Dual localization of receptor-type adenylate cyclases and cAMP response protein 3 unveils the presence of two putative signaling microdomains in Trypanosoma cruzi.
Dual localization of receptor-type adenylate cyclases and cAMP response protein 3 unveils the presence of two putative signaling microdomains in Trypanosoma cruzi.
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克氏锥虫是恰加斯病的病原，恰加斯病是美洲致残和过早死亡的主要原因。这种寄生虫一生在三角虫和哺乳动物宿主之间度过，在不同的发育阶段之间过渡，以应对微环境的变化。在克氏锥虫中驱动分化的第二信使中，cAMP已被证明介导细胞分裂和对渗透胁迫的反应，但这一信号通路在克氏锥虫中仍未被充分探索。腺苷酸环化酶（ACs）催化ATP转化为cAMP。它们包括一个多基因家族，编码克氏锥虫中假定的受体型ACs。通过蛋白质序列比对，我们将其分为5组，并从每组中选择一个具有代表性的成员（TcAC1-TcAC5）进行定位研究。我们在克氏锥虫中表达了每个蛋白的ha标记版本，并进行了免疫荧光分析。在鞭毛远端区域和收缩液泡复合体（CVC）中观察到一种特殊的TcAC1和TcAC2的双定位，并通过基因互补在酵母中证实了它们的酶活性。此外，TcAC1过表达的寄生虫表现出细胞生成增加，宿主细胞入侵缺陷和细胞内复制减少，突出了该蛋白在克氏锥虫生命周期中的重要性。这些突变体对低渗胁迫的耐受性更强，在体外细胞生成过程中表现出更高的粘附能力，而在破坏tac1定位后恢复了野生型表型。最后，发现TcAC1与cAMP反应蛋白3 （TcCARP3）相互作用，在鞭毛尖端和CVC中与该蛋白共定位。我们在克氏锥虫中发现了cAMP信号通路的三个组成部分（TcAC1、TcAC2和TcCARP3）具有双重定位：鞭毛远端结构域和CVC，它们分别参与细胞粘附和渗透调节。我们发现了tac1在两个细胞过程以及元胞形成中的作用的证据。我们的数据表明，tcac通过在膜微域中合成cAMP作为信号传感器和传感器。我们提出了一个模型，在这个模型中，tcac感知到triatomine后肠中的恶劣条件（营养剥夺、酸性pH、渗透胁迫、离子组成、疏水相互作用）并变得活跃。cAMP的合成会在细胞生成完成之前触发细胞粘附，同时介导寄生虫对渗透应激的反应。这些结果揭示了camp介导的克氏t细胞分化的驱动机制，同时对tacs的激活及其下游组分的作用提出了新的问题。
Trypanosoma cruzi is the etiologic agent of Chagas disease, a leading cause of disability and premature death in the Americas. This parasite spends its life between a triatomine insect and a mammalian host, transitioning between developmental stages in response to microenvironmental changes. Among the second messengers driving differentiation in T. cruzi, cAMP has been shown to mediate metacyclogenesis and response to osmotic stress, but this signaling pathway remains largely unexplored in this parasite. Adenylate cyclases (ACs) catalyze the conversion of ATP to cAMP. They comprise a multigene family encoding putative receptor-type ACs in T. cruzi. Using protein sequence alignment, we classified them into five groups and chose a representative member from each group to study their localization (TcAC1–TcAC5). We expressed an HA-tagged version of each protein in T. cruzi and performed immunofluorescence analysis. A peculiar dual localization of TcAC1 and TcAC2 was observed in the flagellar distal domain and in the contractile vacuole complex (CVC), and their enzymatic activity was confirmed by gene complementation in yeast. Furthermore, TcAC1 overexpressing parasites showed an increased metacyclogenesis, a defect in host cell invasion, and a reduced intracellular replication, highlighting the importance of this protein throughout T. cruzi life cycle. These mutants were more tolerant to hypoosmotic stress and showed a higher adhesion capacity during in vitro metacyclogenesis, whereas the wild-type phenotype was restored after disrupting TcAC1 localization. Finally, TcAC1 was found to interact with cAMP response protein 3 (TcCARP3), co-localizing with this protein in the flagellar tip and CVC. We identified three components of the cAMP signaling pathway (TcAC1, TcAC2, and TcCARP3) with dual localization in Trypanosoma cruzi: the flagellar distal domain and the CVC, structures involved in cell adhesion and osmoregulation, respectively. We found evidence on the role of TcAC1 in both cellular processes, as well as in metacyclogenesis. Our data suggest that TcACs act as signal sensors and transducers through cAMP synthesis in membrane microdomains. We propose a model in which TcACs sense the harsh conditions in the triatomine hindgut (nutrient deprivation, acidic pH, osmotic stress, ionic composition, hydrophobic interactions) and become active. Synthesis of cAMP then triggers cell adhesion prior completion of metacyclogenesis, while mediating a response to osmotic stress in the parasite. These results shed light into the mechanisms driving cAMP-mediated cell differentiation in T. cruzi, while raising new questions on the activation of TcACs and the role of downstream components of this pathway.
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影响因子：
3.3
作者：
Durante IM;Butenko A;Rašková V;Charyyeva A;Svobodová M;Yurchenko V;Hashimi H;Lukeš J
通讯作者：
Lukeš J
影响因子：
3.3
作者：
Chiurillo, Miguel A.;Lander, Noelia;Docampo, Roberto
通讯作者：
Docampo, Roberto
影响因子：
4.1
作者：
Diaz-Benjumea, Rocio;Laxman, Sunil;Rascón, Ana
通讯作者：
Rascón, Ana
影响因子：
1.5
作者：
Hamedi, Afsaneh;Botelho, Larisse;Salmon, Didier
通讯作者：
Salmon, Didier
影响因子：
16.6
作者：
Bachmaier, Sabine;Giacomelli, Giacomo;Calvo-Alvarez, Estefania;Vieira, Larissa Rezende;Van Den Abbeele, Jan;Aristodemou, Aris;Lorentzen, Esben;Gould, Matthew;Brennand, Ana;Dupuy, Jean-Wiliam;Forne, Ignasi;Imhof, Axel;Bramkamp, Marc;Salmon, Didier;Rotureau, Brice;Boshart, Michael
通讯作者：
Boshart, Michael