Possible Chemotherapeutic Potential of Inhibiting N-Alpha Terminal Acetylation Activities to Combat Trypanosome Infections

Abstract

New anti-trypanosome drugs focusing on N-alpha terminal acetylation (Nt-acetylation) interference are necessary scientific inputs because currently, many of the drugs in use are unacceptably toxic; moreover, resistance is emerging. Nt-acetylation transfers an acetyl molecule to the N-alpha terminal of a protein by enzymes called N-alpha terminal acetyltransferases (Nats). Nats are grouped according to their amino acid sequence at the N-terminus where they acetylate. It is conserved in all kingdoms of life, and in humans, approximately 80% of proteins are thought to be Nt-acetylated. NatA-NatF and NatH identified in humans, and NatG has been observed in plants. Nats play critical roles in several cellular processes and integrity and have been suggested as possible drug targets to control different cancer diseases. NatA and NatC have been partially characterized in trypanosomes and shown to be essential for parasite viability. Biologically, the way parasites program their lives is embedded in their unique organelles, metabolic pathways, gene regulation, epigenetic gene activities, and many virulence factors including surface molecules. These characteristics and the different protein-coding genes involved could be Nt-acetylated, and the inhibition of Nats can deny the ability of trypanosomes to survive in any environment because many proteins can be simultaneously affected.

1. Introduction

Protein N-alpha terminal acetylation (Nt-acetylation) is an essential process carried out by N-alpha acetyl transferase (Nat) enzymes and can be useful as a drug target for controlling infectious diseases, including those caused by trypanosomes. Trypanosoma parasites (Trypanosoma cruzi and Trypanosoma brucei) affect millions of people in both developing and developed countries, according to the World Health Organization 2023 (WHO 2023) and as suggested elsewhere [1]. Moreover, tropical neglected diseases other than malaria and tuberculosis result in up to 112 million disability-adjusted life-years (DALYs) [2]; see also Table 1. Trypanosomes have unique biological features and alternate between vertebrates and invertebrates during their life cycles [3, 4]. In addition, they have many developmental stages, and Trypanosoma cruzi enters the host cell, in contrast to Trypanosoma brucei, which is extracellular [3, 4].

Disease	Pathogenic agent	Vector	People infected (inf) and at risk in millions (M)	Deaths and new cases/year	Economic loss/year
Malaria	Plasmodium falciparum	Female Anopheles mosquito	About 50% of the world population at risk	0.619 M with 80% deaths in children, 0.247 M cases	Reduced economic growth by about 1.3%
Schistosomiasis	Schistosoma species	Snail species	200 M (inf), 700 M at risk	11,792 deaths	About US $5.5 billion
* Chagas disease	Trypanosoma cruzi	Triatomines bugs	About 6-7 M (inf), 75 M at risk	About 0.012 M deaths, 0.3 M new cases.	About US $7.19 billion
* Leishmaniasis	Leishmania species	Phlebotomine sand flies	12 M (inf), 350 M at risk	0.03 M deaths, 1 M cases.	75% of affected households
* Sleeping Sickness	Trypanosoma brucei	Tsetse flies	0.11 M (inf), 70 M at risk	About 0.5 M deaths, 2.800 new cases	US $1.5 billion
River Blindness (Onchocerchiasis)	Filarial parasite Onchocerca volvulus	Simulium blackflies	18 M (inf) and 0.27 M blinded, 220 M at risk	About 40.000 new blind	US $3.2–6.9 billion
Lymphatic Filariasis	Wucheria bancrofti, Brugia malayi, Brugia timori	Mosquitos in the genera; Culex, Anopheles, Mansonia and Aedes	51 M (inf), over 882 M at risk	40 M disfigured, about 36 M cases	US $5.8 billion

Table 1.

Selected parasites and tropical diseases. *parasitic diseases caused by kinetoplastids (source; WHO 2023, 2022, 2021 reports).

To date, no vaccine or effective drug is available to treat diseases caused by these two parasitic organisms, although some attempts have been made [5, 6]. The genome sequences produced and published [7, 8, 9, 10, 11] have greatly improved our understanding of molecular biology of trypanosomes. Thus, high-throughput drug target identification using RNA interference is currently advancing [12]. Furthermore, integrative genomic and bioinformatics mining is possible, and can be used to facilitate target selection for new anti-Trypanosoma activities [13, 14, 15, 16].

Other than mining the genome to fish what is druggable, a given parasite protein or organelle information such as mitochondrion [17], different from human protein/machinery, essential for parasite way of life, the genome sequences of trypanosomes (T. brucei and T. cruzi) have enabled identification of homologs/orthologs of genes in other organisms that have been validated as drug targets. Of interest are genes encoding lysine acetyltransferases/histone acetyltransferases, and pipeline N-alpha terminal acetyltransferases (Nats). Acetyltransferases are powerful enzymes responsible for protein acetylation (post-translational modifications) and are important for cellular integrity [18]. In the absence of this, cells may not perform biological tasks. Histone acetyltransferases (Hats), also known as lysine acetyltransferases (Lats), transfer acetyl from acetyl-CoA to the lysine residues of a protein sequence, known as N^ε acetylation. It is a reversible process mediated by deacetylases (HDACs), in contrast to N^α acetylation, see Figure 1. The biological significance of histone acetylation has attracted much attention because it correlates with chromatin structure [19, 20, 21]. An imbalance between HAT and HDAC activity is associated with carcinogenesis [22]. Many small inhibitors that target HDAC are in use or are under clinical trials to control cancer [23, 24].

Figure 1.

Protein acetylation. A. Lysine/histone acetyltransferase (HATs), a reversible process by histone deacetylases (HDs/HDACs). B. N-alpha terminal acetyltransferase (NAT), thought to be non-reversible process.

Protein Nt-acetylation is thought to be a permanent modification carried out by Nats, which are enzymes that transfer acetyl moieties to the N-alpha terminal amino acids of the protein sequence [25]. In humans, seven Nats, NatA-NatF, and NatH, have been identified [26]. The groups of Nats are justified depending on their substrate preferences [26]. It is common for different Nats to have a catalytic subunit that contains an acetyl-CoA structure and one or more auxiliary subunits with different molecular weights [27]. In most cases, these subunits co-sediment with ribosomes [28].

