲的外圍，藍色是由一種，被稱為肌動蛋白之蛋白質組成的宿主細胞“骨架”。

Toxoplasma gondii parasites infecting a host cell. The purple is the host cell's and parasites' nucleii, the white shows the periphery of the parasites, and the blue is the host cell's "skeleton" made of a protein called actin.

Apicomplexan parasites are a common cause of disease, infecting hundreds of millions of people each year.

頂複門寄生蟲(一群細胞內的寄生蟲)是疾病的一種常見原因，每年感染數億人。

They are responsible for spreading malaria; cryptosporidiosis – a severe childhood diarrheal disease; and toxoplasmosis – a disease that endangers immune compromised people and fetuses, and is the reason why pregnant women are told to avoid changing cat litter.

它們是導致傳播瘧疾(隱孢子蟲病，一種嚴重之兒童時期的腹瀉病)及弓漿蟲病(一種危及免疫受損者與胎兒的疾病)的原因，因此也是孕婦被告知，避免更換貓砂的原因。

Apicomplexan parasites are very good at infecting humans and many other animals, and persisting inside of them. The more that researchers can learn about how apicomplexans infect hosts, the better they will be able to develop effective treatments against the parasites.

頂複門寄生蟲非常擅長感染人類及許多其他動物，且持續存在於它們體內。研究人員們能瞭解越多，有關頂複門菌如何感染宿主，將越能開發出，針對此些寄生蟲的有效療法。

To this end, researchers in Whitehead Institute Member Sebastian Lourido’s lab, led by graduate student Christopher Giuliano, have now completed a genome-wide screen of the apicomplexan parasite Toxoplasma gondii (T. gondii), which causes toxoplasmosis, during its infection of mice.

為此，美國懷特海德研究所成員，Sebastian Lourido實驗室的研究人員們，在研究生Christopher Giuliano的帶領下。目前，已經完成有關，在感染小鼠期間，引發弓漿蟲病之頂複門寄生蟲─弓漿蟲(T. gondii)的全基因體篩選。

This screen shows how important each gene is for the parasite’s ability to infect a host, providing clues to genes’ functions. In the journal Nature Microbiology on July 8, the researchers share their approach for tracing lineages of parasites in a live host, and some specific findings of interest—including a possible anti-parasitic drug target.

該篩選顯示了，每一基因對寄生蟲感染宿主的能力是如何重要，而為基因功能提供了諸多線索。在(2024年)7月8日的《自然•微生物學》雜誌上，此些研究人員分享了，他們追蹤活宿主體內之寄生蟲種系的方法，及一些令人感興趣的具體發現，包括一種可能之抗寄生蟲藥物的標的。

Researchers in Lourido’s lab previously developed a screen to test the function of every T. gondii gene in cells in a dish in 2016. They used CRISPR gene editing technology to make mutant parasites in which each lineage had one gene inactivated.

於Lourido實驗室的研究人員們，先前於2016年研發了一種篩選方法，來測試培養皿中，弓漿蟲每一基因於細胞中的功能。他們使用群聚、規律性間隔開的短迴文結構複製(CRISPR：Clustered Regularly Interspaced Short Palindromic Repeat)的基因編輯技術，來產生每一種系具有一基因，遭鈍化的突變寄生蟲。

The researchers could then assess the importance of each gene to a parasite’s fitness, or ability to thrive, based on how well the mutants missing that gene did. If a mutant died off, this implied that its inactivated gene is essential for the parasite’s survival.

然後，此些研究人員能根據，缺少那基因之突變體表現怎麼樣，來評估每一基因對寄生蟲的健康，或快速生長之能力的重要性。倘若突變體死亡，這意味著其遭鈍化的基因，對此寄生蟲的存活至關重要。

This screen taught the researchers a lot about T. gondii’s biology but faced a common limitation: the parasites were studied in a dish rather than a live host. Cell culture provides an easier way to study parasites, but the conditions are not the same as what parasites face in an animal host. A host’s body is a more complex and dynamic environment, so it may require parasites to rely on genes that they don’t need in the artificial setting of cell culture.

