The central dogma of biology, as described by Francis Crick (Crick, 1970), provided a solid comprehension of the genetic flow followed by most cells, despite its limitations. The idea that genetic information flows from DNA to RNA molecules via transcription and from RNA to proteins via translation is still being vastly explored in molecular biology practices (Li and Xie, 2011; Liu et al., 2018; Schneider-Poetsch and Yoshida, 2018). The latest advances in genomics, transcriptomics and proteomics have enabled assessing levels of gene and protein expression in cells under different conditions (Kim et al., 2010; Zhang et al., 2014; Segundo-Val and Sanz-Lozano, 2016; Aslam et al., 2017; Hung and Weng, 2017). Considering that several mechanisms are involved in transcription and translation regulation, inferences can be made to extrapolate one type of data to predict the other, even though there is no trivial relationship between levels of transcripts and proteins (Jansen et al., 2002; Greenbaum et al., 2003; De Sousa Abreu et al., 2009; Buccitelli and Selbach, 2020).

In most eukaryotes, known regulatory mechanisms present monocistronic mRNAs with transcription regulated by individual promoters, enhancers and transcription factors (Cramer, 2019) resulting in highly regulated RNA production. Some of these mechanisms, however, are not present in trypanosomatids such as Leishmania, which exclusively utilizes posttranscriptional regulation (Martínez-Calvillo et al., 2004; Ivens et al., 2005; Clayton, 2016; De Pablos et al., 2016; Clayton, 2019). The absence of gene-specific transcription regulation raises questions about the relevance of quantifying mRNA in these organisms: How could the quantification of mRNA provide insight on the orchestration of the phenotype in different scenarios? What could be the relevance of measuring mRNA levels in these organisms? How is mRNA information relevant for overall expression analysis?

Leishmania is a protozoan parasite and causative agent of leishmaniases, a group of diseases characterized by cutaneous, mucocutaneous or visceral lesions. Leishmaniases is caused by at least 20 species of the Leishmania genus, affecting approximately 0.7 to 1 million people every year in nearly 100 endemic countries (Burza et al., 2018). According to the World Health Organization (WHO), leishmaniases are emerging tropical neglected diseases for which new treatments should be prioritized (Burza et al., 2018).

The parasite presents two different life forms: the promastigote and the amastigote ( Figure 1 ). Drastic changes in pH, temperature and nutrient availability are related to differentiation (Gupta et al., 2001); these changes challenge the parasite’s ability to adapt under a lack of regulation at the transcriptional level. The promastigote forms present a motile elongated body with an apparent flagellum, live and multiply within the digestive tract of sandflies (25°C and pH ~7.0) in a microenvironment containing insect gut nutrients, digestive enzymes associated with its microbiota and saliva components (Kamhawi, 2006; Dostálová and Volf, 2012). In this environment, the promastigote undergoes a series of morphological changes that culminate in differentiation into metacyclic forms, which are the infectious and nonreplicative stage. This process includes the differentiation of procyclic promastigotes into nectomonad promastigotes, leptomonad promastigotes and metacyclic promastigotes (Sacks and Perkins, 1984; Bates and Tetley, 1993; Serafim et al., 2012). Metacyclic promastigotes are defined as highly infective, rapidly swimming, nonproliferative and present a long flagellum that allows motility to infect a mammalian host (Sacks and Perkins, 1984). Metacyclic promastigotes infect the host during the sandfly blood meal and differentiate into amastigotes once inside host cells. Additionally, new insight into parasite development have been described and have led to a revised Leishmania life cycle (Bates, 2018). Furthermore, metacyclic promastigote forms are able to dedifferentiate in the sandfly, enhancing parasite population growth through a second blood meal and providing a greater disease transmission potential (Serafim et al., 2018).

Once inside mammalian host cells, metacyclic promastigotes differentiate into amastigotes. The amastigote stage of Leishmania displays a rounded cell body with a nonapparent flagellum (Howells and Gardener, 1972; Gardener et al., 1973; Hentzer and Kobayasi, 1977). These life forms live and multiply inside the host phagocytic compartment (36°C and pH ~5.0).

The advent of high-throughput technologies has improved the analysis of genomes, transcriptomes and proteomes; however, establishing a correlation among these profiles is still a challenge. Most studies focus individually on mRNA or protein expression, as performing both can be time-consuming, expensive and demands trained personnel. Despite that, it is worth noting that general trends in groups of genes observed in transcriptomes or proteomes are not predictive of individual gene or protein expression. Therefore, the present review aims to discuss the correlation between mRNA and protein levels in the Leishmania life cycle, mainly with regard to metacyclogenesis and amastigote differentiation.