Nt-acetylation is thought to be present in all kingdoms of life and has been suggested to determine cell fate and survival [29]. At the molecular level, the biological importance of Nt-acetylation has been shown to have a variety of effects on its substrates, including i) protein complex formation, ii) protein targeting, iii) protein degradation, and iv) inhibition of post-translational ER translocation [26, 30]. In cancer biology, Nt-acetylation is being investigated as a possible chemotherapeutic [30, 31]. The first design and synthesis of Nat inhibitors has recently been reported, indicating that Nat could be selectively inhibited, highlighting the possibility of developing drug-like properties for functional investigation and treatment [32].

Nat studies, particularly the identification of substrates in which they acetylate in trypanosomes, have just begun [33, 34, 35]. Only NatA and NatC have been experimentally and partially examined in trypanosomes [34, 36]. Interestingly, the data indicated that the acetylate substrates of the proteins were similar to those observed in humans and yeast [25, 37]. Furthermore, the predicted substrate sequences in trypanosomes, as assessed [34], are present in the genes used by the parasite during infection, suggesting that trypanosomes Nats activities could be involved in parasite-host interaction and that inhibition of the enzymes could block parasite infectivity. The predicted acetylation state in trypanosomes could be as high as 70–80 percent [34], suggesting that the cellular integrity of parasites or their lives greatly depends on this post-translational modification. Thus, the chance of organisms causing infectious diseases to survive is limited if the Nats or pathways leading to the production of acetyl-CoA are inhibited. Downstream/upstream effects may simultaneously affect hundreds to thousands of proteins, denying their ability to execute their biological functions, thereby causing the death of pathogens.

In this chapter, I highlight trypanosome biology, notably organelles, gene regulation, epigenetic mechanisms, trypanosome population structures, some metabolic characteristics, and virulent factors, such as surface molecules and other recommended drug targets. The question is whether the components of different protein-coding genes essential for parasite survival are Nt-acetylated, providing merits for Nats as novel drug targets. Later, I will summarize information about different Nats, and their significance as analyzed in different organisms, and possibly, similar biological functions are executed in trypanosomes. Finally, I discuss the inhibition of Nats as a new target for interventions to control diseases caused by trypanosomes. This is because the inhibition of Nats can simultaneously affect several proteins in different pathways and multiple surface molecules, and the parasite machinery that causes infections is destroyed.

2. Some highlights of trypanosomes and their biology

Kinetoplastida organisms (T. brucei, T. cruzi, and Leishmania), cause life-threatening infections in humans as reported by WHO 2022 and as suggested elsewhere [38, 39]. Trypanosoma brucei causes human African Trypanosomiasis/sleeping sickness and Nagana in livestock [40]. It is transmitted by the Tsetse fly, Glossina species. In humans, the acute form is caused by T. brucei rhodesiense, which is transmitted by savanna species of tsetse flies, confined to East African countries, and lethal if not treated. The chronic form is caused by T. brucei gambiense, mainly found in Central and Western African countries, and is transmitted by riverine tsetse [40]. In later stages, both species can cross the brain barrier and cause neuropsychiatric-related problems, leading to death [40, 41]. Several species of T. brucei infect animals, notably T. brucei (infects all domestic animals and wild animals), T. congolense (burden on domestic animals and many wild animals), T. vivax (domestic animals and ruminants), T. simiae (infects domestic and wild pigs), and T. evansi (mainly infects camels) [42].

T. cruzi, common in Latin American countries and the Southern part of the USA, causes American Trypanosomiasis/Chagas disease [43]. The disease has two forms: an asymptomatic short acute phase, and, when not treated, it develops into a chronic phase decades later. It is mainly transmitted by the reduviid bug (triatomine bug) and through other sources such as contaminated blood during blood transfusion, organ transplant, congenital transmission, accidental transmission during laboratory work, ingestion of contaminated food, and organ transplantation [44]. Migration from infected to non-endemic regions has increased transmission beyond the American continent [45]. The magnitude of trypanosomes and burden of other parasites and causative agents are summarized in Table 1.

The life cycle of trypanosomes is complex, as they alternate between vertebrates and invertebrate [3, 46]; see Figure 2. During this process, they undergo several morphological changes to adapt to the new environment. It is known that T. cruzi enters the host cell in contrast to T. brucei, which is extracellular, see Figure 2, raising the peculiar biology of the two organisms and how they circumvent immune challenges, as discussed later in this chapter. Mindful of the possible control of Trypanosoma infections, it is important to highlight a few biological features and ask whether the proteins involved have Nt-acetylation signatures.

Figure 2.

Life cycle of trypanosomes. A. T. brucei where numbers are represented as follows: 1. Tsetse fly takes blood meal and injects metacyclic trypomastigotes. 2. Metacyclic trypomastigotes transform into trypomastigotes. 3. Trypomastigotes divide by binary fission. 4. Trypomastigote circulating in the blood. 5. Trypomastigotes ingested by tsetse fly during feeding. 6. Trypomastigotes transform into procyclic trypomastigote in the tsetse fly midgut and divide. 7. Procyclic trypomastigotes transform into epimastigotes. 8. In salivary gland, epimastigotes transform into infective metacyclic trypomastigotes. B. T. cruzi where numbers represented as follows: 1. Triatomine bug takes a blood meal and passes in the feces infective metacyclic trypomastigotes. 2. Metacyclic trypomastigotes penetrate cells and transform into amastigotes 3. Amastigotes multiply inside the cell. 4. Amastigotes transform into trypomastigotes, then come out of the cell into the bloodstream. 5. Triatomine bug takes blood meal and ingests trypomastigotes. 6. In the midgut, trypomastigotes transform into epimastigotes. 7. Epimastigotes divide by binary fusion and multiply in the midgut. 8. In the hint gut, epimastigotes transform into infective metacyclic trypomastigotes.

