Proximity-Dependent Biotinylation and Identification of Flagellar Proteins in Trypanosoma cruzi

Won, Madalyn M.; Baublis, Aaron; Burleigh, Barbara A.

doi:10.1128/msphere.00088-23

Cited by 5 publications

(6 citation statements)

References 66 publications

(101 reference statements)

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“…Using mass spectrometry, they identified 218 flagellum-enriched proteins. Among them, 40 flagellum-enriched proteins were found to occur in all life stages of two parasitic organisms, including homologs of known flagellar proteins from other trypanosome species and proteins specific to the Trypanosoma cruzi lineage [ 140 ]. Studies have shown that phase separation is involved in many important cellular processes such as RNA metabolism, signal transduction, and stress granule assembly.…”

Section: Applications Of Biotin Ligase-based Proximity Marker Technologymentioning

confidence: 99%

The development of proximity labeling technology and its applications in mammals, plants, and microorganisms

Guo,

et al. 2023

Cell Commun Signal

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Protein‒protein, protein‒RNA, and protein‒DNA interaction networks form the basis of cellular regulation and signal transduction, making it crucial to explore these interaction networks to understand complex biological processes. Traditional methods such as affinity purification and yeast two-hybrid assays have been shown to have limitations, as they can only isolate high-affinity molecular interactions under nonphysiological conditions or in vitro. Moreover, these methods have shortcomings for organelle isolation and protein subcellular localization. To address these issues, proximity labeling techniques have been developed. This technology not only overcomes the limitations of traditional methods but also offers unique advantages in studying protein spatial characteristics and molecular interactions within living cells. Currently, this technique not only is indispensable in research on mammalian nucleoprotein interactions but also provides a reliable approach for studying nonmammalian cells, such as plants, parasites and viruses. Given these advantages, this article provides a detailed introduction to the principles of proximity labeling techniques and the development of labeling enzymes. The focus is on summarizing the recent applications of TurboID and miniTurbo in mammals, plants, and microorganisms.

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Section: Applications Of Biotin Ligase-based Proximity Marker Technologymentioning

confidence: 99%

The development of proximity labeling technology and its applications in mammals, plants, and microorganisms

Guo,

et al. 2023

Cell Commun Signal

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“…T. cruzi amastigote maintains elements of a motile 9+2 axoneme such as dynein arms and central pair apparatus, as shown by structural ( Fig. 1A and B ) and biochemical approaches ( 2 - 4 ), arguing in favor of a motile activity. In amastigote, it is commonly reported that the PFR is absent.…”

Section: The Surprisementioning

confidence: 90%

“…1A , arrowhead). This morphological data is strengthened by the presence of some IFT proteins (IFT81, IFT88, and IFT20) identified in amastigote during the flagellum membrane protein SMP1-1 proximity proteome ( 4 ).…”

Section: How Is the Short Motile Flagellum Constructed?mentioning

confidence: 96%

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The hows and whys of amastigote flagellum motility in Trypanosoma cruzi

Alves

Bastin

2023

mBio

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The protist Trypanosoma cruzi exhibits several extracellular stages characterized by the presence of a long and motile flagellum and one intracellular life cycle stage termed amastigote, which possesses a tiny flagellum barely exiting the flagellar pocket. This stage was so far described as replicative but immotile cells. Unexpectedly, the recent work of M. M. Won, T. Krüger, M. Engstler, and B. A. Burleigh (mBio 14:e03556-22, 2023, https://doi.org/10.1128/mbio.03556-22 ) revealed that this short flagellum actually displays beating activity. This commentary explores how such a short flagellum could be constructed and why it could affect the parasite’s survival inside the mammalian host.

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“…In addition, endogenously tagged TcCARP3 exhibited the same dual localization as TcAC1. While this manuscript was in preparation, TcCARP3 was identified by mass spectrometry in a T. cruzi fraction of enriched flagellar proteins obtained using a proximity-dependent biotinylation approach ( 54 ). These authors also reported the localization of this protein at the tip of the flagellum, as confirmed by endogenous tagging and IFA.…”

Section: Discussionmentioning

confidence: 99%

Dual localization of receptor-type adenylate cyclases and cAMP response protein 3 unveils the presence of two putative signaling microdomains in Trypanosoma cruzi

Chiurillo

Carlson

Bertolini

et al. 2023

mBio

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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. IMPORTANCE 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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Proximity-Dependent Biotinylation and Identification of Flagellar Proteins in Trypanosoma cruzi

Cited by 5 publications

References 66 publications

The development of proximity labeling technology and its applications in mammals, plants, and microorganisms

The development of proximity labeling technology and its applications in mammals, plants, and microorganisms

The hows and whys of amastigote flagellum motility 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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