Metacyclogenesis is essentially the process of differentiation from procyclic to metacyclic promastigotes. Throughout this process, procyclic promastigotes differentiate into nectomonad and leptomonad promastigote stages, finally becoming metacyclic promastigotes (Sacks and Perkins, 1984; Bates and Tetley, 1993; Sacks and Kamhawi, 2001; Serafim et al., 2012). At the proteomic level, metacyclic promastigotes present a reduction in the abundance of proteins involved in protein synthesis and an increase in proteins involved in cell motility (Mojtahedi et al., 2008; Amiri-Dashatan et al., 2020b). Additionally, transcriptomic analysis of metacyclogenesis has demonstrated that each promastigote life stage has exclusive differentially expressed transcripts associated with them (Inbar et al., 2017; Coutinho-Abreu et al., 2020).

Overall, the specific conditions that trigger metacyclogenesis have not been fully disclosed. In nature, this process occurs within the midgut of the sandfly and involves stress induction and a lack of nutrients (Bates and Tetley, 1993; Kamhawi, 2006; Bates, 2008). Furthermore, the absence of purines seems to play a role in triggering metacyclogenesis (Serafim et al., 2012). Binding to the midgut of the insect is an essential process for promastigote development and this binding capability is strictly stage specific: it is observed in the leptomonad and nectomonad uninfective promastigote forms but it is not common in the metacyclic stage (Pimenta et al., 1992; Wilson et al., 2010). Surface glycoconjugate lipophosphoglycan (LPG) has been described as responsible for host midgut binding in L. major (Pimenta et al., 1992) and it is also hypothesized to be the molecule responsible for midgut binding in other Leishmania species (Sacks and Kamhawi, 2001). Detachment and release from the host midgut occurs as the LPG molecule becomes elongated and modified by transferase enzymes (McConville et al., 1992; Novozhilova and Bovin, 2010). The observed stage-specific ability to bind to the host midgut found in different Leishmania species (Pimenta et al., 1992; Wilson et al., 2010) is a finding that might indicate that the expression pattern of mRNAs and proteins involved in LPG synthesis is accordingly regulated.

Since the host midgut microenvironment plays an important role in metacyclogenesis, questions on the reliability of cultured promastigotes as a model, compared to sandfly-derived promastigotes, have emerged (Alcolea et al., 2016). The insect microenvironment contains several digestive enzymes and a microbiota. Some stage-specific molecules have been identified to play a role in protecting the parasite from proteolytic activity found within the insect midgut (Sacks and Kamhawi, 2001). Other molecules present in the saliva of the insect are also crucial to the development of infection in the mammal and are capable of suppressing the host immune response and determining the fate of the infection (Katz et al., 2000; Andrade et al., 2007). Transcriptomic comparisons among sandfly and culture-derived metacyclic promastigotes have revealed an overall transcriptional similarity, with specific differences in transcripts associated with nutrient stress (such as amino acid transport, fatty acid biosynthesis, catabolism of ketone bodies, and protein recycling via autophagy) that appear upregulated in cultured metacyclic promastigotes (Inbar et al., 2017).

To assess correlations between mRNA and protein levels in metacyclogenesis, we analyzed data from 6 different studies, 3 of which contained transcriptomic data (Almeida et al., 2004; Dillon et al., 2015; Inbar et al., 2017) and 3 of which contained proteomic data (Mojtahedi et al., 2008; Amiri-Dashatan et al., 2020a; Amiri-Dashatan et al., 2020b) from L. major and L. tropica, differentiating from procyclic to metacyclic promastigotes. To standardize our comparisons, we used the L. major Friedlin genome as a reference for gene IDs. Even though there are obvious differences in transcriptomic and proteomic profile between these two Leishmania species, the authors decided to use gene IDs of L. major since it is the most complete genomic database among Leishmania species. In cases in which the reference genome was another, we obtained syntenic orthologs of the L. major Friedlin genome using TryTrip database (www.tritrypdb.org). It is also worth noting that our search for studies upon which to base the present work retrieved a significantly smaller number of proteomic and transcriptomic studies of Leishmania metacyclogenesis in comparison to those focusing on amastigote differentiation. All analyzed data was statistically verified by each individual study then, the authors of this review did not interfere with or perform own statistical analysis of the data used. It is important to take into consideration that different studies may have presented different statistical thresholds to determine the own differentially expressed genes and proteins. Because of this limitation, the results observed here should be interpreted as proof-of-concept, since they were obtained from an extensive literature review and manual ad-hoc curation. The analysis was performed using a simple search tool in a table containing raw data from every analyzed study to find correlation between protein and mRNA trends. There is a consideration that some genes in the Leishmania genome can present the same coding region but differ in UTRs (Rastrojo et al., 2019). This aspect of the Leishmania genome organization is very complex and not discussed within the analyzed studies that were based solely on coding regions.

