Reporter gene systems: A powerful tool for Leishmania studies

Highlights

• •A reporter is a tool for the detection and quantification of a molecule of interest.
• •The main usage in Leishmania studies is for the subcellular localization of proteins.
• •Other usages are drug screening, gene expression, and disease progression studies.
• •GFP and its derivatives are the most popular used reporters in Leishmania studies.
• •Reporters may interfere with the native expression of the molecule.

Abstract

Protozoan parasites of the genus Leishmania are responsible for leishmaniases, one of the most important anthropozoonotic diseases affecting millions of people worldwide. To date, there are no approved vaccines against leishmaniases for humans. At present, available treatment options lack specificity, which may lead to drug resistance and often cause adverse effects. Genomic analysis of Leishmania spp. revealed that most of the annotated genes encode hypothetical proteins, yet the functions of those proteins are still unknown. Characterization of these proteins is, hence, of utmost importance for the discovery of new therapeutic targets against leishmaniases. Reporter gene systems, or reporters, are powerful tools that enable the detection and measurement of targeted gene expression when introduced to a biological system. Over the years, numerous expression systems containing various reporters have been employed in characterizing several novel genes essential for parasite development. Such systems can be used to predict the subcellular localization of targeted proteins, screen antileishmanial drugs, and monitor the progression of infection within the vector and vertebrate hosts, among other uses. Therefore, it is critical to comprehend the available reporter gene expression systems to choose the most suitable for each study.

Graphical abstract

1. Introduction

1.1. Leishmania

Protozoan parasites of the genus Leishmania are causative agents of one of the most important vector-borne neglected tropical diseases known as leishmaniases. There are over 20 Leishmania species and 90 sandfly species involved in both zoonotic and anthroponotic transmissions (WHO, 2022). Leishmaniases are endemic in 98 countries, affecting more than 350 million people around the globe (Alvar et al., 2012; Burza et al., 2018). These diseases present a broad spectrum of clinical manifestations, ranging from self-healing cutaneous and mucocutaneous lesions to potentially fatal visceral forms (Hong et al., 2020).

The life cycle of Leishmania parasites alternates between two main distinct morphological forms within two different hosts, the obligate intracellular amastigote form in the mammalian host and the motile promastigote form in the invertebrate host. In brief, a phlebotomine sandfly acquires macrophages infected with Leishmania amastigotes during a blood meal. The amastigotes differentiate into procyclic promastigotes in the midgut of the sandfly, which then differentiate into infective metacyclic promastigotes. In the next blood meal, an infected sandfly regurgitates metacyclic promastigotes into the tegument of the mammalian host. The parasites are then internalized by various cell types, mainly by macrophages, where they differentiate into amastigotes, completing the life cycle of the parasites (Gossage et al., 2003; Teixeira et al., 2013).

During its life cycle, parasites are exposed to several stressors including acidic pH, increased temperature, and changes in nutrient availability, which may affect both mRNA and protein expression levels (Zilberstein, 2021). In particular, the expression of proteins involved in stress response is crucial for the parasites to overcome these environmental changes and for the regulation of parasite differentiation (Cloutier et al., 2012). Furthermore, the regulation of mRNA levels was found to have a pivotal role at the beginning of the differentiation process, whereas translation and post-translational regulations seem to be more important in later stages (Lahav et al., 2011).

In most eukaryotes, gene expression is controlled at the transcription level by cis-acting promoters and trans-acting transcription factors. Nonetheless, Leishmania lacks the regulation of transcription, so as the canonical core promoter elements and transcription factors. Instead, the parasites’ protein-coding genes are arranged and transcribed into long polycistronic transcription units (PTUs) of functionally unrelated genes (Clayton, 2002). In Leishmania, gene transcription is initiated by RNA polymerase II and the maturation of mRNA occurs by coupled trans-splicing and polyadenylation (Ullu et al., 1993; Mair et al., 2000; Liang et al., 2003). In the absence of transcriptional control, Leishmania regulates its gene expression by post-transcriptional and post-translational modifications, with the involvement of RNA-binding proteins which regulate splicing, mRNA transport, and modulation of mRNA translation and decay (Clayton, 2019).

1.2. Reporter gene expression system

A reporter gene expression system, or reporter, encodes a protein or a peptide tag that can be distinguished and measured readily over a host cell background (Alam & Cook, 1990). When reporters are introduced into a host cell, they provide easily measurable signals upon expression (Wood, 1995). Depending on the nature of the experiment and adaptability of the assay to the appropriate detection tools, a reporter system should 1) be absent from the host cell, 2) be easily distinguishable from the endogenous cell background, 3) not affect the physiology of the host cell, and 4) the detection and quantification should be ideally effortless, sensitive, and cost-effective.

