The Apetala 2 factors, originally identified in plants, have expanded into the Apicomplexa phylum [15]. The ApiAP2 family has been the subject of ongoing studies and has been identified as an important class of transcriptional regulators that act as transcriptional activators or silencers in Plasmodium [19,76,77,78,79,80,81]. A total of 27 factors with AP2 domains were identified, and these factors do not have homologs in the human genome or the transmitting Anopheles vectors. In-depth studies that individually examine the function of these transcription factors, such as the recent paper by Russell and colleagues [82], are of great importance for strengthening and developing anti-malarial strategies. P. falciparum appears to utilize transcription factors from the ApiAP2 family to regulate gene expression, and at least 80% of protein-coding transcripts are regulated during the intraerythrocytic cycle [19]. Consequently, proteins belonging to the AP2 family play central roles in the genetic regulation of these parasites and assist in maintaining the complex life cycle of this apicomplexan. The transcription factors ApiAP2 that were individually approached in their function in the parasite’s life cycle within the vertebrate host organism are AP2-L [76], SIP2 [83], AP2-I [84], AP2SP3-TEL [85], AP2-O [86], AP2-EXP [81], AP2-G [87,88], AP2-G2 [89], AP2-G3 [78,90], AP2-G4 [71], and AP2-G5 [91]. The AP2-L factor is known to be essential for the development of the parasite within hepatocytes, and as of now, there are no records of another transcription factor (TF) acting during the hepatic phase of Plasmodium [76]. Described as recognizing motifs AATTTCC, the ortholog of PF3D7_0730300 (gene ID code in www.PlasmoDB.org) encodes a 1272-amino acid protein with two AP2 domains. Studies on transcriptional and histone modifications have reported that AP2-L is overexpressed in sporozoite stages in P. falciparum [92], although its expression also occurs in the trophozoite and schizont stages [76,92]. The knockout of AP2-L in the P. berghei model of infection does not affect the invasion of sporozoites into hepatocytes; however, it causes disruptions in growth and cell division after 24 and 36 h, respectively [78]. Despite the hepatic phase of the parasite being a potent target for malaria infection drugs and vaccines, genetic regulation during this stage is not yet well elucidated.

During the phase of merozoite invasion into erythrocytes within the vertebrate host, the parasite expresses numerous surface proteins to facilitate this process. The transcription factor AP2-I, along with bromodomain protein 1 (PfBDP1) [93], is closely associated with the control of the expression of these genes [84]. This AP2-I protein is detected in the nuclei of trophozoites and schizonts but is absent in newly invaded forms (ring stage), and its coding gene seems to possess the capability for autoregulation, a feature attributed to the inclusion of its own DNA-binding motif among its regulatory targets, as elucidated by Santos et al. in 2017 [84].

Due to its importance for the intraerythrocytic developmental stage, Oladejo et al. (2023) conducted in silico predictions of various molecules with potential inhibitors of the binding of the AP2-I domain to its DNA-binding motif. Five compounds were considered suitable for post-docking studies, with the molecule MCULE-7146940834 being indicated as the main candidate. Experimental preclinical validations are required to assess its effectiveness as an AP2-I inhibitor [94].

The transcription factor PfSIP2 (PF3D7_0604100) is associated with heterochromatin formation and genome integrity rather than transcriptional regulation. During schizogony, the 60 kDa PfSIP2 protein binds to SPE2 motifs in the N-terminal portion using only one of its two DNA-binding domains, the D1 domain, and its deletion is refractory [19,83]. The recognition sequence of PfSIP2 is located upstream of subtelomeric var genes in UpsB regions associated with telomeres [19]. In an in-silico study, approximately 700 sites were targeted by PfSIP2, of which 94% correspond to two distinct regions of subtelomeric heterochromatin, one to tandem sequences located upstream of this region in var genes, and the other associated with 2/3 of repetitive elements within telomeric regions (TAREs). These results suggest a multifunctional role for this protein and its potential role in heterochromatin formation [83]. It is important to highlight that in the case of both P. falciparum and the murine species P. berghei, the SIP2 transcription factor cannot be deleted, indicating its essential role in the intraerythrocytic cycle of the parasite [83,95]. This warrants further studies for finding inhibitors of PfSIP2.

