Currently, there is no vaccine available to prevent giardiasis in humans, while an unrefined veterinary vaccine only reduces the symptoms and duration of cyst shedding in cats and dogs ^[1][21]. Commonly used drugs for the treatment of giardiasis, such as nitroimidazoles ^[2][3][22,23] and benzimidazoles, can cause mild to serious side effects, and treatment failures with metronidazole (MTZ) have been reported ^[2][4][22,24]. In recent years, proteomic approaches have gained popularity as a way of finding new targets to enable the development of safer effective drugs.

Several proteomic studies have aimed to investigate the biochemical aspects of the parasite ^[5][18], the significant role of proteins expressed in the completion of the life cycle ^[6][25], the antigenic surface proteins in the intestinal lumen ^[7][20], the differentially expressed proteins implicated in the mechanism of action resistance to drugs ^[8][9][26,27], and new potential chemotherapy agents ^[10][11][19,28]. Since the life cycle of G. duodenalis alternates between cysts and trophozoites, it is crucial to compare the proteomes of trophozoite encystment at different stages. The change in morphology, followed by the modification of protein expression levels, is vital for encystment, thus implying that the proteins involved in this process may be key targets for vaccine and drug development.

One powerful proteomics tool with which to estimate stage-specific protein abundance is isobaric tags for relative and absolute quantitation (iTRAQ), an advanced multiplexing technique that assists in identifying and quantifying proteins simultaneously ^[12][29]. Lingdan et al. (2012) examined the differential expression of soluble proteins during G. duodenalis sporozoite encystation where the trophozoites and cysts were isolated from faeces. High-performance liquid chromatography (HPLC) was then used to fractionate the isobarically tagged proteins for further proteomic analyses using a database search ^[6][25]. As a result, 63 proteins were quantified by iTRAQ labelling, and these labelled proteins were then classified as cytoskeletal proteins, metabolic enzymes, cell-cycle-specific kinases, and stress resistance proteins by using MS analyses. In addition, significant differences in the expression of seven proteins in the trophozoites and cysts that are associated with encystation have also been reported in several studies ^[13][14][30,31]. In particular, Lingdan and his colleagues described the role of these seven proteins dissolved in the life cycle of G. duodenalis, raising their potential as likely targets for the development of vaccines and chemotherapies that inhibit the transmission of G. duodenalis into the epithelial cells of hosts ^[6][25].

Other studies have examined the potential of using repurposed drugs. Camerini et al. (2017) investigated the use of the anticancer drug 6-(7-nitro-2,1,3-benzoxadiazole-4-ylthio) hexanol (NBDHEX) to find protein targets other than phosphate dehydrogenase ^[10][19]. To identify the proteins potentially targeted by NBDHEX in G. duodenalis trophozoites, Camerini and her co-researchers performed a bottom-up proteomic study using a combination of SDS-PAGE, Western blot, and a mass spectrometry analysis, and detected several fluorescent protein bands in NBDHEX-treated samples, with only one or two cysteines found to be specifically NDBHEX-modified in each protein. For instance, modified Cys137 and Cys140, discovered in thioredoxin reductase, gTrxR, and Cys347, of gα-TUB structure proteins, were covalently bound to NBDHEX, suggesting that the functions of many of these protein targets were inhibited when treated with NBDHEX. The study also found that NBDHEX killed G. duodenalis trophozoites at a dose five times lower than that of MTZ (NBDHEX IC50:0.3 ± 0.1 mM; MTZ IC50: 1.5 ± 0.1 mM), thus supporting the idea that this drug agent could be a good option for treatment-refractory giardiasis in the future ^[15][32].

Cell surface proteins have been identified to be the source of antigens in the intestinal lumen between two genetic assemblages (A and B) of G. duodenalis, which may inform vaccine development. A study by Langford et al. (2002) highlighted that surface proteins might be crucial targets of protective IgA responses, and they identified several biotin-labelled proteins from total cell lysates of G. duodenalis WB strain (assemblage A) trophozoites and G. duodenalis GS/M (assemblage B) trophozoites using mass spectrometry ^[16][33]. Another protein analysis by Davids et al. (2019) led to the identification of 86 proteins in assemblage A, 51 proteins in assemblage B, and 27 proteins in both assemblages, for which 15 and 6 proteins from each group were annotated as variant surface proteins (VSPs), respectively ^[7][17][20,34]. A surface proteome analysis of these proteins, using a multiplex beads immunoassay, identified several conserved antigens present on the surface of the trophozoite, namely α1-giardin, α11-giardin, β-giardin, and γ-giardin, making these antigens suitable candidates for human vaccine development.