該篩選使此些研究人員得悉，諸多有關弓漿蟲的生物學。不過，面臨了一個共同的限制：此些寄生蟲是在培養皿中，而不是在活宿主中被研究。細胞培養提供了一種，較簡單研究寄生蟲的方法。不過，此環境與寄生蟲在動物宿主中，所面臨的環境不相同。宿主的身體是一種，更複雜且動態的環境。因此，寄生蟲可能需要仰賴它們，在細胞培養之人工環境中，不需要的基因。

To overcome this limitation, researchers in Lourido’s lab figured out how to repeat the T. gondii genome-wide screen, which their colleagues in the lab had previously done in cell culture, in live mice. This was a massive undertaking, which required solving various technical challenges and running a large number of parallel experiments.

為了克服此限制，於Lourido實驗室的研究人員們想出了，如何重複弓漿蟲全基因體的篩選，他們於該實驗室的同僚們，先前曾在活小鼠的細胞培養中，進行過這種篩選。這是一項，需要解決各種技術挑戰，並進行大量平行實驗的艱鉅任務。

 T. gondii has around eight thousand genes, so the researchers performed pooled experiments, with each mouse getting infected by many different mutants—but not so many as to overwhelm the mouse. This meant that the researchers needed a way to more closely monitor the trajectories of mutants in the mouse.

弓漿蟲具有大約八千個基因，因而此些研究人員進行了諸多，具有每一小鼠遭許多不同突變體感染，不過不會多到使小鼠受不了的匯集實驗。這意味著，此些研究人員需要一種，更密切監視小鼠中，諸多突變體發展軌跡的方法。

They needed to track the lineages of parasites that carried the same mutation over time, as this would allow them to see how different replicate lineages of a particular mutant performed.

隨著時間推移，他們需要追蹤攜帶了相同突變體的寄生蟲種系。因為，這將使他們得以瞭解，特定突變體的不同複製種系如何表現。

The researchers added barcodes to the CRISPR tools that inactivated a gene of interest in the parasite. When they harvested the parasites’ descendants, the barcode would identify the lineage, distinguishing replicate parasites that had been mutated in the same way.

此些研究人員將諸多條碼添加到，鈍化了寄生蟲中，一個重要基因的CRISPR諸工具中。當他們獲得此些寄生蟲的後代時，條碼能識別種系，區分已經以相同方式被突變的複製寄生蟲。

This allowed the researchers to use a population-based analytical approach to rule out false results and decrease experimental noise. Then they could draw conclusions about how well each lineage did. Lineage tracing allowed them to map how different populations of parasites traveled throughout the host’s body, and whether some populations grew better in one organ versus another.

這使得此些研究人員得以使用一種，以種群為基礎的分析法，來排除錯誤結果及減少實驗的干擾。然後，他們能得出有關每一種系，表現怎麼樣的結論。種系追蹤使他們得以繪製，不同寄生蟲的種群，如何在整個宿主體內的傳播圖，及某些種群在一個器官中，是否比在另一個器官中，生長得更佳。

The researchers found 237 genes that contribute to the parasite’s fitness more in a live host than in cell culture. Many of these were not previously known to be important for the parasite’s fitness. The genes identified in the current screen are active in different parts of the parasite, and affect diverse aspects of its interactions with a host.

此些研究人員發現了237個，促使此些寄生蟲，於活宿主內比在細胞培養中，更健康的基因。其中許多先前並未被知曉，對寄生蟲的健康是重要的。於目前篩選中，被確認的此些基因，在寄生蟲的不同部位是活躍的，且影響其與宿主交互作用的各種層面。

The researchers also found instances in which parasite fitness in a live host increased when a gene was inactivated; these genes may be, for example, related to signals that the host immune system uses to detect the parasites. Next, the researchers followed up on several of the fitness-improving genes that stuck out as of particular interest.

