How can proteomics overhaul our understanding of Leishmania biology?

KEYWORDS:

• Leishmania
• protozoa
• proteomics
• mass spectrometry
• evolution

1. Introduction

In the post-genomic era, genomic and transcriptomic techniques have significantly advanced our understanding of the fundamental genetic aspects of many human diseases. However, these techniques have a narrow scope in the study of cellular processes of disease mechanisms. For instance, gene- or transcript-based studies are totally blind to the profound implications of post-translational modifications (PTMs) in normal cellular physiology and in pathology. Events such as alternative splicing, somatic recombination, proteolytic cleavage, single amino acid polymorphisms and PTMs could lead to over 100 protein isoforms per gene [1]. More than 95% of currently known drug targets are proteins, of which about 55% are enzymes [2]. The functional expression of proteins in cells and tissues and the catalytic activity of enzymes are modulated by various molecular mechanisms that operate at the protein level – such as protein-protein interactions, co- and post-translational modifications and cellular localization, on which genomic and transcriptomic methods can provide very little to no information. As proteins serve as the work horses of the cell, proteomics, the large-scale study of proteins in an organism, has recently gained significant traction in the biological sciences as a powerful methodology for molecular-level characterization of the processes of life and disease development.

Proteomics has a special place in the study of the protozoan parasites Leishmania spp., the etiological agents of the complex Neglected Tropical Disease (NTD) leishmaniasis. The disease is endemic in over 90 countries in the tropics, sub-tropics, and southern Europe, affecting the lives of over 200 million people with about 1.5 million new cases and an estimated 20,000 to 50,000 deaths annually [3]. At least 20 different species of the genus Leishmania have been identified as causing disease in humans, with distinct species leading to different clinical manifestations ranging from various forms of cutaneous lesions to the deadly visceral form. An ancient lineage in the evolution of eukaryotes unicellular Leishmania spp., unlike higher eukaryotes, lack promoter-mediated gene expression and regulation. Instead, they rely on post-transcriptional mechanisms such as mRNA processing, translation, and degradation for regulating nuclear gene expression [4]. Proteins are at the centre of all these crucial processes and recent developments in proteomic technologies have the potential to transform our understanding of the biology of Leishmania spp. Herein we discuss the unique strengths and scope of some of these proteomic methodologies.

2. The curious case of life cycle differentiation, stress adaptation, and PTMs of Leishmania

During their life cycle Leishmania spp. undergo several morphological transformations, ranging from the multiple promastigote forms in the phlebotomine sandfly host to an amastigote form in the mammalian host. These transformations reflect the adaptation of the parasite to the environmental changes experienced in the vertebrate and invertebrate hosts. Characterizing the underlying biochemical changes in the parasite during this differentiation may provide valuable information for antileishmanial drug development strategies. A handful of proteomic mass spectrometry (MS) studies that compared the proteomes of the different life cycle stages of the Leishmania parasites have provided information on many differentially expressed proteins [5]. However, further studies are required to establish whether the observed differences are the cause or the effect of the life cycle differentiation. Similarly, the extraordinary ability of Leishmania spp. to adapt to environmental stresses also calls for in-depth study. Molecular-level characterization of stress adaptation in Leishmania spp. will shed light into not only the unknown biology of the parasite, but also into the mechanisms of drug resistance. Using a combination of BONCAT (bioorthogonal noncanonical amino acid tagging) metabolic labeling and quantitative proteomic MS, we recently showed that whilst nutrient deprivation causes a repression of global protein synthesis in L. mexicana, a set of starvation-responsive proteins are preferentially synthesized [6]. In principle, the BONCAT-quantitative proteomic MS combination could be employed for accurate measurement of perturbations in the Leishmania proteome induced by a variety of stress conditions. Although highly powerful, the method provides measurement of only the newly synthesized proteome (NSP). Recent developments in proteomic MS technologies, such as the mPDP (multiplexed proteome dynamic profiling) [7] that exploits a combination of dynamic SILAC (stable isotope labeling by amino acids in cell culture) labeling and isobaric mass tagging, have the potential to simultaneously profile the effects of perturbing conditions on both the mature (preexisting) proteome and the NSP.