2.1 Some unique biological characteristics of trypanosomes

Unique features in trypanosomes make the study of organisms interesting, although it could be problematic given the unusual biochemical and morphological characteristics, leaving alone genetic traits. Trypanosomatids (T. cruzi and T. brucei) have unique organelles such as kinetoplasts (containing kinetoplast DNA) [47] and glycosomes (containing glycolytic enzymes) [48], in contrast to mammals, which have the enzymes in the cytosol. There is also an acidocalcisome (high concentrations of calcium and sodium ions, with most proteins thought to be transporters) [49, 50]. Flagellar pockets (perform endocytic activities) [51], reservosomes (accumulate endocytosed macromolecules) rich in cysteine proteinase [52], and RNA granules (sites for storage, processing, and degradation of RNA) [53]. Furthermore, mitochondria, which surround the kinetoplast, provide characteristic signals and generate reactive oxygen species [54]. Additionally, there are mitochondrial calcium ions and reaction species, which are an overload of calcium ions, leading to the generation of oxygen reaction species, important to parasites [55], and contractile vacuoles, which are important in osmoregulation [56].

The different organelle structures in Trypanosomatids, useful in functional analyses, have been presented using fluorescent-tagged proteins [54]. Indeed, some basic cell biology of Trypanosoma parasites have been examined elsewhere [50, 57, 58]. It is time to profile the proteomic components of different trypanosome organelles and assess the Nt-acetylation state. I imagine that some of the proteins constituting trypanosome organelles could be highly Nt-acetylated, and that inhibition of Nats may interfere with their normal functions. In addition to organelles, the regulation of nuclear gene expression is completely different from that in other eukaryotes, as briefly discussed in Section 2.2.

2.2 Gene regulation

The complexity of the life cycle of trypanosomes dictates that they must undergo several transformations to acquaint themselves with environmental cues, which include temperature changes, immune challenges from the host, drug pressure, and the possibility of acquiring nutrients. Moreover, in the same host, they ought to transform from the proliferation stage to the nondividing stage, see Figure 2. Therefore, drastic measures must be taken to coordinate gene expression. In contrast to other eukaryotes, gene expression in trypanosomes is very unusual [58], and they regulate their genes post-transcriptionally; that is, genes are organized in long polycistronic units and transcribed by RNA polymerase II [58, 59], see Figure 3. As examined previously [60], it is intriguing that some Variant Surface Glycoprotein (VSG) genes in T. brucei are transcribed by RNA polymerase I. VSG is a key antigen used by T. brucei to survive in mammalian hosts because of its capacity to vary to confuse host immune challenges; thus, they escape by switching the dense protective coat of VSG [61]. Furthermore, the parasite is equipped with a repertoire of VSG, about 15, which direct VSG transcription [62], and only one VSG gene is expressed at a time from the telomeric expression site (ES). Indeed, antigen variation is common in many parasites, and understanding the proteins and pathways leading to survival mechanisms requires further investigation, although this has been discussed recently [62].

Figure 3.

Trans -splicing and polycistronic transcription.

On another note, how trypanosomes regulate their gene could depend on ribosomal RNA-binding proteins (RBPs) since they act as modulators of mRNA abundance [63], leave alone promoting cell differentiation [64, 65]. Disruption of RBP33 in trypanosomes results in overexpression of approximately 40% of all annotated genes in the genome [66]. Moreover, the knockout of three RBPs, TcZC3H39, TcZC3H29, and TcZC3HTTP, in T. cruzi using CRISPR-Cas9, resulted in morphological changes of the parasite, suggesting the significance of RBPs in parasite survival [67]. Additionally, the RNA-binding protein DRBD18 has been implicated in the export of a subset of mRNA [68]. The processed RNAs should be transported from the nucleus to the cytoplasm, and the way trypanosomes transport their mRNA from the nucleus deviates from that of other eukaryotes as previously examined [69]. Taken together, the proteins involved in mRNA turnover may be interesting targets for therapeutic interventions. Therefore, it is time to profile the mRNA binding proteins, determine possible pathways in which they are engaged, and assess whether they have Nt-acetylation signatures and what consequences they may have on parasite survival if acetylation states are denied. This may aid in developing new strategies for intervention by inhibiting Nats.

The advancement of new technologies, such as RNA-seq, has improved our knowledge of the transcriptome of kinetoplastids [70, 71]. Furthermore, a multi-step control of gene expression, providing new information about T. cruzi proliferative proteins, was presented [72], highlighting T. cruzi cell cycle-regulated genes. In other studies, T. cruzi transcriptomics has revealed many upregulated genes, such as mucin-associated proteins involved in infection, providing potential drug targets to control T. cruzi infection [73, 74]. Interestingly, using single-cell sequencing, detailed biological events during parasite development, especially gene expression patterns and identification of putative gene regulators, are now possible [75]. However, direct sequencing of the T. brucei transcriptome has identified 32 novel long non-coding RNA (lncRNAs), which are stage-specifically expressed [76], informing the scientific community of the complexity of the parasite transcriptome, which includes alternative transcriptional start and stop sites. In summary, it can be hypothesized that proteins involved in the proliferation of trypanosomes could be Nt-acetylated and, collectively, their functional capabilities, if interfered with by inhibiting Nats, could deny trypanosomatids survival strategies.

2.3 Highlight of epigenetics

As part of gene regulation, there is growing evidence that epigenetics (control of gene activity without changing the DNA sequence) is a key player in the way trypanosomes program or aid their survival capabilities [77, 78]. DNA methylation is the most common epigenetic mechanism, where a methyl molecule is attached to the DNA building blocks, causing the gene to be silenced/turned off [79, 80]. Histone modification is another important epigenetic marker; for example, methyl or acetyl groups are added or removed, and this determines the turning off or on of a gene, or one of the genes, and any errors caused, can lead to normal or abnormal gene activity [19, 81]. A detailed review highlighting epigenetic marks in kinetoplastids was presented [21], recommending multiprotein complexes involved in chromatin machinery as possible drug targets. The question is whether the components of these different protein complexes are Nt-acetylated. If so, the underlying cellular programming will be revealed, and guilty Nats will be used as possible drug targets for control strategies. Much as gene regulation in trypanosomes is being discovered, it is important to bear in mind Trypanosomatids population structures to drive trypanosome control.