此些研究人員也發現了諸多，當一個基因遭鈍化時，於活宿主內，寄生蟲健康提升的實例。譬如，此些基因可能與宿主免疫系統，用來發現此些寄生蟲的訊號有關。接下來，此些研究人員進行追蹤了，針對幾個在特別重要時，突出之改善健康的基因。

One gene that stuck out was GTP cyclohydrolase I (GCH), which codes for an enzyme involved in the production of the essential nutrient folate. Apicomplexans rely on folate, and so the researchers wanted to understand GCH’s role in securing it for the parasite.

一個突出的基因是一種，為涉及產生不可或缺營養素葉酸之酶，指定遺傳碼的第一型5'-三磷酸鳥苷(GTP：Guanosine 5'-Triphosphate)環水解酶(GCH)。頂複門菌依賴葉酸，因此這些研究人員希望瞭解，GCH在確保寄生蟲獲得葉酸上的角色。

Cell culture media contains high levels of folate, and in this nutrient-rich environment, GCH is not essential. However, in a live mouse, the parasite must both scavenge folate and synthesize it using the metabolic pathway containing GCH. Lourido and Giuliano uncovered new details of how that pathway works.

細胞培養基具有高水平的葉酸。因此，在此營養豐富的環境中，GCH並非必需的。不過，在活小鼠中，寄生蟲必須清除葉酸並利用具有GCH的代謝途徑，這兩者合成葉酸。Lourido及Giuliano揭露了，那途徑如何運作的新細節

Although previously GCH’s role was not fully understood, the importance of folate for apicomplexans is a well-known vulnerability that has been used to design anti-parasitic therapies. The anti-folate drug pyrimethamine was commonly used to treat malaria, but many parasites have developed resistance to it.

雖然，先前GCH的角色不完全被瞭解。不過，葉酸對頂複門菌的重要性是一種，已經被用於設計抗寄生蟲療法，眾所周知的弱點。這種抗葉酸藥物，乙胺嘧啶(商品名達拉匹林)普遍被用於治療瘧疾。不過，許多寄生蟲已經對其產生抗藥性。

Some drug-resistant apicomplexans have increased the number of GCH gene copies that they have, suggesting that they may be using GCH-mediated folate synthesis to overcome pyrimethamine. The researchers found that combining a GCH inhibitor with pyrimethamine increased the efficacy of the drug against the parasites.

一些抗藥性的頂複門菌已經增加GCH基因的複製數，這暗示它們可能利用GCH介導的葉酸合成，來抑制乙胺嘧啶。此些研究人員發現，使GCH抑制劑與乙胺嘧啶結合，提升了此藥物對抗此些寄生蟲的功效。

The GCH inhibitor was also effective on its own. Unfortunately, the currently available GCH inhibitor targets mammalian as well as parasitic folate pathways, and so is not safe for use in animals. Giuliano and colleagues are working on developing a GCH inhibitor that is parasite-specific as a possible therapy.

GCH抑制劑對其自身也有效。不巧的是，目前可資用的GCH抑制劑，除了寄生蟲的葉酸途徑之外，也鎖定哺乳動物。因此，供使用於動物中是不安全的。Giuliano及同僚們正致力於開發一種，對寄生蟲有效之作為一種可能療法的GCH抑制劑。

“There was an entire half of the folate metabolism pathway that previously looked like it wasn't important for parasites, simply because people add so much folate to cell culture media,” Giuliano says. “This is a good example of what can be missed in cell culture experiments, and what’s particularly exciting is that the finding has led us to a new drug candidate.”

Giuliano宣稱：「有一整半的葉酸代謝途徑，先前看起來對寄生蟲並不重要。只是因為，人們在細胞培養基中，添加了太多葉酸。這是在諸多細胞培養實驗中，會被漏掉的一個好例子。特別令人振奮的是，此發現已經引領我們朝向一種新的候選藥物。」

Another gene of interest was RASP1. The researchers determined that RASP1 is not involved in initial infection attempts, but is needed if the parasites fail and need to mount a second attempt. They found that RASP1 is needed to reload an organelle of the parasites called a rhoptry that the parasites use to breach and reprogram host cells. Without RASP1, the parasites could only deploy one set of rhoptries, and so could only attempt one invasion.