Table 1 shows differentially expressed mRNAs and proteins according to transcriptomic and proteomic studies comparing procyclic and metacyclic promastigotes. For most genes, correlations between protein and mRNA levels were detected, whereas some genes show opposite trends in comparisons. We analyzed genes within the following biological groups: stress response, mitochondria, gene expression, energy metabolism and cell signaling. Hypothetical proteins and other biological functions were also considered ( Table 1 ). The genes were grouped to facilitate the analysis. Gene Ontology was originally characterized in the respective study (Almeida et al., 2004; Mojtahedi et al., 2008; Dillon et al., 2015; Inbar et al., 2017; Amiri-Dashatan et al., 2020a; Amiri-Dashatan et al., 2020b).

For the stress response, two peroxidases (LmjF.26.0800 and LmjF.15.1040) appeared to be upregulated at both the mRNA and protein levels in metacyclic promastigotes in comparison to procyclic promastigote forms. This may represent an adaptation of the parasite during metacyclogenesis, preparing to infect mammalian cells. A putative gene (LmjF.12.1130 - NADH/flavin oxidoreductase) and a heat shock protein (LmjF.33.2390 - TRAP1/HSP75) did not present correlation between protein and mRNA levels, with both showing a decrease in transcript levels in metacyclic promastigotes and an increase in protein levels ( Table 1 ).

During metacyclogenesis, correlation between mRNA and protein levels was observed for genes encoding for mitochondrial proteins: two cytochrome c oxidase subunits (LmjF.26.1710 and LmjF.25.1130) appeared downregulated in metacyclic promastigotes compared to procyclic promastigotes ( Table 1 ).

The most expressive finding for metacyclogenesis was for gene expression-related genes. Of the 11 related genes found in our analysis, 8 (over 72%) were found to consistently be downregulated (both mRNA and proteins) in metacyclic promastigotes. This category included elongation factors (LmjF.18.0740, LmjF.36.0180, LmjF.34.0840), RNA helicases (LmjF.21.1552, LmjF.35.3100), histone H3 (LmjF.10.0870), guanosine monophosphate (GMP) reductase (LmjF.17.0725) and the protein transport protein SEC13 (LmjF.32.0050). This downregulation in gene expression-related proteins is in accordance with the previously demonstrated reduction in transcription and translation in metacyclic promastigotes (Kloehn et al., 2015).

Proteins involved in cell energy metabolism, such as phosphoglycerate kinase (PGKC) (LmjF.20.0100) and pyruvate kinase (LmjF.35.0030), were increased in both mRNA and protein levels during metacyclogenesis ( Table 1 ). On the other hand, ATP-dependent 6-phosphofructokinase (PFK) (LmjF.29.2510), a main regulator of glycolysis (Mor et al., 2011), showed decreased levels during metacyclogenesis in L. tropica ( Table 1 ), possibly indicating a reduction in glycolytic activity. These data, with reduced levels of cytochrome c oxidase subunits V and VII, corroborate the hypothesis that cell respiration is affected upon metacyclogenesis. Additionally, greater energy consumption has been shown to occur in procyclic promastigotes of L. mexicana than in metacyclic promastigotes due to a higher replication rate (Costa et al., 2011).

Interestingly, glutamate dehydrogenase (GDH) (LmjF.28.2910) shows opposite trends in different Leishmania species In L. major, both mRNA and protein levels of GDH increased in metacyclics in comparison to procyclic promastigotes. In L. tropica, however, there was a decrease in GDH (LmjF.28.2910) during metacyclogenesis at both protein and mRNA levels. Nonetheless, another GDH (LmjF.15.1010) presented no correlation when we compared L. major and L. tropica data, with decreased mRNA levels but increased protein levels ( Table 1 ).