2.4 Trypanosoma cruzi and Trypanosoma brucei genetic diversity and clinical implications

It is known that T. cruzi species are heterogeneous and recognized as T. cruzi I and T. cruzi II, which is further divided into six to seven discrete typing units (DTUs) based on the nucleotide sequence [82, 83, 84]. The detailed aspects of different DTUs and their significance for parasite survival in both human and insect hosts are discussed elsewhere [83]. T. cruzi undergoes genetic exchange [85] and mixed infections occur. Hybrid lineages [86] are common; notably, TcIII-TcIV is suggested to have arisen because of ancestral recombination between TcI and TcII, and TcV-TcVI arose because of recombination between TcII and TcII [85, 87]. The population structure complexities of T. cruzi strains may be regarded as multiclonal [88], as those isolated from insects may differ from clinical samples [88]. For parasites assessed in chronic Chagas-infected patients and non-human hosts, TcII/VI was associated with domestic cycles and patients with chronic Chagas disease, and the predominant genotype was documented as TcII [89]. TcI was also observed in two human and triatomine samples [89]. This finding further confirmed the genetic polymorphism of T. cruzi bloodstream populations in Chagas disease, showing a TcII prevalence of 74.3% [89]. Geographically, TcI is common in North Amazon areas and is linked to cardiomyopathy in these countries [88], whereas TcVI is common in South American countries and is associated with megacolon, megaoesophagus, and cardia-related complications [90, 91]. Recently, epidemiological data on DTUs have been presented [92], suggesting that, DTUs are widespread across the American continent. In terms of treatment, the DTUs can no longer be ignored, and with no proper drugs available, T. cruzi genetic polymorphisms add to another layer of the parasite life complexities, which may be programmed by Nt-acetylation; therefore, deeper analyses of Nats are warranted.

For T. brucei, the population structure has implications in determining epidemiology [93]. Moreover, in another study, stable genotypes rather than new genotypes maintained endemicity [94]. Genetic exchange in T. brucei was suggested to differ from that in T. cruzi and appears to involve meiotic cell division, but not hybridization [95]. However, recent studies have suggested widespread hybridization, which involves human infective and non-human infective trypanosomes of T. brucei, circulating in sub-Saharan African countries with a risk of generating serious virulent Trypanosoma brucei strains [96]. This might dramatically increase T. brucei infection in the population, with further demand for new chemotherapeutics [97]. Given the different population structures of trypanosomes, it is worth perusing Nats as possible drag targets, because they are likely to cut across, and may be useful in the fight against all trypanosome strains and structures.

2.5 Trypanosomes virulent factors

It is worth mentioning some key virulence factors trypanosomes use to circumvent the human immune system. The virulence factors-coding proteins could be Nt-acetylated in large numbers in trypanosomatids. For T. cruzi to evade the host, the parasite expresses on its surface, several members of protein families:

2.5.1 Trans sialidase (TS)

Trans sialidase (TS) superfamily has about 1430 gene members [98]. TS activity involves the transfer of sialic acid from host glycoconjugates to parasite mucins present in the plasma membrane of trypomastigotes. The inability of T. cruzi to synthesize monosaccharide sialic acid forces the parasite to scavenge it from the infected host via TS activity [9]. Therefore, sialylation in T. cruzi is crucial for its viability and propagation into the host [99].

2.5.2 Mucin

T. cruzi is coated with a series of glycoproteins, which mainly belong to the mucin (TcMUC) family, used by the parasite for protection and in infectivity [100, 101]. The family comprises approximately 863 genes, of which 201 are pseudogenes [9].

2.5.3 Mucin-associated surface proteins (MASPs) family

The genome sequence revealed the MASPs family, where the name is derived because of its proximity to TcMUC [9]. Structurally, members of the MASPs family are similar to TcMUC, and their sequences are different [9, 102]. In total, the genome has discovered about 1400 member gene families encoding MASP [9]. There were variations among the strains [103]. Experimental evidence suggests that MASPs are simultaneously expressed throughout the parasite life cycle, that is in epimastigote, trypomastigote, and amastigote stages [104]. Moreover, in trypomastigotes, the bias of multiple MASP variants toward a particular MASP subgroup was revealed [102]. The plethora of MASP and the second largest gene family in T. cruzi, have been presented [105], suggesting that MASP is involved in mechanisms of host–parasite interactions. Furthermore, it is necessary for the infection process, as shown by analyzing mucin-associated surface protein 49 (MASP49) [106].

2.5.4 TcGP63 family

T. cruzi zinc-metalloprotease (TcGP63) has many copies in its genome [9]. It is expressed at all life stages of the parasite, its expression is regulated during parasite differentiation, the individual isoforms of TcGP63 play a role in host cell infection [107, 108]. Two key TcGP63 families, TcGP63-I and TcGP63-II, have been identified and have 5–10 and 62 gene copies, respectively [109]. While TcGP63-I is attached to the parasite by a C-terminal glycosylphosphatidylinositol (GPI) anchor signal, TcGP63-II does not have a GPI-anchor signal [110].

2.5.5 Amastin family

Amastin is mainly expressed in amastigotes, the mammalian stage of the parasite, during the life cycle. They are groups of transmembrane glycoproteins possessing two distinct subfamilies: β- and δ-amastins, which are thought to play a role in proton or ion trafficking across the membrane [111] and in T. cruzi infection [112]. A total of 14 copies of amastin genes have been identified in the parasite [111].

2.5.6 TcTASV family

T. cruzi Trypomastigote Alanine Serine Valine-rich protein (TcTASV), expressed mainly in trypomastigotes, is a family with 40 members [113] and possibly plays a significant role in host-parasite interactions, thus being a significant virulence factor [114].

2.5.7 Cruzipain family

Cruzipains are major papain-like cysteine proteases expressed mainly in epimastigotes and amastigote bodies, in contrast to the trypomastigote form where they are expressed only in the flagellar pocket [115]. Later, cruzipains were grouped into two families and four subtypes [116]. In addition, 67 genes encoding cruzipain have been identified in different T. cruzi strains [116].

Cruzipain has been implicated in host-parasite interactions [117]. Some detailed characteristics of the T. cruzi surface molecules have been discussed elsewhere [98]. Taken together, T. cruzi employs many strategies to differentiate, survive, evade the immune system, and infect its host. These strategies, especially the modulation of virulence potentials, are carried out, among others, by surface molecules [118], the majority of which are probably Nt-acetylated, and inhibition of Nats could abolish infection.

