Non-coding RNAs as emerging players in Leishmania development and host-parasite interactions

Eyson QuicenoZemfira N Karamysheva^*

• Texas Tech University Health Sciences Center, Lubbock, United States

Leishmaniasis is a neglected disease, widespread throughout the world. It represents a major global health challenge due to its economic and social implications. It is caused by protozoan parasites of the genus Leishmania (1,2). Unlike most eukaryotes, Leishmania has an atypical genome, characterized by its high plasticity (3)(4)(5)(6), the absence of introns, polycistronic constitutive transcription of its genes and lack of gene regulation at the transcriptional level (4,(7)(8)(9). Because of this, the regulation of gene expression in Leishmania and trypanosomatids in general occurs at the post-transcriptional level. Recent studies have found that non-coding RNAs (ncRNAs) play an important role in these regulatory mechanisms in trypanosomatids, however, the precise function and mechanisms associated with them are poorly understood (10)(11)(12)(13)(14).In general, ncRNAs are a class of RNA transcripts that are not translated into proteins but serve essential regulatory functions in a variety of biological processes. ncRNA are typically categorized based on length or functions. Based on length, they are classified into small ncRNAs (<200 nucleotides) and long ncRNAs (>200 nucleotides) (15)(16)(17)(18). Based on function, they are divided into two major categories: 1) housekeeping ncRNAs (ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs) and telomerase RNA (TERC); these ncRNAs are ubiquitously expressed and participate in fundamental cellular activities and 2) regulatory ncRNAs (microRNAs (miRNAs), small interfering RNAs (siRNAs), PIWI-interacting RNAs (piRNAs), tRNA-derived fragments (tRFs), and tRNA halves (tiRNAs), enhancer RNAs (eRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs)); these are involved in fine-tuning gene expression at multiple levels -epigenetic, transcriptional, and post-transcriptional (17)(18)(19)(20).Similar to other eukaryotes, Leishmania parasites carry same types of housekeeping ncRNAs. A diverse repertoire of regulatory ncRNAs has been identified across various Leishmania species including siRNAs (21)(22)(23), tRNA-and rRNA-derived small RNAs (23,24), snoRNAs (25)(26)(27)(28)(29), and lncRNAs (13,30,31). Although many Leishmania species lost the canonical RNA interference (RNAi)Deleted: pathway, however, several species in the Viannia subgenus (Leishmania braziliensis and Leishmania guyanensis) (32) retain a functional RNAi machinery capable of producing siRNAs (21,22). Importantly, while canonical miRNAs have not been identified in Leishmania, some in silico studies have proposed the existence of miRNA-like molecules with potential regulatory function (33)(34)(35). However, these findings remain speculative and require in vitro validation before any definitive conclusions can be drawn. Complementing these predictions, various RNA-seq datasets have revealed a wide range of ncRNAs encoded in the Leishmania genome. In many cases these transcripts remain functionally uncharacterized; they have been identified in species such as L. braziliensis (14,(35)(36)(37), L. amazonensis (38,39), L. major (25-27, 35, 40), L. donovani (28,29,35,40), L. infantum (30,31), L. mexicana (41).Across a wide range of organisms, ncRNAs have been implicated in mRNA processing, mRNA stability and emerge as key players in a variety of regulatory processes, such as DNA replication, chromosome maintenance, transcriptional regulation, translation, protein stability, the translocation of regulatory proteins and host-parasite interactions (40). Understanding the role and mechanisms of ncRNAs actions in Leishmania and host may lead to new avenues in the search for strategies to control leishmaniasis. Leishmania parasites are characterized by the absence of classic genetic control at the transcriptional level, therefore, one of the main roles of ncRNAs could be at the post-transcriptional level (Figure 1A), through regulation of mRNA stability, processing, transport, and degradation; these processes at the post-transcriptional level have been seen in other protozoa (42,43). Recent evidence suggests a significant presence of regulatory ncRNAs derived from untranslated regions (UTRs) of mRNAs (40). RNA-Binding Proteins (RBPs) are central to post-transcriptional regulation (44). Over 2,400 