In addition to changes in protein abundance, stage-specific phosphorylation events have been reported in Leishmania spp. [8], pointing to functional regulatory role for phosphorylation in the organism. The presence of many protein kinases and protein phosphatases in the Leishmania genome hints the potential role of reversible protein phosphorylation on modulating protein activity and cell signaling in the organism. Glycosylation [9,10], methylation [10], and acetylation [10] were also reported in Leishmania spp. Similarly, bioinformatics has revealed that the Leishmania genome harbors several E1, E2, and E3 ligases as well as many deubiquitinating cysteine peptidases (DUBs) [11]. Using activity-based protein profiling and MS, a recent study identified six active DUBs in L. mexicana [12] providing the first protein-level evidence for active DUBs in Leishmania spp. Also, in L. mexicana 28 enzymes of the ubiquitination pathway have recently been characterized, of which many are found to play important roles in life cycle progression or in vivo infection [11]. In the absence of transcriptional regulation in Leishmania spp., it is likely that the different PTMs serve crucial roles in differentiation, stress adaptation, and interaction with the host. At present, however, the study of PTMs is underdeveloped in the Leishmania spp. Large-scale proteomic MS discovery studies are required to identify the complete repertoire of PTMs, establish the cross-talks between the different PTMs and to elucidate their biological functions in the organism.

In addition to profiling PTMs, our ability to accurately determine the localization of proteins within the different specialized cell compartments and structures in the parasite will provide valuable information on the protein function and the elusive biology of Leishmania spp. Toward this, microscopy methods are typically employed. Although highly useful, these are limited by low-throughput and availability of specific fluorescent reporters of individual proteins. Although not yet reported in Leishmania spp, the spatial proteomic MS technology hyperLOPIT (hyperplexed localization of organelle proteins by isotope tagging) [13] holds great potential for high-throughput characterization of steady-state subcellular localization of Leishmania proteins and may provide a comprehensive understanding of the proteomic organization in the parasite as recently reported in the case of Toxoplasma gondii [14].

3. Characterizing Leishmania-host interactions

The development and severity of clinical manifestations of leishmaniasis depend not only on the Leishmania spp. but on the many factors pertaining to the susceptible individual such as malnutrition, comorbidities and the state of the immune system. Leishmania spp. parasites have co-evolved with humans in endemic areas and this positive selection pressure has contributed to pathogen survival by latent infection. The development of post-Kala-azar dermal leishmaniasis (PKDL) in some visceral leishmaniasis (VL) patients after recovery from the VL, and the development of mucosal lesions in some individuals after several years or even decades of developing primary cutaneous lesions, are indicative of the ability of the parasite to persist within the host even after successful treatment of the initial clinical condition. Clearly, despite years of research, many aspects of the Leishmania-host interaction remain poorly understood. In principle, all antileishmanials that work purely by targeting a parasite protein are likely to eventually fail as the extraordinary genetic plasticity of Leishmania spp. will confer fitness gains enabling the parasite to effectively evolve toward drug-resistant phenotypes. Therefore, an alternative strategy of host-directed therapeutic development has been proposed to tackle this issue. However, in the first place this requires better understanding of the Leishmania-host interaction.

Proteomic MS has enabled detection of changes in the host protein expression profiles in macrophage in vitro infection models [15,16], murine and human cutaneous lesions [17,18] and in the serum of infected individuals [19]. Proteomic MS studies have demonstrated that the Leishmania infection causes a species-specific reprogramming of major metabolic pathways in the host cell [16]. Similarly, Leishmania spp. may cause epigenetic reprogramming in the host cell by altering DNA methylation or histone post-translational modifications [20]. However, further studies are required to profile and understand these processes in greater depth. It is anticipated that in the coming years proteomic technologies will shed more light on the molecular interactions and impact of intracellular Leishmania spp. infection on the host cell.

4. Chemical proteomics and thermal proteome profiling

The use of synthetic chemical probes in combination with proteomic MS, known as chemical proteomics, has emerged as a powerful approach for unraveling protein function in various biological systems. Chemical proteomics workflows typically rely on a suitable quantitative proteomic method such as SILAC, iTRAQ (isobaric tags for relative and absolute quantitation) or TMT (tandem mass tag) labeling or LFQ (label-free quantification) for quantitative comparison of the different conditions of the study. Chemical proteomics in Leishmania spp., although in its infancy, has showed glimpses of its unique capabilities. For instance, in a seminal study, Wyllie et al. employed SILAC-based chemical proteomics to identify a set of L. donovani cyclin-dependent kinases (CDKs) as molecular targets of their antileishmanial preclinical candidate compound [21]. Another noteworthy study involves global profiling of substrates of L. donovani N-myristoyltransferase (NMT) using metabolic incorporation with a clickable myristic acid analog and LFQ proteomic MS [22]. With further development in the chemical probing of specific Leishmania proteins, the field of chemical proteomics could significantly contribute to increasing our understanding of the parasite’s biology.