For T. brucei, the main surface molecule used by the parasite to survive is the variant surface glycoprotein (VSG), which encodes approximately 806 genes [119]. The detailed strategies for how T. brucei uses VSG to survive in the host have been presented [120]. However, in the insect stage, T. brucei forms (procyclic), and the VSG coat is replaced by procyclic acidic repetitive protein (PARP) and glutamic acid-rich protein (GARP) [121].

Arguably, various ways in which trypanosomes circumvent environmental cues and propagate may significantly depend on the surface molecules, which are in their hundreds. If these molecules are Nt-acetylated, which was predicted to be the case [34], then the simultaneous inhibition of their activities is possible by interfering with Nats. As mentioned before, Nt-acetylation is a post-translational modification of a protein, which can be measured by proteomics.

3. Proteomics

Trypanosomes differ in species and in the diseases they cause. Integration of different data, such as genomics, transcriptomics, and proteomics, may provide a better picture of parasites´ ways of life. Trypanosome genomic data [8, 9] and comparative genomic information [122] have made it possible to identify possible drug targets for individual trypanosomes or drug targets shared by the two organisms. Indeed, it is now possible for scientists to examine integrated functional genomics in a database, the so-called TriTrypDB [123], and examine the molecular insights of kinetoplastid organisms (T. brucei, T. cruzi, and leishmania). To improve the gain, protein studies may be of huge benefit since gene expression in trypanosomes appears to be posttranscriptional, rendering nucleic acid studies less informative [124], yet most drugs target proteins.

3.1 Proteomic efforts to identify new drug targets in trypanosomes

In trypanosomes, proteomic studies using mass spectrometry have revealed proteins that are highly expressed in different developmental stages and those expressed in some distinct pathways, proteins observed to be virulent factors, and many more, which could be used for drug development or as vaccine candidates [125, 126]. The fundamental question is to what extent these proteins are post-translationally modified, especially with respect to Nt-acetylation.

4. Post-translational modification of proteins in trypanosomes

The functionality of proteins in a cell is partly attributed to post-translational modifications. Proteins released from the ribosome often undergo post-translational modifications such as phosphorylation [127], protein glycosylation [126, 128], SUMOylation (attachment of small ubiquitin-like modifier) [126], N-terminal methylation [129, 130], and lysine acetylation. Before Nt-acetylation is discussed in detail as a possible chemotherapeutic, it is important to highlight some common strategies employed to eradicate trypanosomatid infections.

5. Control and eradication strategies for Trypanosoma infections

No vaccine is available to control infections caused by trypanosomatids. Attempts to find an effective vaccine thus far are far long from ideal due to parasite antigenic variations and T. cruzi hides inside the cell [6, 131]. However, genetic editing using CRISPR–Cas9 is a promising tool, that is useful for assessing vaccine candidates, as demonstrated in Leishmania major, a parasite closely related to trypanosomes [132]. Controlling vectors to disrupt trypanosome transmission has not yet succeeded [133, 134]. Therefore, chemotherapeutics is a better option. Unfortunately, for T. cruzi, there are only two drugs in the market, Benznidazole and Nifurtimox, which have different efficacies for different parasite life cycle stages [135, 136]. Moreover, the benefits of these drugs to human health are problematic owing to their adverse side effects [135, 137].

For T. brucei, pentamidine is used as the first-line drug for early symptoms of West African trypanosomiasis caused by T. brucei gambiense [138]. Eflornithine is used as the first-line drug for the second stage of West African trypanosomiasis, Suramin is the drug of choice for early T. brucei rhodesiense infection in East African trypanosomiasis. Melarsoprol can cross the blood-brain barrier and is used in the management of central nervous system symptoms in the second stage of T. brucei rhodesiense infections [139]. Generally, current drugs are far from ideal when it comes to the late or chronic stage [140, 141]. However, Fexinidazole and Acroziborole are promising drugs against African trypanosomiasis, and already, Fexinidazole has been approved for the treatment of T. b. gambiense and clinical trial is ongoing for T. b. rhodesiense [6]. Fexinidazole is also undergoing clinical trials for the treatment of Chagas disease and oxarbazole is in phase I clinical trial [6].

The efforts to find new drug targets against trypanosome infections are advancing, notably interference with the trypanothione metabolism pathway [142], calcium homeostasis, nucleoside analog targeting purine salvage pathway, and drug combination of low-cost molecules in T. cruzi [143, 144]. Furthermore, it is now possible to perform in silico analysis to identify new drug targets in trypanosomes [14, 145]. Several new molecular targets eligible for clinical trials have been identified [6]. Regardless of the drug targets available, clinical evidence in humans must be presented and convincing. Therefore, a combination therapy with different modes of action is needed. In that aspect, I imagine that interference of Nt-acetylation may provide the answer, where several proteins in different pathways, proteins encoding surface molecules, and others, may be affected at the same time.

6. N-terminal acetylation

One of the most common PTM of proteins, described as regulatory modifications as important as phosphorylation, is protein acetylation [146]. In this process, an acetyl molecule is transferred from acetyl-CoA to the N-terminal or internal residue of a protein by acetyltransferase enzymes. N-alpha terminal acetylation (Nt-acetylation) is common in all kingdoms [147] and is thought to be a permanent protein modification, see Figure 1. It is carried out by N-alpha acetyltransferases (Nats). Nt-acetylation has been poorly investigated in trypanosomes, yet the modification as shown in other organisms, is linked to various biological cellular processes and can determine the fate of the cell by influencing protein folding, activity, protein localization, and many more [148]. Using proteomics, the acetylation state in many organisms has been defined and found to vary. Approximately 80% in humans, 75% in plants, 60% in yeast, and 18% in archaea [149, 150, 151]. Recent evidence suggests that Nt-acetylation is required for bacterial virulence [152, 153]. Nats are categorized according to the amino acid sequence of the N-terminus in which they acetylate. Thus, in human, NatA to NatF and NatH, NatG in the plant has been defined and their known substrates, dictated by the first few amino acid residues at the N-terminal of a protein, are displayed elsewhere [26], see also Table 2.

N-alpha acetyl transferase	Catalytic subunit	Substrate preferences
NatA	NAA10	S, A, T, V, C, G
NatB	NAA20	ME, MD, MN and MQ
NatC	NAA30	MI, ML, MF, MY and MK
NatD	NAA40	SGRGK on Histone H2A and Histone H4
NatE	NAA50	MA, MV, MS and MT
NatF	NAA60	ML, MI, MF, MK and MY
NatG	NAA70	M. A, S, T (in Plant)
NatH	NAA80	DDD, EEE (only on Actin)

Table 2.