Although genome sequences for assemblages A, B, and E have been published ^[18][19][20][35,36,37], little is known about the specific differences in virulence factors between Giardia strains and assemblages upon infection from a proteomics point of view. OTo the best of our knowledge, only two proteomics studies have been published that aimed to analyse the virulence proteins in G. duodenalis: one study investigated G. duodenalis in humans and cockatoos ^[21][11], and the other focused on canine isolates to understand giardiasis in dogs ^[22][38].

The first study, by Emery et al. (2014), presented the findings of a comparative shotgun proteomic study between two different strains of assemblage A of G. duodenalis that may be associated with the virulence of giardiasis in mammals, namely BRIS/95/HEPU/20141 (B-2041) and BRIS/83/HEPU/106 (H-106) ^[21][11]. B-2041 and H-106 were isolated from a wild-caught cockatoo and a diarrheic child in Brisbane, Australia, respectively, the former representing a virulent strain and the latter a control strain. Since both strains were isolated from the same area from the zoonotic assemblage A1, capable of transmitting from animals to humans ^[21][23][11,39], the authors were able to elucidate the disease mechanisms and antigenic variation of Giardia independent of assemblage and geographical variation. They utilised label-free shotgun proteomics by using a gel-based platform (LC-MS/MS) combined with an in-solution platform (filter-aided separation of protein; FASP) to assess the total protein abundance and proteome coverage. According to the study, 1376 proteins were identified in both strains, with a large core of 76.6% common proteins being shared between the two strains ^[24][25][40,41]. B-2041 was found to have a wider range of VSPs than H-106, with some of the VSPs hypothesised to be involved in giardiasis virulence. Interestingly, the authors noted that there were less antibodies specific for Giardia antigens in B-2041 compared to H-106, concluding that the greater the antigenic variation between different strains at the intra-assemblage level, the more diverse the population of the parasite that is capable of evading the host immune responses ^[21][11]. Indeed, the antigenic variation of G. duodenalis becomes a key source of variability in the virulence of different strains of the same assemblage.

Another study, by Coelho et al. (2016), analysed the proteomic mapping of soluble and insoluble protein fractions of trophozoites in canine G. duodenalis using 2D electrophoresis ^[22][38]. The group utilized the BHFC1 strain of G. duodenalis, isolated from dog stool, and identified 187 proteins, 27 of which matched hypothetical proteins, while the remaining ones had been previously annotated. Among the 27 hypothetical proteins, there were 20 soluble proteins and 4 insoluble proteins, and another 3 were found in both soluble and insoluble proteins. From the remaining 160 annotated proteins, the numbers of soluble and insoluble proteins found were 79 and 53, respectively, while another 20 were identified in both proteins. Most of the identified proteins were involved in metabolic processes, catalytic activity, nucleic acid binding, hydrolases, and oxidoreductases ^[22][38]. Additionally, some of these proteins have been related to virulence in other pathogens, namely Candida albicans ^[26][42], Pseudomonas sp. ^[27][43], and Shigella flexneri ^[28][44]. A comparison of the proteins of canine G. duodenalis with proteins of human G. duodenalis may lead to a better understanding of the biology of this parasite as well as of the virulence of giardiasis in different species, which can aid efforts to control zoonotic giardiasis. Notably, despite reports of high rates of G. duodenalis infection in domestic dogs in several countries, far fewer studies have been performed on canine isolates than on human isolates of G. duodenalis, highlighting a significant gap in the understanding of the risk of the transmission of giardiasis from dogs to humans ^[29][30][45,46].