RBPs, including non-poly(A)-binding proteins, form complexes with ncRNAs, suggesting roles in RNA transport and stability in L. mexicana (41).In the absence of transcriptional regulation translational control plays a crucial role in Leishmania gene expression supporting survival and adaptation to dramatically different environments during change of host (45)(46)(47)(48)(49). In eukaryotes ncRNAs play direct roles in modulating protein synthesis, either by interacting with ribosomes, regulating the availability of mRNAs for translation or governing modifications of ribosomal proteins (50). rRNAs facilitate the peptidyl transfer reaction during protein synthesis (51). Recent studies support importance of rRNA modifications by snoRNAs in L. major (25). snoRNAs are organized in gene clusters containing both C/D and H/ACA types, guiding rRNA modifications like 2'-O-methylation (Nm) and pseudouridylation (25,27). These modifications occur in conserved rRNA domains and are critical for rRNA maturation, stability and mRNA translation.In L. infantum, a class of lncRNAs (300-600 nucleotides) was mainly identified in amastigotes, showing a preferential association with the small ribosomal subunit (40S) (30). These findings indicate a possible role in regulation at the translation level, although a direct effect on translational initiation has not been demonstrated. In L. braziliensis, the lncRNA45 was functionally characterized, demonstrating possible roles in RNA processing and modulation of translation rates, either enhancing or impairing protein synthesis (13). Additionally, a small ncRNA called ncRNA97, was found to be preferentially expressed in the amastigote form of L. braziliensis (14). This ncRNA modulates gene expression through the control of the stability of the mRNAs that are involved in metacyclogenesis and responses to nutritional stress, indicating a role in developmental adaptation.These examples underscore the intricate and multifaceted roles of ncRNAs in regulating gene expression in Leishmania parasites. However, the number of examples is limited, so the specific functions and mechanisms are still areas to be explored. Leishmania, being an intracellular pathogen, has machinery that allows it to adapt and survive the hostile environment within the hosts. One of the main mechanisms of Leishmania to alter the host's responses favoring parasite survival involves host transcriptome remodeling that includes modulating the expression of both coding RNAs and ncRNAs such as miRNAs (52)(53)(54)(55)(56)(57)(58)(59)(60)(61)(62)(63)(64)(65)(66)(67), lncRNAs (62,(68)(69)(70)(71)(72) and circRNAs levels (73,74), (Figure 1B). Interestingly, 30% of differentially expressed transcripts in infected macrophages correspond to lncRNAs supporting their importance to control macrophage function during infection (68).Leishmania modulates host immune responses via alteration of host ncRNA expression profiles, affecting processes such as apoptosis, phagocytosis, and immune signaling (65,66,68,75). L. donovani infection in CD4+ T cells upregulates certain miRNAs (miR-6994-5p, miR-5128, miR-7093-3p, miR-574-5p and miR-7235) which interferes with the expression of the pro-inflammatory cytokine IFN-γ (52). Moreover, the downregulation of miR-340-5p, miR-93-3p, let 7j, 486a-3p and miR-3473f promotes the differentiation of macrophages towards a Th2 phenotype, favoring the survival of the parasite. In L. braziliensis the upregulation of miR-2940-3p and miR-5100 caused suppression of TNF and NF-κB pathways, reducing inflammatory responses (53). L. amazonensis parasites are able to change the TLR signaling pathways by modulating the expression level of miRNA-let-7e (54). The upregulation of this miRNA decreases the inflammatory response of host cells. Also, L. amazonensis induces an upregulation of miR-294 and miR-721 in macrophages (55). This upregulation causes a repression of inducible nitric oxide synthase (NOS2) leading to reduced production of nitric oxide and subsequent decrease in the capability of the macrophage to kill the parasites. L donovani and L. major causes an upregulation of miR-210 in macrophages under hypoxic conditions, leading to downregulation of pro-inflammatory cytokines and enhancing parasite survival (56,57).L. infantum infection has been shown to alter the expression of numerous lncRNAs in human neutrophils, leading to the impairment of key antimicrobial responses such as phagocytosis and