Reporters can either be detected indirectly or directly. In general, indirect methods involve intermediate detection steps, which could be advantageous in many cases if such steps result in an increased detection sensitivity. However, indirect methods are usually slower and less reproducible when compared to direct methods, which are faster and more robust.

A required step for implementing reporters in any living system is to introduce exogenous genetic materials into the targeted cells by either transient or stable DNA transfection. Stable transfection refers to a DNA construct that is integrated into the genome of the host cell and does not require the continuous application of drug pressure for maintenance. Transient transfection is referred to as an extrachromosomal circular DNA, or episome, that is maintained at a high copy number in the presence of drug pressure but may be lost in the absence of the drug (Kapler et al., 1990; Wilson & Patient, 1991; Joshi et al., 1995). Nevertheless, there are some studies showing persistent episomal expression in the absence of drug pressure for months (Kelly et al., 1992; Singh et al., 2009; Sharma et al., 2019).

In the past decades, numerous genetic tools including enzymatic, fluorescent, and epitope-based systems have been used to study Leishmania (Fig. 1) (Duncan et al., 2017). Such advancements enabled the investigation of targeted gene expression and function, as well as the subcellular localization of the encoded protein of interest using various tags. However, the results obtained from manipulating heterologous reporter gene must be carefully analyzed since the introduction of exogenous sequences may interfere with the endogenous sequences, which then could result in misleading observations and conclusions (Zhang et al., 2003; Folgueira & Requena, 2007; Kovtun et al., 2010; Goldman-Pinkovich et al., 2016; Espada et al., 2021).

Fig. 1.

Timeline of the application of reporters in Leishmania. Enzymatic systems (annotated in red) include chloramphenicol acetyltransferase (CAT), β-galactosidase (β-gal), luciferase (LUC), β-lactamase (β-lac), and haloalkane dehalogenase (Halo). Fluorescent systems (annotated in green) include green fluorescent protein (GFP), yellow fluorescent protein (YFP), Discosoma red fluorescent protein (DsRed), mCherry, infrared fluorescent protein (iRFP), and mNeonGreen (mNG). Epitope-based systems (annotated in purple) include hemagglutinin-tag (HA-tag), biotin-tag, and FLAG-tag.

In this review, we grouped reporters into enzymatic, fluorescent, and epitope-based systems to discuss the application of each reporter in Leishmania studies (Fig. 2), as well as highlight their advantages and disadvantages (Table 1). Thereupon, we hope to assist future studies in choosing the most suitable reporters for each intended objective.

Fig. 2.

Schematic representation of enzymatic, fluorescent, and epitope-based reporter systems applied in Leishmania studies. Enzymatic reporters (red) involve enzyme-dependent reactions. Fluorescent reporters (green) exhibit fluorescence when exposed to light sources. Epitope-based reporters (purple) are composed of short amino acid sequences.

Table 1.

Advantages and limitations of various reporter systems used in Leishmania studies.

