ABSTRACT
Development of the malaria parasite, Plasmodium falciparum, is regulated by a limited number of sequence-specific transcription factors (TFs). However, the mechanisms by which these TFs recognize genome-wide binding sites is largely unknown. To address TF specificity, we investigated the binding of two TF subsets that either bind CACACA or GTGCAC DNA sequence motifs and further characterized two additional ApiAP2 TFs, PfAP2-G and PfAP2-EXP, which bind unique DNA motifs (GTAC and TGCATGCA). We also interrogated the impact of DNA sequence and chromatin context on P. falciparum TF binding by integrating high-throughput in vitro and in vivo binding assays, DNA shape predictions, epigenetic post-translational modifications, and chromatin accessibility. We found that DNA sequence context minimally impacts binding site selection for paralogous CACACA-binding TFs, while chromatin accessibility, epigenetic patterns, co-factor recruitment, and dimerization correlate with differential binding. In contrast, GTGCAC-binding TFs prefer different DNA sequence context in addition to chromatin dynamics. Finally, we determined that TFs that preferentially bind divergent DNA motifs may bind overlapping genomic regions due to low-affinity binding to other sequence motifs. Our results demonstrate that TF binding site selection relies on a combination of DNA sequence and chromatin features, thereby contributing to the complexity of P. falciparum gene regulatory mechanisms.
ABSTRACT
Bacterial lipoproteins are membrane-associated proteins with a characteristic acylated N-terminal cysteine residue anchoring C-terminal globular domains to the membrane surface. While all lipoproteins are modified with acyl chains, the number, length, and position can vary depending on host. The acylation pattern also alters ligand recognition by the Toll-like receptor 2 (TLR2) protein family, a signaling system that is central to bacterial surveillance and innate immunity. In select Listeria monocytogenes isolates carrying certain plasmids, copper exposure converts the lipoprotein chemotype into a weak TLR2 ligand through expression of the enzyme lipoprotein intramolecular acyltransferase (Lit). In this study, we identify the response regulator (CopR) from a heavy metal-sensing two-component system as the transcription factor that integrates external copper levels with lipoprotein structural modifications. We show that phosphorylated CopR controls the expression of three distinct transcripts within the plasmid cassette encoding Lit2, prolipoprotein diacylglyceryl transferase (Lgt2), putative copper resistance determinants, and itself (the CopRS two-component system). CopR recognizes a direct repeat half-site consensus motif (TCTACACA) separated by 3 bp that overlaps the -35 promoter element. Target gene expression and lipoprotein conversion were not observed in the absence of the response regulator, indicating that CopR phosphorylation is the dominant mechanism of regulation. IMPORTANCE Copper is a frontline antimicrobial used to limit bacterial growth in multiple settings. Here, we demonstrate how the response regulator CopR from a plasmid-borne two-component system in the opportunistic pathogen L. monocytogenes directly induces lipoprotein remodeling in tandem with copper resistance genes due to extracellular copper stress. Activation of CopR by phosphorylation converts the lipoprotein chemotype from a high- to low-immunostimulatory TLR2 ligand. The two-component system-mediated coregulation of copper resistance determinants, in tandem with lipoprotein biosynthesis demonstrated here in L. monocytogenes, may be a common feature of transmissible copper resistance cassettes found in other Firmicutes.
