Insect Stage-Specific Receptor Adenylate Cyclases Are Localized to Distinct Subdomains of the Trypanosoma brucei Flagellar Membrane

Abstract

cIncreasing evidence indicates that the Trypanosoma brucei flagellum (synonymous with cilium) plays important roles in hostparasite interactions. Several studies have identified virulence factors and signaling proteins in the flagellar membrane of bloodstream-stage T. brucei, but less is known about flagellar membrane proteins in procyclic, insect-stage parasites. Here we report on the identification of several receptor-type flagellar adenylate cyclases (ACs) that are specifically upregulated in procyclic T. brucei parasites. Identification of insect stage-specific ACs is novel, as previously studied ACs were constitutively expressed or confined to bloodstream-stage parasites. We show that procyclic stage-specific ACs are glycosylated, surface-exposed proteins that dimerize and possess catalytic activity. We used gene-specific tags to examine the distribution of individual AC isoforms. All ACs examined localized to the flagellum. Notably, however, while some ACs were distributed along the length of the flagellum, others specifically localized to the flagellum tip. These are the first transmembrane domain proteins to be localized specifically at the flagellum tip in T. brucei, emphasizing that the flagellum membrane is organized into specific subdomains. Deletion analysis reveals that C-terminal sequences are critical for targeting ACs to the flagellum, and sequence comparisons suggest that differential subflagellar localization might be specified by isoform-specific C termini. Our combined results suggest insect stage-specific roles for a subset of flagellar adenylate cyclases and support a microdomain model for flagellar cyclic AMP (cAMP) signaling in T. brucei. In this model, cAMP production is compartmentalized through differential localization of individual ACs, thereby allowing diverse cellular responses to be controlled by a common signaling molecule.

The Social Life of African Trypanosomes

“…The same phenotype was observed after dual knockdown of ACP1&2 [38]; these two ACs are closely related, but diverge at their C-termini and are differentially localised to the tip and entire flagellum, respectively [37]. At first sight it might seem puzzling that the stock used by Lopez et al [38] produced fewer than half the number of radial projections seen by Imhof et al [28], although both were derived from T. b. were 5-fold more abundant in late procyclic forms [26].…”

“…between early and late procyclic forms [26]. In addition, proteomics and quantitative reverse transcription PCR (qRT-PCR) identified 6 AC, designated ACP1-6, that were detected in procyclic forms but not bloodstream forms [37]. In the latter study, it is not known if the procyclic forms were early, late or a mixture of the two.…”

“…Trypanosomal ACs have the capacity to dimerise [37,39], but it is not yet known if AC1 and AC6 are part of a single complex that operates as a functional unit. In this context, other ACs that are stage-regulated between early and late procyclic forms also merit further investigation.…”

Protein diversity in discrete structures at the distal tip of the trypanosome flagellum

“…In contrast, there is a dearth of information about the distal tip of the flagellum/cilium. This is the site of assembly of components delivered by the intraflagellar transport system (2), of localization of soluble signaling molecules (3,4), and of distinct mechanical properties critical for flagellar beat (5). These unique tip features are reflected by a very discrete ultrastructure (6)(7)(8)(9).…”

The languages of parasite communication

“…Negative regulators of SoMo have also been identified: knocking down the procyclic form-specific adenylate cyclases ACP1/2 and ACP6 result in a hypersocial phenotype, with the production of more radial projections than in communities expressing wild-type levels [21]. Interestingly, ACP1 and ACP6 both localise to the flagellar tip [22]. Taken together with the phenotype of the PDEB1 knockdown, these data indicate that increases in local concentrations of cAMP inhibit SoMo.…”