Reporter system	Vector	Species Ref	Advantage	Limitation
CAT	pALT1–1, pSP65CATA, pX-CAT70 pLaΔnCAT, pLarΔnCAT, pTEX-cat	L. amazonensis1, L. braziliensis2 L. donovani3; 4 , L. enriettii2 L. infantum5, L. major2, L. mexicana3	Relatively efficient and sensitive. Can be fused to other proteins and retain the activity.	Time consuming. May influence regulatory mechanisms of genes. May require a harsh cell lysis step. May limit imaging contrast. Some systems present a short half-life. May not be possible for repeated measurements.
β-lac	pIR1SAT-β-LAC	L. amazonensis6, L. donovani7 L. major6
β-gal	pSSU-int:β-gal, pX-β-gal, p6.5-β-gal pLEXSY-Hyg2-lacZ	L. amazonensis8; 9; 10, L. infantum10 L. major8; 11; 12, L. mexicana12; 13
LUC	pSPYNEOαLUC pSPαLUC, pLucLink 2.0-Luc, 4pKSNeo-Luc8, pF4x1.HYG, pX63NEO-3Luc, pGL-αNEOαLUC, pGM-αNEOαLUC, pIR1SAT-LUC, YNEOαLUC, pCRm-Luc-DRG, pGUH1, pFHLTG, pSPαLUC, pLEXSY-PAC-PpyRE9h, pSSU-NanoLuc, pSSU-NanoLuc-PEST, pSSU-PRE9, pSSU-RedLuc, pSSU-Luc2, pPLOTv1nanoLuc	L. amazonensis14; 15, L. braziliensis16 L. donovani17; 18; 19; 20; 21; 22; 23; 24 L. infantum25; 26; 27; 28, L. major19, L. mexicana29
HaloTag	pPLOTv1-Halo-tag, pLPOT-Halo-tag	L. mexicana29; 30
GFP/YFP	pXG-GFP+, pSSU-int: eGFP, pXGFP5, p6.5-eGFP, pPLOTv1, pIR1PhleoGFP+, pLEXSY-EGFP, pNUS-H1-GFP, pRP-GFP pLENTv1-eGFP, pLAC-eGFP, pALTNEO/ANT, pLENTv2-eYFP, pX-GFP-glmS	L. amazonensis9; 17; 31; 32; 33 L. braziliensis34; 35, L. donovani36; 37; 38 L. infantum39, L. major12; 36; 39 L. mexicana12; 34, L. tarentolae39; 40; 41	Low toxicity. Photostability. Relatively easy imaging. No requirement of substrate, permeabilization, and fixation of cells. Direct detection.	May lose fluorescence during fixation. May affect protein expression, folding, functionality, and/or subcellular targeting due to size of the tag and/or tagging site.
mNG	pLPOT, pPLOTv1	L. braziliensis42, L. mexicana29; 42; 52
DsRed	pKSNEO-DsRed, pLEXSY-DsRed, pIR1SAT-DsRed2, pLAC-DsRed, pF4X1.HYG-DsRed2	L. amazonensis17; 43, L. donovani44 L. major45; 46, L. tarentolae40
mCherry	pIR1SAT-mCherry, pLEXSY-mCherry, pXG-mCherry, pLEXSY-CHR, pFL-mCherry, pLPOT-(mCh/puro), pLAC-mCherry, pPLOT- mCherry, pX-mCherry-glmS	L. donovani47, L. infantum48; 49 L. major47; 50; 51, L. mexicana29 L. tarentolae40; 41
iRFP	pLEXSY-IFP1.4-HYG, pLEXSY-iRFP-HYG	L. amazonensis52, L. infantum53
HA-tag	pX63, pLEXSY, PGPAHA, pKSNEO, pXG	L. donovani54; 55; 56; 57, L. mexicana58; 59, L. tarentolae60	Less interference with tagged protein due to smaller size. Commercially available antibodies for detection.	May affect protein expression and/or subcellular targeting. May display cross-reactivity of antibodies.
FLAG-tag	pXGSAT, pXGHyg, pSSU-int	L. amazonensis61; 62, L. donovani63
Biotin tags	pPLOTv1-BirA*, pHyg, pTOR, pX72-Hyg	L. mexicana29; 64; 65

Enzymatic systems: CAT, chloramphenicol acetyltransferase; β-lac, β-lactamase; β-gal, β-galactosidase; LUC, luciferase; Halo, haloalkane dehalogenase. Fluorescent systems: GFP, green fluorescent protein; YFP, yellow fluorescent protein; mNG, neon green; DsRed, Discosoma red Fluorescent protein; mCherry; IFP, infrared fluorescent proteins. Epitope-based systems: HA-tag, Influenza hemagglutinin; FLAG-tag; Biotin tag. ¹Uliana et al., 1996; ²Laban & Wirth, 1989; ³Kelly et al., 1992; ⁴Kelly et al., 1993; ⁵Folgueira & Requena, 2007; ⁶Buckner & Wilson, 2005; ⁷Mandal et al., 2009; ⁸LeBowitz et al., 1990; ⁹Okuno et al., 2003; ¹⁰ da Silva Santos et al., 2019; ¹¹LeBowitz et al., 1991; ¹²Misslitz et al., 2000; ¹³López et al., 1993; ¹⁴Agostino et al., 2020; ¹⁵Rocha et al., 2013; ¹⁶Coelho et al., 2016; ¹⁷Wu et al., 2000; ¹⁸Ashutosh et al., 2005; ¹⁹Ravinder et al., 2012; ²⁰Luque-Ortega et al., 2001; ²¹Roy et al., 2000; ²²Soysa et al., 2014; ²³Yan et al., 2002;  ²⁴Domínguez-Asenjo et al., 2021; ²⁵Sereno et al., 2001; ²⁶ Ramamoorthy et al., 1996; ²⁷Michel et al., 2011;  ²⁸Álvarez-Velilla et al., 2019;  ²⁹Beneke et al., 2017; ³⁰Wang et al., 2020; ³¹Tetaud et al., 2002; ³²Mehta et al., 2008; ³³Chan et al., 2003; ³⁴Bastos et al., 2017; ³⁵Jara et al., 2019; ³⁶Ha et al., 1996; ³⁷Guevara et al., 2001; ³⁸Soysa et al., 2015; ³⁹Bolhassani et al., 2011; ⁴⁰Kushnir et al., 2011; ⁴¹Turra et al., 2021; ⁴²Espada et al., 2021; ⁴³Lecoeur et al., 2010; ⁴⁴Jaiswal et al., 2016; ⁴⁵Kimblin et al., 2008; ⁴⁶Ng et al., 2008;  ⁴⁷Vacchina and Morales, 2014; ⁴⁸Thalhofer et al., 2011; ⁴⁹Zorgi et al., 2020; ⁵⁰Calvo-Álvarez et al., 2012; ⁵¹Vacas et al., 2017; ⁵²Oliveira et al., 2016; ⁵³Calvo-Álvarez et al., 2015; ⁵⁴Descoteaux et al., 1995; ⁵⁵Selvapandiyan et al., 2001; ⁵⁶Shakarian et al., 2010; ⁵⁷Bea et al., 2020; ⁵⁸Gomes et al., 2010; ⁵⁹Ishemgulova et al., 2017; ⁶⁰Légaré et al., 2001; ⁶¹Huynh et al., 2012; ⁶²Laranjeira-Silva et al., 2018; ⁶³Dan-Goor et al., 2013; ⁶⁴Detke, 2007; ⁶⁵Kelly et al., 2020.