Whilst chemical proteomics requires development of specific chemical probes, the alternative thermal proteome profiling (TPP) approach, which neither requires chemical modification of the active compounds nor covalent interaction with the target protein, is gaining popularity [23]. It uses multiplexed quantitative proteomic MS to monitor ligand binding-induced changes in the melting profile of proteins in complex proteomic samples. In combination with TMT-10plex labeling the method has been recently employed by Corpaz-Lopez et al. to identify L. donovani NMT as the most significant target of their potent pyrazolyl sulfonamide antileishmanial compound [24]. Despite the use of highly multiplexed isobaric labeling and high-resolution Orbitrap mass spectrometers, TPP studies, as of writing, report only MS2-based quantifications. It should also be noted that the TPP is limited to soluble proteins and not all protein-ligand interactions will produce a noticeable change in the melting temperature. Nevertheless, as a promising technology, further development in the TPP and its increased application in several laboratories could offer new insights into Leishmania spp. biology.

5. Concluding remarks

Proteomics is arguably the most appropriate method for unraveling the complex biology of Leishmania spp. We have discussed selected applications and the realistic possibilities of proteomic MS technology in the study of Leishmania parasites (Table 1). It is anticipated that further developments in proteomic MS technologies, particularly with improvements in detection sensitivity, improved sample preparation methods and the combination of proteomics with chemical approaches such as activity-based protein profiling and genomic and transcriptomic technologies, will shed more light on the biology of Leishmania spp. and the molecular mechanisms of pathogenesis.

Table 1. Select examples of application and possibilities of proteomic MS in Leishmania spp

Proteomic MS method	Key findings//Possible outcome	Citation
BONCAT-iTRAQ combination	Quantitative profiling of newly synthesized Leishmania proteins (NSPs) under a stress condition	[6]
mPDP	Simultaneous quantitative proteomic MS profiling of modulations in mature proteins and NSPs under a perturbation	[7]
hyperLOPIT	High-throughput characterization of subcellular localization of thousands of proteins	[13]
MDLC-MS proteomics	Differential protein expression in CBA macrophages resistant to L. major but susceptible to L. amazonensis upon infection with the two species	[15]
iTRAQ quantitative proteomics	Metabolic reprogramming in THP-1 macrophages upon infection with L. donovani	[16]
LFQ MS proteomics	Differentially modulated proteins in murine cutaneous lesions following infection with L. major and L. amazonensis	[17]
2DE followed by MALDI-ToF/ToF MS	Differentially modulated proteins in cutaneous lesions in humans following infection with L. braziliensis	[18]
2DE and 2D-DIGE followed by MALDI-ToF/ToF MS	Differentially expressed plasma proteins in L. donovani infected individuals	[19]
Combination of chemical probing and SILAC quantitative proteomic MS	Identification of molecular targets of antileishmanial pre-clinical candidates	[21]
Combination of metabolic incorporation of a clickable myristic acid analogue and LFQ proteomic MS	Profiling of substrates of L. donovani N-myristoyltransferase	[22]
TPP	Identification of L. donovani N-myristoyltransferase as the molecular target of an antileishmanial compound	[24]

6. Expert opinion

Genome editing technologies such as CRISPR/Cas9 and Di-Cre are now increasingly being developed and applied for probing the essentiality of select Leishmania genes [25]. Similarly, the opposite gain-of-function approach using COSMID overexpression library generation and sequencing, termed Cos-Seq [26], is also catching up, particularly for applications such as drug target profiling in Leishmania spp. However, the special dependency of protein-based post-transcriptional mechanisms of gene expression regulation in Leishmania spp. calls for integrated applications of these gene manipulation approaches with robust and sensitive proteomic methods for unraveling the unique eukaryotic biology of this evolutionarily distant parasite.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Reviewer disclosures

Peer reviewers in this manuscript have no relevant financial or other relationships to disclose.

Additional information

Funding

K. Kalesh and P.W. Denny are funded, in part, by UK Research and Innovation via the Global Challenges Research Fund under grant agreement ‘A Global Network for Neglected Tropical Diseases’ grant number MR/P027989/1.