Adhesion of
Crithidia fasciculata
promotes a rapid change in developmental fate driven by cAMP signaling
Authors: Denecke Shane , Malfara Madeline F. , Hodges Kelly R. , Holmes Nikki A. , Williams Andre R. , Gallagher-Teske Julia H. , Pascarella Julia M. , Daniels Abigail M. , Sterk Geert Jan , Leurs Rob , Ruthel Gordon , Hoang Rachel , Povelones Megan L. , Povelones Michael 
Published: September 2024

ABSTRACT

Trypanosomatids are single-celled parasites responsible for human and animal disease. Typically, colonization of an insect host is required for transmission. Stable attachment of parasites to insect tissues
via
their single flagellum coincides with differentiation and morphological changes. Although attachment is a conserved stage in trypanosomatid life cycles, the molecular mechanisms are not well understood. To study this process, we elaborate upon an
in vitro
model in which the swimming form of the trypanosomatid
Crithidia fasciculata
rapidly differentiates following adhesion to artificial substrates. Live imaging of cells transitioning from swimming to attached shows parasites undergoing a defined sequence of events, including an initial adhesion near the base of the flagellum immediately followed by flagellar shortening, cell rounding, and the formation of a hemidesmosome-like attachment plaque between the tip of the shortened flagellum and the substrate. Quantitative proteomics of swimming versus attached parasites suggests differential regulation of cyclic adenosine monophosphate (cAMP)-based signaling proteins. We have localized two of these proteins to the flagellum of swimming
C. fasciculata
; however, both are absent from the shortened flagellum of attached cells. Pharmacological inhibition of cAMP phosphodiesterases increased cAMP levels in the cell and prevented attachment. Further, treatment with inhibitor did not affect the growth rate of either swimming or established attached cells, indicating that its effect is limited to a critical window during the early stages of adhesion. These data suggest that cAMP signaling is required for attachment of
C. fasciculata
and that flagellar signaling domains may be reorganized during differentiation and attachment.

IMPORTANCE

Trypanosomatid parasites cause significant disease burden worldwide and require insect vectors for transmission. In the insect, parasites attach to tissues, sometimes dividing as attached cells or producing motile, infectious forms. The significance and cellular mechanisms of attachment are relatively unexplored. Here, we exploit a model trypanosomatid that attaches robustly to artificial surfaces to better understand this process. This attachment recapitulates that observed
in vivo
and can be used to define the stages and morphological features of attachment as well as conditions that impact attachment efficiency. We have identified proteins that are enriched in either swimming or attached parasites, supporting a role for the cyclic AMP signaling pathway in the transition from swimming to attached. As this pathway has already been implicated in environmental sensing and developmental transitions in trypanosomatids, our data provide new insights into activities required for parasite survival in their insect hosts.