2. Enzymatic systems

Over the years, enzymatic systems have been widely used due to their relatively high sensitivity and lower cost compared to other systems (Table 1) (Pardy, 1993; Jiang et al., 2008; Guo et al., 2019). In Leishmania, enzymatic reporters have contributed to a better understanding of the gene expression and physiology of the parasite (Uliana et al., 1996). Besides, these systems have been used as a tool for parasite tracking and drug screening (Buckner & Wilson, 2005; Reimão et al., 2013; Agostino et al., 2020; Domínguez-Asenjo et al., 2021).

Nonetheless, the application of enzymatic systems presents some disadvantages. Disadvantages include the requirement of time-consuming assays, as some detection methods may require specific lysis steps, the potential influence on regulatory mechanisms of a target gene, and the single analysis limitation since repeated measurement of the enzymatic activities may not be possible (Table 1) (Dube et al., 2009).

2.1. Chloramphenicol acetyltransferase

Chloramphenicol acetyltransferase (CAT) is an enzyme that confers resistance against the antibiotic chloramphenicol (Miyamura, 1964). CAT catalyzes the acetyl-CoA-dependent acetylation of chloramphenicol resulting in chloramphenicol 3-acetate (AcCAM), which has no antibiotic activity (Shaw, 1967). Labeled CAT substrate or product can be detected by radioactive (Gorman et al., 1982) or fluorescent (Young et al., 1991) techniques. As an alternative, anti-CAT antibodies can be used for CAT detection by enzyme-linked immunosorbent assay (ELISA) (Smale, 2010).

The use of the CAT system in Leishmania was first reported in 1989 by Laban and Wirth (Laban & Wirth, 1989) (Fig. 1). The authors constructed a hybrid plasmid containing the bacterial cat gene and an intergenic region of the α-tubulin gene cluster containing polyadenylation and splice acceptor sites. After the transfection of L. enriettii, L. braziliensis, and L. major with this plasmid, CAT activity was detected by Thin Layer Chromatography (TLC) using radiolabeled chloramphenicol (Laban & Wirth, 1989). Subsequently, CAT was used in studies looking for DNA sequences involved in L. enriettii trans-splicing (Curotto de Lafaille et al., 1992) and metabolic studies for functional analysis of gene products involved in infection virulence and drug resistance of the parasite (Kelly et al., 1992; Kelly et al., 1993). CAT was also utilized in the characterization of the promoter region of the ribosomal RNA (rRNA) genes in L. amazonensis where they produced the reporters from an RNA polymerase I transcription (Uliana et al., 1996).

One of the main advantages of using CAT in Leishmania is that no ortholog of CAT is found in eukaryotes. However, detection and quantification of the CAT activity require laborious procedures. Besides, CAT was found to be less efficient than luciferase, another commonly used reporter, when used for studying weak eukaryotic gene promoters (Williams et al., 1989). Along with the previous observations, the presence of CAT sequences may also interfere with regulatory mechanisms associated to expression of Leishmania genes (Folgueira & Requena, 2007).

2.1.1. β-lactamase

β-lactamase (β-lac) is a member of a large and structurally diverse family of enzymes that produce chromogens from the hydrolyzes of beta-lactam antibiotics, such as penicillin and cephalosporin (Ambler, 1980; Abraham & Chain, 1988) among other substrates (Lebedev et al., 2018). These chromogens can be detected by flow cytometry, fluorescence microscopy, and colorimetric assays (Zlokarnik, 2000; Knapp et al., 2003).

Although β-lac is not the most utilized among the available enzymatic reporters in Leishmania studies, it has been used in several drug screening analyses (Buckner & Wilson, 2005; Mandal et al., 2009) revealing the potential of β-lac for high throughput screening of antiparasitic drugs.

One of the main advantages of β-lac is its relatively sensitive detection, as some assays can detect as few as 50 molecules of chromogen within a cell (Zlokarnik et al., 1998). Also, there is no ortholog of β-lac in eukaryotes and does not appear to be cytotoxic when overexpressed in cells (Gao et al., 2003). Nevertheless, just like other enzyme-based reporters, β-lac involves a laborious detection process (Dube et al., 2009).