Showing major Nats and their substrates. Note that NatB, C, E and F, maintain methionine, that is, methionine is not removed by methionine aminopeptidase enzyme.

Generally, each Nat has a catalytic subunit (containing an acetyl-CoA structure), and some have one or more auxiliary subunits [154]. The auxiliary subunits are thought to influence the catalytic subunit activity. To date, only the catalytic subunit and no auxiliary subunits have been identified for NatD (NAA40) [155], NatF (NAA60) [150], NatG (NAA70) [156], and NatH (NAA80) [157]. NatB and NatC have the catalytic subunits NAA20 and NAA30, with one and two auxiliary subunits, respectively [158, 159]. NatA has the catalytic subunit NAA10 and the commonly known auxiliary subunit NAA15, whereas NatE has the catalytic subunit NAA50 [26]. However, recent evidence suggests that NAA50, together with huntingtin-interacting protein K (HYPK) forms part of the NatA complex [160], yet NAA50 is also part of the NatE complex together with NAA10 and NAA15, displaying distinct substrate specificity [160]. It is known that NatA to NatF are conserved from yeast to humans and co-sediment with the ribosome [160], suggesting a co-translational function. In humans, NatA has the largest pool of substrates at about 31–38%, followed by NatB at approximately 21%, and NatCEF at 21% [18]. To date, little is known about the preferences of Nats and their substrates in parasites, especially in trypanosomatids. Therefore, I will briefly mention some known molecular significance of Nat, as analyzed in other organisms, and contemplate that similar biological functions may be true for trypanosomes.

6.1 NatA

As mentioned previously, NatA is the most common Nat in both humans and plants [26, 161], and it acetylates co-translationally small amino acids, notably serine, glycine, alanine, threonine, and cystine, although post-translationally, it acetylates proteins with acidic side chains [162]. NatA is implicated in the regulation of gene transcription and cell motility [163] and other evidence indicates that NatA altered gene expression, notably, sub-telomeric genes, and nuclear genes encoding mitochondrial ribosomal proteins [164]. Moreover, the significance of NatA and its developmental roles in cells have also been discussed [165]. Additionally, the biological significance of NatA in mammals, plants, yeast, C. elegans, and prokaryotes has been presented elsewhere [160, 162]. In cancer biology, excess or deficiency of NatA is associated with various tumor diseases, such as liver, lung, colon, breast, and prostate cancers [166].

Nats´ studies in trypanosomatids, especially the identification of substrates, have not been paid attention to, although an attempt was made by proteomics to understand the acetylation state of different parasite life cycle stages in T. cruzi [35]. In addition, partial characterization and substrate profiling of NatA in trypanosomes have been reported [34]. Interestingly, these data indicate that the proteins acetylate substrates as has been shown in humans and yeast [34]. Given the diverse cellular functions of NatA, it is tempting to believe that its inhibition kills the parasites, as demonstrated in T. brucei [36], suggesting its chemotherapeutic potential. Thus far, it is unknown which proteins are acetylated by NatA in trypanosomatids.

6.2 NatB

NatB is the second most abundant Nat in eukaryotes, and is conserved from yeast to humans [159, 167]. It acetylates Met-Asn-, Met-Asp-, Met-Gln-, and Met-Glu-starting N-terminus [159]. In cancer, NatB promotes hepatocyte carcinoma [168] and has been proposed as a drug target to combat liver cancer [169]. One of the key roles of NatB is its involvement in mitochondrial inheritance and the organization of the actin cytoskeleton [170]. Considering that NatB participates in actin cytoskeleton function and if inhibited, can disrupt the cell proliferative signaling pathway, one can imagine that similar functions are also true for trypanosomes. Actin, as a component of the cytoskeleton, performs a wide range of cellular functions, including the regulation of endocytosis and intracellular trafficking in trypanosomes [171], which are important for parasite survival. It will be of interest to assess whether these sets of actin-binding proteins are Nt-acetylated.

NatB is involved in viral polymerase activity [172]. In trypanosomes, protein-coding genes are transcribed together as long polycistronic precursor RNAs [173], which is unusual. It is of interest to assess whether the different subunits of RNA polymerase in trypanosomes have NatB signatures or any other Nats. In plants, NatB has been associated with multiple stress responses [174]. Given the way trypanosomatids survive, they must adapt to several environmental stresses by directing their cellular machinery to transform into different life cycle stages in both vertebrates and invertebrates. Therefore, NatB may be a key player in these biological events and substrate screening is worth investigating. NatB predominantly affects protein folding and plays a key role in cellular homeostasis [164]. In trypanosomatids, redox balance [175], mitochondrial calcium ion uptake [176], biogenesis and metabolic homeostasis [177], and pH homeostasis of organelle (acidocalcisome) [49] are all important homeostasis processes necessary for parasite survival. The cellular protein components responsible for homeostasis, are likely to be Nt-acetylated, providing grounds to assess inhibitory effects of the guilty Nats on parasite viability, which will aid the recommendation of Nats as possible new therapeutic targets.

6.3 NatC

Like NatB, NatC acetylates proteins with no methionine removed; that is, NatC acetylates protein N-termini starting with methionine, followed by a hydrophobic or amphipathic amino acid (ML-MI-, MF-MW-, MV-, MM, MH-, and MK-) [158]. NatC complex is composed of a catalytic subunit NAA30, and two auxiliary subunits NAA35 and NAA38 [158, 178]. NatC-mediated Nt-acetylation is essential for proper cell growth and development in eukaryotes as suggested elsewhere [37, 178]. It is also required for mitochondrial function [158]. NatC plays a significant role in photosynthesis in plants [179]. Little is known about NatC in trypanosomes. An attempt to characterize the protein in T. cruzi was made, and substrate preferences were profiled [34]. Using the RNAi technique on T. brucei equivalent, NatC was observed to be essential for parasite growth, suggesting that NatC knockdown in trypanosomes may affect infection [34].