A putative lipophosphoglycan biosynthetic protein 3 (LPG3) is reportedly downregulated in metacyclic forms of L. tropica and L. major (Inbar et al., 2017; Amiri-Dashatan et al., 2020a). LPG is a protein responsible for insect midgut binding and also acts as a virulence factor in mammalian hosts. This supports the idea that regulation of LPG expression is stage-specific, as is the ability of Leishmania to bind to the host midgut. (Pimenta et al., 1992; Sacks and Kamhawi, 2001; Wilson et al., 2010). Metacyclic promastigotes, however, present upregulation of transferase mRNAs, in accordance with the previous identification of elongated LPG in this specific phase (Rowton et al., 1995). Adding to this hypothesis, mRNA for the glycosyltransferase gene, which is involved in elongation and modification of LPG, appears to be upregulated in nectomonads (Coutinho-Abreu et al., 2020). Other transferases (galactosyl and mannosyl-transferases) are also upregulated in nectomonad and metacyclic promastigotes, indicating that these stages of metacyclogenesis detach from the midgut, in accordance with what is known about the parasite life cycle.

Overall, of 46 common genes found in our independent analysis of transcriptomic and proteomic data, more than 60.9% (28 genes) presented a positive correlation between mRNA and protein levels in metacyclogenesis; the other 39.1% (18 genes) exhibited contrary trends ( Table 1 ). Based on these findings, the data obtained from each individual study supports the hypothesis that transcriptional differences exist between procyclic and metacyclic forms in the Leishmania life cycle, even though the genome is mostly constitutively expressed.

Amastigote differentiation or amastigogenesis is the process by which metacyclic promastigotes differentiate into amastigotes inside phagocytic cells. Amastigotes live and replicate inside mammalian host cells in the compartment called parasitophorous vacuoles (PVs) or phagolysosomes ( Figure 1 ), which is the most drastic change in environmental conditions faced by the parasite in its life cycle, involving pH, temperature, and nutrient availability changes, as previously described (Gupta et al., 2001). This environmental alteration causes some observable consequences on parasite gene expression, even though the vast majority of genes appear to be constitutively expressed (Cohen-Freue et al., 2007). An overall reduction in RNA content is observed in amastigotes compared to promastigotes (Shapira et al., 1988). Indeed, most differences in mRNA abundance are observed when comparing the amastigote and procyclic promastigote stages (Inbar et al., 2017). A study showed that RNA abundance seems to play an important role in the early stages of amastigogenesis, but later in the process, posttranscriptional and translational mechanisms act to regulate gene expression (Lahav et al., 2011). The same study found that approximately 20-30% of genes presented a correlation between mRNA and protein levels during differentiation, most of which were up/downregulated in the early stages of differentiation (Lahav et al., 2011).

Amastigogenesis can be induced in vitro for Leishmania species by subjecting promastigotes to environmental conditions that mimic the inside of the mammalian host cell (37°C, pH ~ 5.0) for over 5 h (Barak et al., 2005; Zilberstein, 2020). In this context, temperature seems to play an important role in altering the gene expression and morphology of the parasite, inducing differentiation (Zilberstein and Shapira, 1994; Kramer et al., 2008; Alcolea et al., 2010b; Zilberstein, 2020). Although axenic amastigotes have been proven to be good models for amastigote studies, they lack complex host signaling and downstream effects (Saar et al., 1998; Gupta et al., 2001; Barak et al., 2005). RNA expression studies have revealed transcriptional differences between axenic amastigotes and intracellular amastigotes related to metabolic processes, surface proteins, intracellular transport and response to oxidative stress (Rochette et al., 2009).

A remarkable change in amastigote morphology is the flagellum structure. The paraflagellar rod structure is known to compose the flagellum in kinetoplastids during specific flagellated life stages; its absence has been observed in amastigotes (Portman and Gull, 2010). Accordingly, several studies have identified upregulation of paraflagellar rod protein 1 and 2 mRNAs (PRF1 and PRF2) in promastigotes of approximately 10- to 15-fold higher than in amastigotes (Moore et al., 1996; Mishra et al., 2003). PRF genes encode a component of the paraflagellar rod.