2.1.2. β-galactosidase

β-galactosidase (β-gal) is a family of enzymes that catalyze the hydrolysis of β-galactosides to monosaccharides. Among the β-gal products, some are chromogens, which can be directly detected by flow cytometer, spectrophotometer, and fluorescent assays (Rotman et al., 1963; James et al., 1996; Knapp et al., 2003).

The use of the β-gal system in Leishmania was first reported in 1990 by LeBowitz and colleagues (Fig. 1). The authors inserted β-gal into a Leishmania expression vector where they observed a better transient transfection efficiency and higher detection sensitivity than CAT (LeBowitz et al., 1990). The same group expressed β-gal in L. mexicana to evaluate the host immune response to parasite infection (López et al., 1993). β-gal was also utilized for examining the effects of 3′-untranslated regions (3′-UTRs) on the expression of genes encoding three classes of major surface protease (gp63) of L. chagasi (Ramamoorthy et al., 1995). Later, another group of researchers generated a plasmid containing the intergenic region of the L. mexicana cysteine proteinase B gene, encompassing the processing signals required for high expression in the amastigote stage, to promote β-gal expression at this life cycle stage (Misslitz et al., 2000). Leishmania expressing β-gal have also been used in several studies as an alternative model of in vitro drug screening analyses (Okuno et al., 2003; da Silva Santos et al., 2019).

Advantages of β-gal include its relatively low cost, easy detection and user safety because it does not require radioactive detection methods or laborious preparation steps. Moreover, β-gal activity can be quantified using commercially available histochemical stains in fixed cells (Nolan et al., 1988; LeBowitz et al., 1991). It can also be used in monitoring biological events in live mammalian cells (Campbell, 2004). However, the limitation of β-gal include its large size (116 kDa per monomer) and endogenous expression in some mammalian cell lines, such as macrophages (Campbell, 2004).

2.1.3. Luciferase

Luciferase (LUC) is an oxidative enzyme that transforms chemical energy in bioluminescence. It was discovered for the first time in Pyrophorus noctilucus, a species of click beetle (Dubois, 1886). Later, LUC activity also loomed in other organisms such as Renilla (Rluc) and firefly (Fluc) (Bhaumik & Gambhir, 2002). LUC oxidizes luciferin in an ATP-dependent reaction and generates bioluminescent signals that can be detected by using an illuminometer, microscope, scintillation counter, or cytometer (de Wet et al., 1987). Over the years, LUC has been modified by codon optimization and mutagenesis to express a brighter variant for bioluminescence microscopy (Branchini et al., 2010; Ogoh et al., 2020).

LUC is mainly used in Leishmania studies to measure parasite burden and in drug screening analyses (Roy et al., 2000; Sereno et al., 2001; Ashutosh et al., 2005; Michel et al., 2011; Ravinder et al., 2012; Mendes Costa et al., 2019), including studies on persistent visceral infections caused by L. donovani and L. infantum (Álvarez-Velilla et al., 2019; Domínguez-Asenjo et al., 2021). Besides, it was also used to demonstrate the role of the 3′UTR of Leishmania amastin in stage-specific gene expression (Wu et al., 2000).

Advances in the LUC system led to the development of a dual-reporter system, Fluc/Rluc. This dual system allowed the evaluation of gene expression of post-transcriptional regulation in response to purine starvation in L. donovani and other kinetoplastids (Soysa et al., 2014). Furthermore, another study compared three modified LUCs, RedLuc (RL), NanoLuc (NL), and NanoLuc-PEST (NLP), demonstrating their suitability for evaluating cutaneous leishmaniasis progression in animal models (Agostino et al., 2020).

One of the main advantages of the LUC is that it allows parasite quantification in living hosts, which reduces the use of animals through in vivo imaging system (IVIS) (Thalhofer et al., 2010). On the contrary, LUC requires the presence of cofactors such as ATP, metal ions, as well as chemical substrates for the enzymatic reaction, which could produce background signals (Neefjes et al., 2021).

2.1.4. Haloalkane dehalogenase

HaloTag is an enzyme that derives from a bacterial hydrolase, haloalkane dehalogenase (Los & Wood, 2007). HaloTag covalently binds to a variety of synthetic ligands that comprise a chloroalkane linker attached to fluorescent dyes, affinity handles, or solid surfaces. The covalent bond between the tag and the chloroalkane linker is irreversible, highly specific, and forms rapidly under physiological conditions (Los & Wood, 2007). Proteins of interest fused to HaloTag can be purified or detected by fluorescence microscope or western blot, or other detection methods depending on the chosen ligand and experimental approach (Los et al., 2008; England et al., 2015).

Despite its infrequent use in Leishmania studies, HaloTag was included as an option for tagging the gene of interest using the T7 RNA polymerase-dependent CRISPR-Cas9 toolkit system (Beneke et al., 2017). Interestingly, HaloTag was the only attainable choice among other fluorophores for tagging SPEF1 in L. mexicana FAZ5 knockout line for flagellum movement studies (Wang et al., 2020).