6.4 NatD

NatD (NAA40) is a highly selective Nat that specifically acetylates histones H2A (Ser-Gly-Arg-) and H4 (Ser-Gly-Gly-) [155, 180]. Histone Nt-acetylation by NAA40 has been shown to greatly affect gene expression, chromatin function, aging, metabolic processes and cancer [181]. It seems that NAA40 does not exist in trypanosomes because no human NatD ortholog has been detected [35], raising the question of whether NatD is irrelevant in cellular programming in these organisms. However, NAA40 is also conserved from yeast to humans [155].

6.5 NatE

NatE (NAA50) acetylates substrates, including Met-starting N-termini: Met-Ala-, Met-Lys-, Met-Met-, Met-Phe-, Met-Ser-, Met-Thr-, Met-Tyr-, and Met-Val [182]. Although NAA50 interacts with NAA10, its enzymatic activity differs [183, 184]. In cancer, NAA50 regulates the cell cycle and proliferation in lung adenocarcinoma [185]. However, in plants NAA50 has been reported to play crucial roles in growth [186], development, and regulation of stress responses [187]. It remains to be seen whether NatE is present in trypanosomes and whether substrate profiles are like those in other organisms.

6.6 NatF

The NatF (NAA60) substrates are listed in Table 2. Different studies indicate that NatF is found only in higher eukaryotes, localizes to the Golgi membranes, acetylates transmembrane proteins, and is probably involved in Golgi structural organization [188, 189]. Additionally, the dual behavior of NatF as a histone acetyltransferase (Hat) and as Nat has been reported [190], indicating that NatF plays a role in epigenetics. The presence of NatF in T. cruzi has also been suggested [35]. Given the uniqueness of gene transcription in trypanosomes, protein post-translational events may be crucial for determining protein characteristics to execute their biological functions. These data indicate that trypanosomes histone are heavily modified post-translationally, including Nt-acetylation [21, 191]. Therefore, if NatF substrates are proven and linked to histone Nt-acetylation in trypanosomes, then molecular insights into gene regulation in these organisms will be discovered, providing a new approach for controlling trypanosomiasis.

6.7 NatG

NatG (NAA70) is only found in plants, acetylating M-, A-, S-, and T-starting-termini as substrates [156]. It is of interest to assess whether NatG is present in any Trypanosoma strain, and its significance investigated.

6.8 NatH

NatH (NAA80) is widespread, has been identified only in animals [157], and is not associated with the ribosome as for NatA-E; moreover, it acts post-translationally. It acetylates ß- and γ-actin substrates with acidic N-termini DDDI and EEEI, respectively [192, 193]. NatH plays a vital role in cytoskeletal organization and cell motility and acts post-translationally [157]. Furthermore, it is important to maintain the structural integrity of the Golgi apparatus in cells [194]. Similarly, if NatH exists in trypanosomes, one could speculate that the components of proteins involved in the Golgi apparatus, cytoskeleton organization, and proteins aiding motility in the two parasites may be Nt-acetylated by NatH. Thus, if proven, it can be a useful target for preventing parasite survival.

7. Remarks

Genome sequencing of trypanosomes has contributed to the molecular insights of these organisms. Moreover, comparative genomics has enabled identification of protein-coding genes shared between the two organisms. Given the posttranscriptional gene regulation mechanisms in trypanosomatids, PTM of protein is a necessary tool that is useful for studying and identifying relevant proteins that are Nt-acetylated for possible chemotherapeutic intervention to control trypanosomiasis.

The study of Nt-acetylation in parasitic organisms, such as malaria [195], Toxoplasma gondii [196], and Leishmania [197], has just begun. In trypanosomatids, only a handful of studies have been documented [34, 35], indicating that Nt-acetylation is common in these organisms and their inhibition interferes with parasite growth and survival [34, 36]. At the infant stage, a comparative acetylome in parasites is now possible [198], providing grounds to identify acetylated proteins specific to individual parasites or those shared between them.

As presented in Section 2.1, Nt-acetylation is involved in the integrity of organelle structure and function, notably in mitochondria, ribosome biogenesis, Golgi integrity, chromosome condensation, and viability [199]. Trypanosomes have unique organelles, as mentioned in Section 2.1, and the maintenance of these organelles may partly depend on Nt-acetylation. Furthermore, components of proteins involved in gene regulation, proteins involved in epigenetic machinery, and hundreds of surface protein-coding genes (molecules) used by the parasite to survive in different environments are all unique features for possible chemotherapeutic attacks against these pathogens. All these characteristics may be influenced by Nt-acetylation, and if Nats are inhibited, the survival strategies of trypanosomes will be denied. Therefore, I hypothesized that the cellular integrity and survival of trypanosomes in different environments may depend on Nt-acetylation. Nats may function as biological sensors in these organisms, where some Nats may be switched on and others shut down, depending on temperature and environmental cues. That said, I imagine that inhibition of Nats may simultaneously affect hundreds to thousands of proteins needed for parasite survival, ultimately killing the pathogen and abolishing the infection.

Thus far, much as suspected Nats in trypanosomatids are not characterized nor detailed substrates known, a gene editing tool using CRISPR-Cas9 can now be employed to assess the functional significance of suspected Nats. To date, the CRISPR-Cas9 method has been tested and used to assess gene functions [200], and the technology is being improved for Plasmodium falciparum, the causative agent of malaria [201]. Specifically, for Trypanosomes, CRISPR-Cas9 has been performed and is being used for the functional characterization and interrogation of none of the Nat proteins [202, 203, 204].

However, even if CRISPR-Cas9 is used to knock out already known Nats, identification and substrate screening of different Nats in trypanosomes will be an important move to unravel the molecular insight of parasite biology. Furthermore, the regulation of these Nats will be tested. Alternatively, what happens if Nats or pathways that lead to the production of acetyl-CoA are inhibited? For the latter, might this mean that all Nats´ activities may be simultaneously affected? The answer to these questions will provide deeper knowledge of parasite behavior and strengthen the protein Nt-acetylation capability as a tool to sustain trypanosomes survival, thus providing a strong basis for Nats as potential new drug targets. Collectively, scientific communities should pay attention to Nt-acetylation, which likely cuts across all species, causing infectious diseases for eventual eradication, particularly neglected tropical diseases, as viewed by the WHO. Specifically, it is important to highlight the role of Nt-acetylation in trypanosomatid biology and the potential therapeutic implications of targeting Nt-acetylation in trypanosomes.