Another change involves the transport of sugars in promastigote and amastigote metabolism. It is known that the main carbon source for promastigotes is sugar and amino acids but that amastigotes mainly utilize amino acids and fatty acids (Krassner, 1969; Krassner and Flory, 1972; Hart and Coombs, 1982; Kuile and Opperdoes, 1992; Opperdoes and Coombs, 2007). A transcriptomic analysis confirmed that levels of different sugar and amino acid transporter genes are upregulated in procyclic promastigotes in comparison to amastigotes (Inbar et al., 2017). In metacyclogenesis, the mRNA levels of these transporters further increased from procyclic promastigotes to neptomonads, reaching their peak in the metacyclic stage (Inbar et al., 2017). This is strong evidence that observable metabolic changes during the Leishmania life cycle can be detected at the transcriptomic level.

Moreover, there are similarities between the transcriptional profile of metacyclic promastigotes and amastigotes (for example, in comparison to procyclics, metacyclic promastigotes present an upregulation of amastin-like proteins - known to be characteristic of the amastigote phase of Leishmania), suggesting that metacyclogenesis is a process that “prepares” the parasite for infection (Inbar et al., 2017).

To assess correlations between mRNA and protein levels in amastigogenesis, we analyzed data from 8 articles involving five Leishmania species: 5 contained transcriptomic data (Almeida et al., 2004; Holzer et al., 2006; Leifso et al., 2007; Saxena et al., 2007; Alcolea et al., 2010a) and 3 proteomic data (Walker et al., 2006; Leifso et al., 2007; Brotherton et al., 2010). These studies obtained Leishmania amastigotes in different ways: recovered from BALB/c mice lesions (Almeida et al., 2004; Holzer et al., 2006, and Leifso et al., 2007), macrophage lysis (Alcolea et al., 2010a) and in vitro cultivation of axenic amastigotes (Walker et al., 2006; Saxena et al., 2007 and Brotherton et al., 2010). The analysis was performed in a similar manner as previously described for metacyclogenesis.

In these works, differentially expressed mRNAs and proteins were compared and correlations were established ( Table 2 ). Each gene listed in Table 2 was identified as differentially expressed in the analyzed proteomic or transcriptomic studies comparing promastigote to amastigote differentiation. Following the same trend observed for promastigotes, a positive correlation between mRNA and protein levels was found for most genes in Table 2 although some genes presented opposite levels. We analyzed genes within the following biological groups: stress response, gene expression, energy metabolism, cell signaling and proliferation. Hypothetical proteins and other biological functions were also considered ( Table 2 ).

Stress response proteins appeared upregulated in amastigotes compared to promastigotes in response to the drastic environmental changes that trigger differentiation and the hostile acidic PV environment. mRNA and protein levels of stress response proteins, such as HSP70 (LmjF.28.2780) and Chaperone protein DNAJ homolog – JDP7 (LmjF.32.1940), presented a positive correlation and were consistently upregulated in amastigotes. On the other hand, superoxide dismutase (LmjF.32.1820) showed opposite mRNA and protein levels, with a decrease in mRNA but an increase in protein. It is interesting to note that some HSPs (LmjF.28.2780 and LmjF.33.0312) presented correlations but that others (LmjF.28.2781 and LmjF.36.2030) did not. Stress inducible protein 1 (STI1) transcripts are heat inducible in L. major promastigotes, with increased transcript levels when subjected to an increased temperature (Webb et al., 1997). Our analysis revealed a decrease in both the mRNA and protein content of an STI1 homolog in amastigotes of L. mexicana and L. donovani (mRNA data) and L. infantum (protein data).

Gene expression-related genes appeared mostly downregulated, with mRNA and protein levels of 10 out of 14 genes being consistently downregulated in amastigotes, suggesting that gene expression is reduced in this life stage. In this category, we observed a polyA-binding protein (LmjF.35.5040), an RNA upstream-binding protein (UBP2) (LmjF.25.0490), and several histones. Histone H4 was the most notable observation, with 6 different genes being downregulated at both the mRNA and protein levels in amastigotes (LmjF.02.0020, LmjF.06.0010, LmjF.31.3180, LmjF.35.1310, LmjF.36.0020 and LmjF.25.2450). Reduced mRNA and protein levels histone 2B (LmjF.19.0030) also was detected in amastigotes, but histone 3 (LmjF.10.0970) mRNA and protein levels did not correlate.