The main advantage of HaloTag is that the ligand can be chosen among endless options according to the experimental approach and available materials. When fluorescent molecules are used as ligands, the fluorophores can be rapidly interchanged among various dyes, enabling a temporal analysis (Los & Wood, 2007). Nonetheless, it is relatively high cost and due to its newness, there is still limited research into its potential applications (England et al., 2015).

2.2. Fluorescent systems

The discovery and development of fluorescent proteins have revolutionized the field of cellular and molecular biology as they enable direct visualization of tagged proteins and quantification of gene and/or protein expression levels. Inevitably, three scientists who pioneered GFP, Osamu Shimomura, Martin Chalfie, and Roger Y. Tsien, were awarded the 2008 Nobel Prize in Chemistry (Shimomura et al., 1962; Chalfie et al., 1994; Heim et al., 1994; Nobelstiftelsen, 2008). Numerous studies over the last decade have demonstrated the application of genetically encoded fluorescent proteins in Leishmania studies. These reporters can be directly detected by fluorescence microscopy, flow cytometry, or spectrofluorometry.

Fluorescent proteins present several advantages such as low toxicity to the study model and allows direct detection without the need for substrates (Chalfie et al., 1994) (Table 1). On the other hand, several limitations include the potential loss of fluorescence during the fixation process and interference with tagged-protein expression, folding, functionality, and/or subcellular targeting (Chalfie et al., 1994; Palmer & Freeman, 2004; Swenson et al., 2007) (Table 1). In regards to the functional interference, in locus reinsertion of a fluorescent protein-tagged PF16 in the L. braziliensis PF16 knockout line showed only partial recovery of wild-type flagellar motility phenotype (Espada et al., 2021). Likewise, regarding the subcellular targeting interference, the amino acid permease 3 (aap3) was located on the surface membrane of L. donovani promastigotes by tagging aap3 with fluorescent proteins at first (Shaked-Mishan et al., 2006). Ten years later, using specific anti-aap3 antibodies, the same group uncovered that aap3 was not only found on the surface of promastigotes but also on glycosomal membranes (Goldman-Pinkovich et al., 2016).

Besides, there have been other concerns regarding the use of fluorescent proteins in protein expression studies. Such as, depending on which terminal the fluorescent protein tag is fused to, it could also affect the tagged protein expression, folding and/or subcellular targeting (de Marco, 2004; Palmer & Freeman, 2004; Kovtun et al., 2010; Tanz et al., 2013).

2.2.1. Green fluorescent proteins

GFP is a green fluorescent protein of 239 amino acids with a molecular weight of ∼27 kDa. GFP is unarguably the most widely used fluorescent protein and it was first isolated from the jellyfish Aequorea victoria (Shimomura et al., 1962). An enhanced brighter version of GFP (EGFP) was engineered to improve detection sensitivity (Heim et al., 1995; Cormack et al., 1996). The use of GFP in Leishmania was first reported in 1996 by Ha and his colleagues (Ha et al., 1996) (Fig. 1).

One of the most frequent applications of this system in Leishmania is for protein subcellular localization. For instance, GFP was used for localization of arginase (Roberts et al., 2004; da Silva et al., 2008), ABC transporter PRP1 (Coelho et al., 2006), heme transporters LHR1 and LFLVCRb (Huynh et al., 2012; Cabello-Donayre et al., 2020), iron transporters LIT1 and LIR1 (Huynh et al., 2006; Laranjeira-Silva et al., 2018), myosin (Katta et al., 2009), phosphatidylserine transporter LABCG2 (Campos-Salinas et al., 2013), nucleoside triphosphate diphosphohydrolase (Sansom et al., 2014), choline ethanolamine phosphotransferase (Moitra et al., 2021) kinesin LmxKIN29 (Al Kufi et al., 2022), and more.

Expression vectors encoding GFP have been used in various in vitro and in vivo studies (Chen et al., 2000; Misslitz et al., 2000; Guevara et al., 2001; Mehta et al., 2008; Bolhassani et al., 2011; Costa et al., 2011; Pulido et al., 2012; Soysa et al., 2015; Bastos et al., 2017; Alexandre et al., 2020) including drug screening analyses (Kamau et al., 2001; Varela et al., 2009), characterization of ubiquitin domains (Bajaj et al., 2020), and tracking of genetic exchange among parasites (Telittchenko & Descoteaux, 2020). Apart from the direct microscope visualization for parasite infectivity quantification, GFP was also integrated into the 18S ribosomal RNA (rDNA) locus to track parasite quiescence, as a biosensor of the parasites’ proliferative stage (Jara et al., 2019).

