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Evolutionary adaptations of doublet microtubules in trypanosomatid parasites

ABSTRACT: The movement and pathogenicity of trypanosomatid species, the causative agents of trypanosomiasis and leishmaniasis, are dependent on a flagellum containing an axoneme of dynein-bound doublet microtubules (DMTs). Here, we present cryo-EM structures of DMTs from two trypanosomatid species, Leishmania tarentolae and Crithidia fasciculata, at resolutions up to 2.7 Å. The structures reveal 27 trypanosomatid-specific microtubule inner proteins, a specialized dynein-docking complex, and the presence of paralogous proteins that enable higher-order periodicities or proximal-distal patterning. Leveraging the genetic tractability of trypanosomatid species, we quantify the location and contribution of each structure-identified protein to swimming behavior. Our study shows that proper B-tubule closure is critical for flagellar motility, exemplifying how integrating structural identification with systematic gene knockout can dissect individual protein contributions to flagellar motility.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Crithidia Fasciculata

SUBMITTER: Sophe Alvarez

LAB HEAD: Sophie Alvarez

PROVIDER: PXD057610 | Pride | 2025-03-04

REPOSITORIES: Pride

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			Action	DRS
	20230718_RZ_sample2_Crithidia_axoneme.raw	Raw
	F021814.dat	Other

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Project description:Spirochetes are long, thin, motile, helical or flat wave bacteria that are unique among the prokaryotes by having flagella or axial filaments confined to an internal periplasmic space. These flagella are complex organelles that can play major roles in bacterial pathogenicity and are used as propellers allowing bacteria to move through liquids, viscous environments or along surfaces. While most bacteria species use transcriptional regulatory cascades to regulate the synthesis and assembly of their flagella, spirochetes employ an unusual post-transcriptional mechanism. Using next generation sequencing, we characterized a natural mutant of the relapsing fever spirochete Borrelia hermsii lacking the flagellar export apparatus protein FliH. The mutant was non-motile, uncoiled and straight compared to the wild-type spirochetes. The B. hermsii fliH mutant produced only a residual amount of the major flagellin protein FlaB, which was correlated with a reduced number of periplasmic flagella. The amounts of flaB transcript were comparable in the fliH mutant and the wild-type strain. The synthesis of FlaB, the motility and the infectivity of the fliH mutant were rescued by trans-complementation. This report reveals a new function for FliH, and we propose that this regulator of the flagellar export apparatus influences the post-transcriptional processing of the flagella, motility and virulence of the relapsing fever spirochete Borrelia hermsii. Spirochetes are bacteria characterized in part by rotating periplasmic flagella that impart their flat-wave morphology and motility. While other bacteria rely on a transcriptional cascade to regulate the expression of motility genes, spirochetes employ posttranscriptional mechanism(s) that are only partially known. In the present study, we characterize a non-motile mutant of the relapsing fever spirochete Borrelia hermsii that was straight, non-motile and deficient in flagella. We used next generation DNA sequencing of the mutant’s genome, which when compared to the wild-type genome identified a 142 bp deletion in the chromosomal gene encoding the flagellar export apparatus protein FliH. Immunoblot and transcriptome analyses showed that the mutant phenotype was linked to the posttranscriptional deficiency in the synthesis of the major flagellar filament core protein FlaB. The turnover of the residual pool of FlaB produced by the fliH mutant was comparable to the wild-type spirochete while the amount of FlaA was similar to the wild-type level. The non-motile mutant was not infectious in mice and its inoculation did not induce an antibody response. Trans-complementation of the mutant with an intact fliH gene restored the synthesis of FlaB, a normal morphology, motility and infectivity in mice. Therefore, we propose that the flagellar export apparatus protein regulates motility of B. hermsii at the posttranscriptional level by influencing the synthesis of FlaB. Borrelia hermsii wild type vs. motility mutant

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Evolutionary adaptations of doublet microtubules in trypanosomatid parasites

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