nitric oxide production (75). This contributes to immune evasion and the survival of the parasite. In THP-1 macrophages, the infection with L. amazonensis, L. braziliensis, and L. infantum led to a differential expression of different host lncRNAs upon infection suggesting a mechanism by which Leishmania can control macrophages and evade the immune response (68). A test performed on peripheral blood from patients infected with visceral leishmaniasis cured patients; asymptomatic infected individuals and healthy controls showed that L. infantum alters the expression of host lncRNAs (69). These lncRNAs are co-expressed with immune-related protein-coding genes and may regulate immune pathways, potentially influencing the host's ability to respond to infection. Leishmania infection of macrophages leads to downregulation of host lncRNA 7SL RNA, an essential component of the signal recognition particle (SRP), which is responsible for targeting newly synthesized proteins to the endoplasmic reticulum (ER). This generates a downregulation of protein targeting and secretion altering trafficking of immune effectors and antigen presentation, favoring parasite persistence (70).Leishmania influences host cellular metabolism to create a more favorable environment for its survival. miR-210 has been linked to altered L-arginine metabolism, leading to a reduction in nitric acid production. Certain miRNAs (miR-372/373/520d family) are upregulated in human macrophages during infection with L. amazonensis, leading to changes in arginine metabolism and increased polyamine production, which supports parasite survival (58). Inhibiting these miRNAs reduces parasite survival. During infection of bone marrow-derived macrophages with L. amazonensis the miRNAs miR-294 and miR-410 were upregulated; these miRNAs can interfere with the production of L-arginine and the immune response in the macrophages (59). L. donovani also modulates host miRNAs that regulate cholesterol and sphingolipid biosynthesis which are crucial for the parasite's survival (65,66). While the functional impact of lnRNAs and miRNAs is only beginning to be uncovered, host circRNAs have emerged as another relevant class of ncRNAs in leishmaniasis. A recent study found a large number of circRNAs differentially expressed in the serum of patients with leishmaniasis compared with healthy controls (73) while in THP-1 cells infected with L. tropica and L. infantum distinct circRNAs profiles were found depending on the parasite strain (74).In addition to directly modulating host ncRNAs, Leishmania also influences the host environment through the secretion of extracellular vesicles, particularly exosomes (Figure 1B). During infection, those exosomes can modulate the host immune response (76)(77)(78). tRFs and other small RNAs have been detected in exosomes secreted by Leishmania, suggesting a role in intercellular communication and possibly in the modulation of host translation (23,24). These vesicles can modulate immune responses in different ways. It has been found that the parasite is able to release exosomes in sand flies and host cells, which can stimulate an inflammatory response leading to exacerbated cutaneous leishmaniasis (79). In vivo studies have demonstrated that treatment of mice with L. donovani exosomes prior to challenge with the parasite exacerbates infection, promotes IL-10 and inhibits TNFα production (80). These findings indicate that Leishmania exosomes, with their ncRNA cargo, are predominantly immunosuppressive and play a significant role in shaping the host immune response to favor parasite survival.Together, the modulation of host miRNAs, lncRNAs and circRNAs as well as the production of exosomes in the parasite uncovers the strategy by which Leishmania manipulates host immune responses and cellular functions for its own benefit. While progress has been made in studying how the parasite alters host ncRNAs, the possible functions of Leishmania's own ncRNAs (beyond those In conclusion, the study of ncRNAs in protozoan parasites is still in 325 its early stages compared to higher eukaryotes. However, the The ncRNAs in black correspond to those validated with RT-qPCR, while in purple correspond to those that have not yet been validated. In the description of ncRNAs, "hsa" refers to ncRNAs from humans (Homo sapiens) while "mmu" refers to ncRNAs from mice (Mus musculus).Biorender software was used to create this figure under an academic license.