Over the years, advancements in genetic engineering enabled the development of various GFP-derived fluorescent proteins covering much of the visible light spectrum (Ormö et al., 1996; Sawano & Miyawaki, 2000; Ai et al., 2007; Ai et al., 2008). For instance, YFP is a GFP-derived yellow fluorescent protein derived from GFP by mutagenesis (Ormö et al., 1996), but it is not the most popular choice mainly due to its sensitivity to pH and photobleaching (Nagai et al., 2002). Regardless, YFP was used to determine the localization of L. amazonensis adenine nucleotide translocator (Detke & Elsabrouty, 2008) and it was indicated as an alternative option in another method for tagging proteins of interest in trypanosomatids (Dean et al., 2015). mNeonGreen (mNG) is a green/yellow fluorescent protein of 237 amino acids with a molecular weight of ∼26.6 kDa, derived from Branchiostoma lanceolatum (Shaner et al., 2013). mNG was shown to exhibit greater photostability and brightness than GFP in Caenorhabditis elegans in vivo studies, even when the standard GFP microscope filter was used (Hostettler et al., 2017). Because of these advantages, mNG has become a popular choice in studies of genome editing using the T7 RNA polymerase-dependent CRISPR-Cas9 toolkit system (Beneke et al., 2017; Beneke et al., 2019; Halliday et al., 2019; Halliday et al., 2020; Wang et al., 2020; Baker et al., 2021).

2.2.2. Red fluorescent proteins

DsRed is a GFP-homologous red fluorescent protein derived from Discosoma sp. (Matz et al., 1999). This reporter has been used in vivo infection studies with L. major (Kimblin et al., 2008) and L. amazonensis (Lecoeur et al., 2010) and in vitro studies with L. donovani (Jaiswal et al., 2016). However, DsRed presents a slower chromophore maturation rate then other fluorescent proteins including GFP, which led to the engineering of several DsRed variants to accelerate the maturation process (Bevis & Glick, 2002; Shaner et al., 2005). mCherry is a red fluorescent protein derived from DsRed via directed evolution (Shaner et al., 2004). Compared to DsRed, mCherry exhibits an improved maturation rate, resistance to pH and photobleaching, and higher photostability (Shaner et al., 2005). Nevertheless, it is less bright than EGFP in fixed tissues (Falcy et al., 2020; Shaner et al., 2005). In Leishmania, there have been several studies using mCherry to monitor the course of infection as this reporter is suitable for intravital imaging and enables deeper tissue penetration than GFP (Calvo-Álvarez et al., 2012; Corman et al., 2019). mCherry was also used to evaluate the amastin expression in L. infantum in a stage-specific manner (Zorgi et al., 2020). Besides, mCherry is one of the possible tag options which can be used in the T7 RNA polymerase-dependent CRISPR-Cas9 toolkit system (Beneke et al., 2017), and the glmS ribozyme-based knock-down system of L. tarentolae (Turra et al., 2021).

Infrared fluorescent protein (iRFP) was engineered from bacterial phytochrome photoreceptors (Shu et al., 2009; Filonov et al., 2011) and enables deep tissue penetration for in vivo imaging. Moreover, the fluorescent emission is close to or within the near-infrared regions, fendering off any background interference derived from other organs or tissues (Shu et al., 2009; Oliveira et al., 2016). Hence why iRFP was the reporter of choice in a study to determine parasite load in L. amazonensis infection by in vivo non-invasive imaging methods (Calvo-Álvarez et al., 2015).

2.3. Epitope tags

Epitope tags comprise short amino acid sequences that can be used as reporters for the detection and/or purification of proteins of interest. In most cases, epitope tags are recognized by antibodies, which allow detection and/or purification of the tagged protein via immunofluorescence, immunoblotting, or immunoprecipitation assays. Among various epitopes, FLAG and HA are the most used in Leishmania studies (Einhauer & Jungbauer, 2001; Phan et al., 2009). It is worth mentioning that in Leishmania studies, histidine tail tags (His-tag) were only used for expression of parasites’ recombinant proteins in bacteria (Oliveira et al., 2011; Kaur et al., 2012; Abass et al., 2013; Pereira et al., 2020).

Epitope tags have emerged as alternatives to the larger tags such as fluorescent proteins to reduce potential interference of the tag with the protein of interest expression, localization, and functionality (Lotze et al., 2016). Nonetheless, they still could affect protein expression and function. In parallel, epitope detection often relies on antibodies that may display cross-reactivity (Table 1).

2.3.1. HA-tag

HA-tag is a 9-amino acid long peptide (YPYDVPDYA) sequence derived from the human influenza hemagglutinin molecule. Widely commercialized anti-HA antibodies are commonly used for peptide detection. However, it was shown that HA-tag could affect the stability and activity of the tagged protein (Saiz-Baggetto et al., 2017).

