Mapping regulatory networks underlying Leishmania stage differentiation reveals an essential role for protein degradation in parasite development

Genetic feedback control represents a central paradigm in regulation of biological systems and their response to environmental change. Vector-borne pathogens have evolved complex developmental programs to adapt to very distinct host environments, but the relevance of feedback regulation in stage differentiation remains to be elucidated. Here we address this open question in the trypanosomatid parasite Leishmania that shows constitutive gene transcription, thus providing a unique model system to assess post-transcriptional mechanisms of feedback regulation in the absence of confounding transcriptional control. Using a five-layer integrative systems analysis (from genome to metabolome), we examined hamster-isolated Leishmania donovani amastigotes and culture-derived insect-stage promastigotes. This approach enabled us to rule out genomic adaptation as a driver of parasite stage differentiation, confirm the pivotal role of differential mRNA turnover in stage-specific gene expression, and uncover an unexpectedly broad dynamic range of protein abundance changes that correlated poorly with transcript levels. This discrepancy was attributed to (i) stage-specific translational control, as indicated by alterations in snoRNA expression and changes in rRNA modification they guide, and (ii) differential protein degradation, as revealed by quantitative proteomics of parasites treated with the proteasomal inhibitor lactacystin. Notably, lactacystin treatment stalled the transition of spleen-derived amastigotes into promastigotes in culture, further underscoring the role of proteasomal activity in stage differentiation. Integration of our five-layer systems analysis established the first link between Leishmania development and the expression of highly connected, stage-specific regulatory networks encompassing mRNA turnover, protein translation, phosphorylation, and degradation. These networks engage in complex recursive (self-regulating) interactions involved in post-transcriptional regulation, ribosomal biogenesis, and signal transduction, as well as reciprocal (cross-regulating) interactions, including a network between protein kinases that phosphorylate proteasomal components in a stage-specific manner and proteasomal activities that, in turn, target protein kinases for stage-specific degradation. Our findings provide a powerful experimental framework to dissect the emergent properties of these regulatory feedback loops, offering critical insights into intracellular infection and serving as a blueprint for other vector-borne pathogens that rely on disease-associated developmental transitions.