Disruption of the Putative Ribosome-Binding Motif of a Scaffold Protein Impairs Cytochrome Oxidase Subunit Expression in .

During their parasitic life cycle, through sandflies and vertebrate hosts, parasites confront strikingly different environments, including abrupt changes in pH and temperature, to which they must rapidly adapt. These adaptations include alterations in gene expression, metabolism, and morphology, allowing them to thrive as promastigotes in the sandfly and as intracellular amastigotes in the vertebrate host. A critical aspect of metabolic adaptation to these changes is maintenance of efficient mitochondrial function in the hostile vertebrate environment. Such functions, including generation of ATP, depend upon the expression of many mitochondrial proteins, including subunits of cytochrome oxidase (COX). Significantly, under mammalian temperature conditions, expression of COX subunit IV (LmCOX4) and virulence are dependent upon two copies of , a gene that encodes the ribosome-associated scaffold protein, LACK ( ortholog of RACK1 [receptor for activated C kinase]). Targeted replacement of an endogenous copy with a putative ribosome-binding motif-disrupted variant (LACK→LACK) resulted in thermosensitive parasites that showed diminished LmCOX4 expression, mitochondrial fitness, and replication in macrophages. Surprisingly, despite these phenotypes, LACK associated with monosomes and polysomes and showed no major impairment of global protein synthesis. Collectively, these data suggest that wild-type (WT) LACK orchestrates robust LmCOX4 expression and mitochondrial fitness to ensure parasite virulence, via optimized functional interactions with the ribosome. parasites are trypanosomatid protozoans that persist in infected human hosts to cause a spectrum of pathologies, from cutaneous and mucocutaneous manifestations to visceral leishmaniasis caused by The latter is usually fatal if not treated. Persistence of in the mammalian host depends upon maintaining gene-regulatory programs to support essential parasite metabolic functions. These include expression and assembly of mitochondrial cytochrome oxidase (LmCOX) subunits, important for ATP production. Significantly, under mammalian conditions, WT levels of LmCOX subunits require threshold levels of the ribosome-associated scaffold protein, LACK. Unexpectedly, we find that although disruption of LACK's putative ribosome-binding motif does not grossly perturb ribosome association or global protein synthesis, it nonetheless impairs COX subunit expression, mitochondrial function, and virulence. Our data indicate that the quality of LACK's interaction with ribosomes is critical for LmCOX subunit expression and parasite mitochondrial function in the mammalian host. Collectively, these findings validate LACK's ribosomal interactions as a potential therapeutic target.