In Leishmania, HA-tag has been used to determine the subcellular localization of several molecules, such as lipophosphoglycan 2 (Descoteaux et al., 1995), ABC transporter PGPA (Légaré et al., 2001), centrin (Selvapandiyan et al., 2001), and ATP/GTP binding protein (ALD1) (Ishemgulova et al., 2017). Moreover, HA-tag has been used for protein expression studies of a lipase in L. donovani (Shakarian et al., 2010), a cyclin-dependent kinase in L. mexicana (Gomes et al., 2010), and a small ubiquitin-like modifier in L. donovani (Bea et al., 2020).

2.3.2. FLAG-tag

FLAG-tag (FLAG) is a synthetic 8-amino acid long polypeptide tag (DYKDDDDK) (Hopp et al., 1988). It is an artificial antigen recognized by specific high affinity commercially available monoclonal antibodies. Later, FLAG was genetically modified to generate a triple-FLAG variant (3X-FLAG) with a long repetitive sequence (DYKDHDGDYKDHDIDYKDDDDK) to increase tag affinity to anti-FLAG antibodies (Kabayama et al., 2020).

The main application of FLAG tags in Leishmania is for protein expression analysis and subcellular localization. Examples include characterization of casein kinase (Dan-Goor et al., 2013), Leishmania Heme Response 1 (LHR1) (Huynh et al., 2012), and Leishmania Iron Regulator 1 (Laranjeira-Silva et al., 2018).

2.3.3. Biotin tags

Biotin tags are composed of a minimum 14-amino acid residues (GLNDIFEAQKIEWH) inserted during molecular cloning as a part of the expression vector construct. Biotinylation refers to the enzymatic or chemical process by which a biotin molecule is covalently attached to a lysine residue of the tag (Cull & Schatz, 2000; Barat & Wu, 2007). Biotin tags can be detected either by anti-biotin antibodies or avidin/streptavidin strategies. The use of biotin-tag in Leishmania was first reported in 2007 by Detke (Fig. 1) who described the biotinylation of recombinant proteins by E. coli biotin ligase (BirA) in Leishmania (Detke, 2007).

Apart from its classification as a peptide tag, this system can also be employed as an enzymatic reporter system, as tagging of proteins of interest with BirA triggers biotinylation of other interacting proteins by proximity. When used as an enzymatic reporter, biotinylated proteins can be isolated by affinity capture and analyzed by mass spectrometry (Roux et al., 2012). This approach was employed for the identification of molecular partners of the KHARON cytoskeletal complex in L. mexicana (Kelly et al., 2020). Alongside other reporters, a biotin ligase variant (BirA*) is one of the tag options for the T7 RNA polymerase-dependent CRISPR-Cas9 toolkit system (Beneke et al., 2017).

One of the main advantages of the biotin tags system is its low cross-reactivity, and the strength and specificity of the avidin-biotin interaction (Barat & Wu, 2007). However, the biggest drawback of using BirA as an enzyme-catalyzed proximity labeling approach is its slow kinetics, requiring prolonged labeling times (Branon et al., 2018).

3. Conclusion

The development of various reporters over the years has enabled researchers in gaining valuable insights into the physiology of parasite Leishmania and unraveled their transmission routes to invertebrate and mammalian hosts. Nonetheless, due to numerous alternatives, it is crucial for researchers to select the most suitable expression systems for their study objectives. Besides, considering the advantages and limitations of each expression system, and the materials available, more than one system can also be employed concurrently to overcome the limitations of one or the other systems. For instance, LUC combined with GFP led to stable expression of both reporters and allowed studies of parasite biology, host-parasite interaction, and anti-parasitic drug screening assays (Sharma et al., 2019; Taheri et al., 2015). Alternatively, a chimeric triple reporter fusion protein composed of a LUC, a red fluorescent protein, and a epitope-tag, was used to monitor the progression of infection with L. major (Calvo-Álvarez et al., 2018), and LUC combined with the fluorescent E2 crimson was used to evaluate the virulence of L. donovani by in vivo imaging (Melo et al., 2017).

Continuous efforts should be made in discovering new reporter systems for studying Leishmania, unveiling the potential and limitations of currently used systems, and in advancing detection methods. Such advances in reporter systems will continue to impact downstream applications including but not limited to monitoring the intracellular pathways regarding gene expression, drug discovery, cellular and gene therapy of leishmaniases, and numerous other infectious diseases.

CRediT authorship contribution statement

Romário Lopes Boy: Conceptualization, Writing – original draft, Writing – review & editing. Ahyun Hong: Conceptualization, Writing – original draft, Writing – review & editing. Juliana Ide Aoki: Conceptualization, Writing – review & editing. Lucile Maria Floeter-Winter: Conceptualization, Formal analysis, Supervision. Maria Fernanda Laranjeira-Silva: Conceptualization, Writing – review & editing, Formal analysis, Supervision.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data Availability

• No data was used for the research described in the article.