7.1 Role of Nt-acetylation in trypanosomatids biology

As mentioned previously, the role of Nt-acetylation in trypanosomatid biology has not been extensively studied, but it is believed to play a role in regulating protein stability, activity, folding patterns, degradation, and protein-protein interactions [127]. Here are some of the specific roles of Nt-acetylation in trypanosomatids that have been suggested in the available search results: Degradation mechanism: In yeast, the Nt-acetyl group was first described as a specific degradation signal, where proteins with an acetylated N-terminal methionine, alanine, valine, serine, threonine, or cysteine are targeted for degradation by the E3 ligase Doa104 [18, 205]. Nt-acetylation may also act as a degradation mechanism for trypanosomatids. Protein stability: Nt-acetylation is associated with protein stability in various organisms, including trypanosomatids [18, 205]. It is possible that Nt-acetylation plays a role in regulating the stability of proteins in trypanosomatids. Protein-protein interactions: Nt-acetylation has been shown to mediate protein-protein interactions in yeast and other organisms [18]. Possibly, Nt-acetylation may play a role in regulating protein-protein interactions in trypanosomatids as well. Chemotherapeutic target: Studies on N-terminal acetylation as a possible chemotherapeutic target to fight parasite infections are limited [18, 33]. Understanding the role of Nt-acetylation in trypanosomatids could provide insights into potential therapeutic targets. Overall, the specific role and significance of Nt-acetylation in trypanosomatids have not been extensively explored. Further research is required to elucidate the specific mechanisms and functions of Nt-acetylation in trypanosomatids.

7.2 Potential therapeutic implications of targeting Nt-acetylation in trypanosomatids

The potential therapeutic implications of targeting Nt-acetylation in trypanosomatids are not extensively explored in the available search results. However, there are potential therapeutic implications that can be inferred based on limited information. Drug target identification: Nt-acetyltransferases responsible for protein acetylation could be evaluated as potential drug targets in trypanosomatids. Targeting these enzymes could potentially disrupt protein acetylation processes and affect the stability, activity, or interactions of essential proteins in the parasites. Chemotherapeutic target: Although studies on N-terminal acetylation as a possible chemotherapeutic target in trypanosomatids are limited, understanding the role of Nt-acetylation could provide insights into potential therapeutic strategies [34]. Targeting the Nt-acetylation processes may offer a novel approach for developing drugs against trypanosomatid infections. Protein stability and regulation: Nt-acetylation has been implicated in regulating protein stability and other cellular processes [205]. Targeting Nt-acetylation can potentially disrupt protein stability and affect the survival and growth of trypanosomatids. Epigenetic regulation: Although not directly related to Nt-acetylation, the role of histone modifications in trypanosomatids has been investigated [21]. Understanding the interplay between Nt-acetylation and other epigenetic modifications may provide insights into the regulation of gene expression and potential therapeutic interventions. It is important to note that the potential therapeutic implications mentioned above are speculative and based on limited information. Further research is needed to explore the specific therapeutic potential of targeting Nt-acetylation in trypanosomatids, and to develop effective and safe treatment strategies.

8. Conclusions

There is scant information regarding Nt-acetylation in trypanosomes. However, the limited data presented suggests that it is common in these organisms. Only NatA and NatC are partially characterized in trypanosomatids, which are essential for parasite survival and growth. Therefore, a call to invest in Nats, vital enzymes for biological function, needs attention for possible assault to disrupt the parasite’s potential to cause infections. Trypanosome biology is unique when one considers their organelles, life cycle programming, gene regulation, epigenetic mechanisms, metabolism in different categories, or infection capabilities, where hundreds of surface molecules are employed. I suspect that many protein-coding genes responsible for parasite biology, in one way or another, are Nt-acetylated, or that the secret of trypanosomes survival is shouldered by Nt-acetylation. Therefore, inhibition of one Nat or several Nats can simultaneously affect many essential proteins and virulence factors, rendering no room for trypanosomes capability to survive in any environment given their way of life. With the advancement of gene editing technologies, CRISPR-Cas9 can be used to interrogate Nt-acetylation significance in trypanosomatids, digs into molecular insight, and fills the gaps that are needed for new intervention approaches. This will bring the scientific community closer to assessing Nt-acetylation as a potential new chemotherapeutic agent against trypanosomiasis. Furthermore, a study of Nats in one parasite and its biological significance may be similar in other pathogens; therefore, Nats inhibition may be used to control any organism causing infectious diseases. In addition, divergent fields of human and animal studies, and the concept of addressing many difficult problems facing them, will be brought together under a single theme of “One Health” in both developed and developing worlds. Thus, I may call Nats the “in-heart” possible drug targets to control both poor and wealthy men’s diseases, including animals.

Acknowledgments

Huge appreciation to Professor Björn Andersson, Karolinska Institute, Sweden, for all support rendered for many years. Many thanks to Associate Professor Lena Åslund, Department of Immunology, Genetics and Pathology, Uppsala University for all inputs during the Nt-acetylation study. I appreciate Professor Thomas Arnesen at the University of Bergen, Norway, for his encouragement in the field of N-alpha terminal acetylation. I appreciate all the co-authors for their engagement when we published the Nt-acetylation work. I convey my sincere thanks to Dr. Reginald Austin, affiliated with Gulu University, Uganda, and Muhimbili University of Health and Allied Sciences in Tanzania for all valuable discussions. I thank Professor Johan Bolting, Clinical Pathology, Uppsala Academic Hospital, Sweden for providing moral support. I also thank the pathologists and the staff members working at Clinical Pathology Department, Uppsala Academic Hospital, for all the necessary support. I appreciate Gulu University staff, especially those at the Faculty of Medicine, Faculty of Science, Faculty of Agriculture, and Gulu University leadership for providing space for intellectual discussions. I am grateful to my current and previous students for asking many questions about Nt-acetylation, which gave me positive thoughts. To all my friends and colleagues, I am associating with, in the scientific context and on other platforms, I appreciate all your positive contributions in different aspects, which have helped me to put things together. Finally, I thank my family for their valuable support.

Conflict of interest

The author declares no conflict of interest.