另一個重要的基因是RASP1。此些研究人員確定了，RASP1並不涉及最初的感染企圖。不過，倘若寄生蟲失敗並需要發動第二次企圖，則需要 RASP1。他們發現，需要RASP1來重新裝載一種，寄生蟲用來破壞並重新編碼宿主細胞指令序列，被稱為棒狀體的寄生蟲細胞器。沒有RASP1，寄生蟲只能調動一組棒狀體，因此只能企圖一次入侵。

Identifying the function of RASP1 in infection also demonstrated the importance of studying how parasites interact with different cell types. In cell culture, researchers typically culture parasites in fibroblasts, a connective tissue cell. The researchers found that parasites could invade fibroblasts with or without RASP1, suggesting that this cell type is easy for them to invade.

確認RASP1在感染中的功能也證明了，研究寄生蟲如何與不同細胞類型交互作用的重要性。在細胞培養中，研究人員們通常，在纖維母細胞(一種結締組織細胞)中，培養寄生蟲。此些研究人員發現，具有或沒有RASP1，寄生蟲皆能入侵纖維母細胞。暗示，對它們而言，此細胞類型很容易入侵。

However, when the parasites tried to invade macrophages, an immune cell, those without RASP1 often failed, suggesting that macrophages present the parasites with more of a challenge, requiring multiple attempts. The screen uncovered other probable cell-type specific pathways, which would not have been found using only model cell types in a dish.

不過，當寄生蟲試圖入侵巨噬細胞(一種免疫細胞)時，那些沒有RASP1的往往失敗。暗示，巨噬細胞帶給寄生蟲更大的挑戰，需要多次嘗試。此篩選揭露了其他，於培養皿中，僅使用模型細胞類型，或許不曾被發現之可能細胞類型的特有途徑。

The screen also highlighted a previously unnamed gene that the researchers are calling GRA72. Previous studies suggested that this gene plays a role in the vacuole or protective envelope that the parasite forms around itself. The Lourido lab researchers confirmed this, and discovered additional details of how the absence of GRA72 disrupts the parasite vacuole.

此篩選也強調了一個，目前此些研究人員稱為GRA72，先前未被命名的基因。先前諸多研究暗示，該基因在寄生蟲圍繞其自身形成的液泡或保護封包中，扮演一種角色。Lourido實驗室的研究人員，證實這一點且發現了，缺少GRA72如何擾亂寄生蟲液泡的更多細節。

Lourido, Giuliano, and colleagues hope that their findings will provide new insights into parasite biology and, especially in the case of GCH, lead to new therapies.

Lourido、Giuliano及同僚們希望，他們的研究發現能為寄生蟲生物學，提供新的洞察力(特別是有關GCH)及引領出新的療法。

They intend to continue pulling from the treasure trove of results—their screen identified many other genes of interest that require follow-up—to learn more about apicomplexan parasites and their interactions with mammalian hosts. Lourido says that other researchers in his lab have already used the results of the screen to guide them towards relevant genes and pathways in their own projects.

他們打算繼續，從諸多結果的寶庫(他們的篩選確認了，許多其他需要後續研究的重要基因)獲取資訊，來得悉更多有關頂複門寄生蟲，及其與哺乳動物宿主的交互作用。Lourido表示，於其實驗室的其他研究人員，已經使用該篩選的此些結果來引導他們，在其自己的計畫中，朝向相關的基因及途徑。

“This is an outstanding resource,” says Lourido, who is also an associate professor of biology at MIT. “The results of the screen reveal such a broader spectrum of ways in which the parasites are interacting with hosts, and enrich our perception of the parasites’ abilities and vulnerabilities.”

也是美國麻省理工學院生物學副教授的Lourido宣稱：「這是一項重要的資源。該篩選的此些結果揭露了，此些寄生蟲與宿主交互作用，如此廣泛的方式，並豐富了我們有關寄生蟲之能耐及脆弱性的洞察力。」

網址：https://wi.mit.edu/news/genome-wide-screen-live-hosts-reveals-new-secrets-parasite-infection

翻譯：許東榮