Experimentally controlled downregulation of the histone chaperone FACT inPlasmodium bergheireveals that it is critical to male gamete fertility

Abstract

Human FACT (facilitates chromatin transcription) consists of the proteins SPT16 and SSRP1 and acts as a histone chaperone in the (dis)assembly of nucleosome (and thereby chromatin) structure during transcription and DNA replication. We identified a Plasmodium berghei protein, termed FACT-L, with homology to the SPT16 subunit of FACT. Epitope tagging of FACT-L showed nuclear localization with high expression in the nuclei of (activated) male gametocytes. The gene encoding FACT-L could not be deleted indicating an essential role during blood-stage development. Using a ‘promoter-swap’ approach whereby the fact-l promoter was replaced by an ‘asexual blood stage-specific’ promoter that is silent in gametocytes, transcription of fact-l in promoter-swap mutant gametocytes was downregulated compared with wild-type gametocytes. These mutant male gametocytes showed delayed DNA replication and gamete formation. Male gamete fertility was strongly reduced while female gamete fertility was unaffected; residual ookinetes generated oocysts that arrested early in development and failed to enter sporogony. Therefore FACT is critically involved in the formation of fertile male gametes and parasite transmission. ‘Promoter swapping’ is a powerful approach for the functional analysis of proteins in gametocytes (and beyond) that are essential during asexual blood-stage development.

The development of malaria parasites in the mosquito midgut

“…Further, a plasmodial FACT (facilitates chromatin transcription) protein (termed FACT‐L) is linked to DNA replication in the males (Laurentino et al ., 2011). Downstream of these events, the loss of mitogen‐activated protein kinase Map‐2 and cell division cycle protein CDC20 interferes with chromatin condensation, axoneme motility and cytokinesis during the formation of the male flagellar microgametes (Rangarajan et al ., 2005; Tewari et al ., 2005; Guttery et al ., 2012a).…”

A Knockout Screen of ApiAP2 Genes Reveals Networks of Interacting Transcriptional Regulators Controlling the Plasmodium Life Cycle

“…The overall positive correlation between transcription and translation dynamics (Caro et al., 2014) and between mRNA and protein levels at various stages of the life cycle (Foth et al., 2011, Tarun et al., 2008) suggests that the same is likely true for Plasmodium . Additionally, some promoters can drive expression of a transgene with an intensity and pattern comparable to their endogenous gene, even when removed from the native genomic context (Janse et al., 2006a, Laurentino et al., 2011, Sebastian et al., 2012). However, a variety of alternative control mechanisms have been described in Plasmodium , which at different life cycle stages include changes in nuclear organization, chromatin modifications (Ay et al., 2015), dynamics of RNA degradation (Shock et al., 2007), and posttranscriptional repression (Mair et al., 2006).…”

The ETRAMP Family Member SEP2 Is Expressed throughout Plasmodium berghei Life Cycle and Is Released during Sporozoite Gliding Motility

“…In fact, deletion of siap dramatically affects sporozoite mobility. Unfortunately, the inability to delete the sep2 gene using standard methodologies, did not allow us to study directly the function of SEP2 in sporozoite gliding and invasion, although novel conditional mutagenesis methods [34] may make this possible in the future.…”