For energy metabolism-related proteins, fructose 1,6-bisphosphate aldolase (LmjF.36.1260) and putative 3-ketoacyl-CoA thiolase-like protein (LmjF.31.1630) were the only proteins, of 6 in total, that were upregulated in amastigotes. These are coincidently the only two that did not present mRNA and protein level correlations, as their mRNAs were downregulated. For all 4 other proteins (LmjF.23.0690, LmjF.21.1770, LmjF.30.2970, LmjF.14.1160), both mRNA and protein levels were reduced upon amastigogenesis. The consistent downregulation of glycolytic enzymes (LmjF.30.2970 - glyceraldehyde 3-phosphate dehydrogenase and LmjF.14.1160 – Enolase) is in accordance with what has been described for L. mexicana amastigotes, utilizing less glucose in relation to promastigotes (Hart and Coombs, 1982).

It is also possible to observe some classical developmentally regulated genes, such as several GP63 and a few paraflagellar rod protein genes (PFR2). Both the protein and mRNA levels of these genes decrease during amastigote differentiation, in accordance with what is known about the parasite life cycle, as discussed previously.

One interesting trend unveiled by Table 2 is that of the 30 genes that presented mRNA and protein correlations, 86% (26 genes) showed decreasing expression in amastigotes. This is consistent with what has been reported about a decrease in mRNA and protein content in amastigotes (Shapira et al., 1988). Of the only 4 correlating genes in the table with increased expression during amastigogenesis, 3 are related to the stress response, and the other is a hypothetical protein.

Overall, of 43 common genes found in our analysis of independent transcriptomic and proteomic data, 30 (69.8%) presented positive correlations between mRNA and protein levels during amastigote differentiation; opposite trends in mRNA and protein levels were found for the other 13 genes (30.2%). Once again, this supports the idea that mRNA and protein levels can correlate in the Leishmania life cycle. This finding of over 69% correlation between mRNA and protein trends in amastigote differentiation is larger than the 53% previously observed in combined proteomic and transcriptomic studies of L. infantum differentiation (McNicoll et al., 2006). Our analysis, however, incorporated a larger number of Leishmania species and several independent studies, which might explain the difference in the percentage of mRNA/protein correlations.

It is important to point that this review was based on an extensive literature review and manual ad-hoc curation. No statistical analysis was performed with the data from all the analyzed studies, proposing this reading as a proof-of-concept. Our analyses were obtained from over 4,000 genes in transcriptomic data and over 400 proteins in proteomic data during metacyclogenesis and amastigonesis in Leishmania. Interestingly, only 46 and 43 genes were commonly differentially expressed in metacyclogenesis and amastigogenesis, respectively ( Figure 4 ). Among these, 28 and 30 genes presented correlation between mRNA and protein levels in metacyclogenesis and amastigonesis, respectively ( Figure 4 ). The considered genes that did not present correlation corroborated the idea that it is not always possible to accurately predict protein levels based solely on levels of mRNA, mostly due to extensive posttranscriptional mechanisms regulating gene expression. In particular case of our analysis the lack of correlation might be related to the different Leishmania species being compared that may interfere in establishing such correlations.

The idea that the Leishmania genome is constitutively expressed at the transcriptional level does not necessarily mean that protein levels cannot follow the same trend of the corresponding mRNA to be expressed. Here, we showed that the mRNA and protein levels of several genes increase or decrease concomitantly during metacyclogenesis and amastigogenesis. These differences in mRNA and protein levels might also be used in epidemiological practice and/or research as stage-specific markers to identify, isolate and recognize specific life forms in the Leishmania life cycle.

To date, little research establishing the correlation of mRNA and protein levels in Leishmania has been performed due to the complexity of data handling. The increase in proteomic and transcriptomic data associated with detailed comparative analysis will certainly enrich the understanding of gene expression regulation in trypanosomatids, providing new ways to use molecular biological data in the control and treatment of the disease. Another approaches that may be helpful in obtaining new answers for the Leishmania gene expression questions could be performed through half-lives measurement (Archer et al., 2008) and polysome profiling (Bifeld et al., 2018; Karamysheva et al., 2018). This technique may provide important information on the correlation between mRNA and protein levels in Leishmania, by exclusively analyzing mRNAs that are in fact being translated.

LC, JA, and LF-W contributed to the conception of the review and manuscript revision. LC made the first draft, performed analysis, and designed figures and tables. JA and LF-W added new information to the original manuscript. All authors contributed to the article and approved the submitted version.

The work was supported by São Paulo Research Foundation (FAPESP) - grants #2017/23696-1 (LC), #2021/04422-3 (JA) and #2018/23512-0 (LF-W).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The handling editor LT declared a shared affiliation with the authors at the time of review.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.