PfAP2-Tel, encoded by PF3D7_0622900, has a size of 237 kDa and contains a single 46-amino acid AP2 domain. Despite this AP2 domain showing a smaller size than the average of other AP2 domains in P. falciparum, this domain enables PfAP2-Tel to directly bind to telomeric repeats [85]. Its expression occurs during the blood stage with peaks of enrichment at all 28 telomeres of P. falciparum, and also in important gene families such as var, rifin, stevor, and Pfmc-2TM [85]. This indicates that PfAP2-Tel may have a function related to the maintenance of chromosomal ends. However, this protein is deemed non-essential, given that no change in parasite fitness was observed [96]. Along with PfSIP2 and PfAP2-Tel, PfAP2-HC also has a primary function related to heterochromatin and the end biology of the chromosome [97]. The results regarding the expression pattern of PfAP2-HC, for a better understanding of its function in the parasite’s genome, showed that the protein was detectable in trophozoites and in immature schizonts, but was not enriched in the predicted target motifs CACACA [19]. It was also found that its AP2 domain is dispensable for DNA binding. In order to associate with heterochromatin, PfAP2-HC is dependent on PfHP1 which, like the trimethylation of histone 3 lysine 9 (H3K9me3), is closely associated with condensed chromatin regions [98]. Interestingly, the knockout of PfAP2-HC leads to the almost complete silencing of var genes, suggesting an interaction with chromatin modifiers [99], such as the non-recruiting of SET/HMT proteins to var loci. The efficient blockage of PfAP2-HC may make IRBC less adhesive, given that var genes are suppressed and the corresponding PfEMP1, which mediates cytoadherence to endothelial cells [100], may be not properly expressed. Theoretically, this inhibition would render IRBC vulnerable to splenic clearance.

Gametocytes/gamonts are the forms ingested by the mosquito vector which ensure the transmission of the parasite to this host. These originate from the differentiation of asexual cells and undergo a series of developmental stages, encompassing commitment, conversion, and sexual maturation [101]. ApiAP2-G is the master regulator of sexual commitment [87,88,102], a stage that may occur before schizogony, referred to as the conversion route in the next cycle of the expression of AP2-G. In P. falciparum, it can also occur at the beginning of the ring stage, resulting in the same cycle conversion [88,103]. In parasites that do not express AP2-G, the gene encoding this protein, PF3D7_1222600 (ortholog PBANKA_143750/ PY17X_1440000), is found in a heterochromatic state, regulated by the epigenetic mark H3K9me3, histone deacetylase 2 (PfHda2), and associations with heterochromatin protein 1 (PfHP1) [37,104]. The stabilization of AP2-G expression is achieved by the removal of these bindings through mechanisms involving the protein GDV1 (gametocyte development 1), which acts as a positive regulator of sexual commitment.

Predicted to bind to DNA through the recognition of the motif (Gx)GTACNC [19], it was identified that these binding regions were also present upstream of the AP2-G-encoding gene itself, indicating the existence of the control of its expression through positive feedback [87]. Furthermore, AP2-G appears to be located at the top of a specific transcriptional cascade of gametocytogenesis, in which AP2-G2, AP2-FG, and AP2-O3 are directly influenced by this master regulator [105].

Many genes regulated by PfAP2-G are also targets of the transcription factor PfAP2-I, and studies indicate their possible direct and joint action on these promoters, an important mechanism that increases gene regulation specificity [106]. It is important to note that while AP2-G is continuously observed in P. falciparum erythrocytic stages at low levels, in P. berghei and P. yoelii, its expression occurs only for a short period, suggesting divergence in the sexual development between these species [90,105]. Although the genomic loci where PfAP2-G associates were discovered [106], it is currently not known which factors physically interact with the factor and if PfAP2-G binds as a multimer or heteromer. This lack of information is also true for all other plasmodial AP2 factors.

Another transcription factor that was identified in Plasmodium spp. with important functions during gametogenesis is AP2-G2 (PF3D7_1408200/PBANKA_1034300), which is expressed in sexually committed ring forms and demonstrates protein expression during the trophozoite and schizont stages [107]. Knockout assays revealed that even in the absence of its gene disruption, there is no discernible impact on the sexual commitment phase of the parasites. However, AP2-G2 knockout parasites were unable to develop beyond stage III-type gametocytes, leading to a blockage in transmission to the vector [78,89,107]. Consequently, AP2-G2 appears to assume a critical role after the activation of AP2-G within the cascade of ApiAP2 proteins associated with gametogenesis [87]. ChIP-seq analyses revealed that the binding motif of AP2-G2 was identified in regulatory regions of 1500 genes, and although many of these targets were related to proliferation in the blood stage [89], no negative effects were observed in these genes during the knockout of AP2-G2. Another transcription factor, ApiAP2-L, which is critical for liver stage development, positively regulated AP2-G2 knocked-out gametocytes in P. falciparum and P. berghei, resulting in the inability of these parasites to trigger liver infection [95,107]. Together, these data suggest that AP2-G2 acts as a repressor during the sexual and asexual stages. In contrast, Xu and colleagues (2021) [108] identified that the deletion of PfAP2-G2 in P. falciparum had a repressive effect on the expression of PfMDV-1 (male development gene) in asexual stages, suggesting the possibility of a dual role for this protein as a repressor and activator of specific genes [101]. Furthermore, PfAP2-G2 shares 80% of its binding sites with epigenetic silencing marks H3K36me3, indicating the potential for collaborative interactions between them [107]. A third AP2 factor related to the gametogenesis phase, AP2-G3, appears to act upstream of AP2-G, translating cytosolic signals into the nucleus and influencing AP2-G transcription [90,109]. In P. berghei, this transcription factor is essential for the regulation of genes specific to the formation of female gametocytes and is named AP2-FG for this species [110]. PfAP2-G5, identified by Shang et al. [91], was considered indispensable for the gametocyte development phase as it binds to the regulatory region of PfAP2-G, inhibiting its activation and that of its target genes, preventing the initiation of sexual commitment. According to the authors, the role of AP2-G5 may be involved in various physiological processes beyond gametogenesis, such as parasite–host interaction remodeling, pathogenesis, and others [91]. The blockage or inhibition of the function of PfAP2-G5 may therefore be useful to render parasites into sexual stage forms that can no longer replicate, nor be transmitted, due to its dual role in controlling AP2-G production and gametocyte maturation. This issue has not yet been approached.

Studies conducted with AP2-exp (PF3D7_1466400) suggest that this protein is involved in the asexual cycle of P. falciparum. At the same time, its ortholog is a strong regulator of sporogony in a murine model for P. berghei (PbAP2-Sp). PfAP2-exp appears to be also involved in the regulation of multigene families, including Rif, Stevor, and Pfmc-2tm [81]. This important transcription factor may be related to the control of parasite virulence, as binding sites in the promoters of var genes have been predicted [19]. The association of PfAP2-exp with accessible chromatin regions [99] and its participation in the PfGCN5 acetyltransferase complex [111] are responsible for preferentially acetylating histone 3 at lysine 9 and lysine 14 (K9, K14), which is considered an important mark related to active genes. The presence of the protein is detectable in the nucleus of schizonts in P. falciparum. Apparently, this transcription factor is also involved in the asexual growth of the parasite, as the complete deletion of PfAP2-exp failed [81,99]. This reinforces the hypothesis that PfAP2-exp may also be considered a useful drug target, as recently shown by Russell and colleagues [82].

Another member of the ApiAP2 family, AP2-O (PF3D7_1143100), was the first member of the AP2 family with a function specifically related to the morphology or formation of ookinetes and oocysts, and these functions were characterized in P. berghei (PBANKA_0905900) [76,79]. To date, only a few studies describe the function or active participation of this transcription factor in the asexual phase of the parasite, specifically during the intraerythrocytic stage. Blocking the AP2-O protein through knockout in P. berghei was demonstrated in Anopheles mosquitoes, where the ookinete phenotype was aberrant, showing an inability to invade the mosquito’s midgut [79]. Additional data demonstrated that AP2-O binds to the promoters of most genes dysregulated in the parasite [77,79], and it may have a role in the asexual cycle, influencing the parasite’s growth within erythrocytes [78]. In P. falciparum, AP2-O is involved in the transmission stages and regulation of virulence genes, such as var genes. However, the knockout of this transcription factor is refractory, and the knockdown via a destabilizing domain resulted in no visible phenotype in the erythrocytic stages of the parasite [86], probably due to the leakiness of the type of knockdown used. Recently, another AP2 factor – PfAP2-P - was found to be essential during the blood stage. The factor apparently controlled a vast number of transcripts important not only for egress/invasion but also transcripts from variant family genes and gametocyte developmental markers [112], indicating a very important general role for this factor, at least in P. falciparum. Surely, inhibitors of PfAP2-P would probably have an immediate and huge impact on parasite proliferation, warranting further research on this factor.