Several studies have focused on comparing the proteome changes of G. duodenalis across in vitro encystation to understand the pathogenicity mechanism of Giardia ^[2][31][16,22]. A recent study, by Balan et al. (2021), formed a high-resolution quantitative proteomic analysis of encystation that covered the encystation process through to cyst maturation ^[31][16]. In their quantitative proteomics workflow, Balan et al. (2021) digested the proteins from different stages of G. duodenalis from its in vitro culture into peptides. The peptides were then quantified and characterized by using LC/MS/MS followed by a database search. By comparing the proteins extracted from trophozoites, low-bile primed (LB) trophozoites, and the 16 h post-induction of encystation and mature cysts, the authors identified a total of 3863 proteins across all stages ^[31][16]. In addition, 667 of these proteins had no preceding proteomic data ^{[32][33][34][35]}[12,13,47,48]. The proteins identified by this group were a three-fold increase in the proteins quantified during encystation by Faso et al. (2013) ^[31][32][12,16]. They also determined 15, 9, 8, and 24 proteins unique to trophozoites, LB trophozoites, encystation cysts, and mature cysts, respectively. Their findings showed that each life stage of G. duodenalis has a significant shift in overall protein expression across encystation. For example, proteomic changes during encystation include the downregulation of cell adhesion proteins, which is linked to changes in the cytoskeleton that cause the ventral disc and flagella to disappear ^[36][14].

Increasing interest in host–parasite interactions in pathogenesis has led to the introduction of secretomic studies in G. duodenalis, as secretory proteins have recently been shown to play vital roles in the cross-talk between cells ^[37][49]. Mass spectrometry secretome-based profiling is a powerful strategy with which to determine and characterise the secretory proteins in the parasite, which can be based on two main proteomics workflows: in-solution digestion combined with LC-MS/MS, and in-gel digestion coupled with LC-MS/MS ^[33][38][39][10,13,50]. Duoborg et al. (2018) conducted a quantitative proteomics study on Giardia assemblages A and B to quantify secreted proteins, which may act as the main mediators of giardiasis pathology. In their study, the soluble and cytosolic fractions of the Giardia proteins were extracted from in vitro cultures of two different strains, namely the WB strain (assemblage A) and the GS strain (assemblage B). Two MS techniques were used, Q-Exactive and Orbitrap MS, to identify the proteins. The proteins were then quantified by using intensity-based absolute quantification (iBAQ). A total of 1542 GS proteins and 1641 WB proteins were identified by using Q-Exactive ^[38][10]. The authors concluded that the most abundant proteins secreted by Giardia are cathepsin B cysteine protease and other members of the Giardia family of cysteine-rich proteins. In addition, Duoborg et al. discovered a new virulence factor, Giardia tenascin, which contributes to a novel mechanism of Giardia pathogenesis and was found to be highly abundant in the whole secretome ^[38][10].

Similarly, other studies sought to uncover changes in the upregulation and downregulation of functional proteins, particularly in host–pathogen interactions ^[8][33][40][13,26,51]. A study by Ma’ayeh et al. (2017) characterised the excretory–secretory products (ESPs) of G. duodenalis during the colonisation of intestinal epithelium cells (IECs) ^[33][13]. This study reported that metabolic functions, such as glycolysis, arginine metabolism, phospholipid re-modelling, and the salvation of purines and pyrimidines, were involved as a secretory response when the trophozoites of Giardia interact with the IECs of a host. These results align with findings by Ringqvist et al. (2008) that Giardia releases glycolytic enzymes when it infects a host ^[41][52].

In addition, Ma’ayeh et al. (2017) noted competition in obtaining nutrients between the parasite and host cells, given the similarity of the metabolic proteins released by both the parasite and host ^[33][13]. For instance, enzymes such as ubiquitin-protein ligase (UPL-1) and phospholipase B (PLB) released by the parasite were upregulated as G. duodenalis is unable to perform de novo pyrimidine or lipid synthesis, relying solely on nutrients from the host ^[17][42][34,53]. A functional secretome analysis of parasite-infected IECs showed that G. duodenalis trophozoites initiate cytoskeletal changes as the parasite attaches to IECs very strongly, leaving marks on the cell surface and hence disturbing the arrangement of the actin cytoskeleton ^[43][44][45][54,55,56], especially the protein villin. Consequently, these findings support reports by Bhargava et al. (2015) that villin is cleaved during Giardia infection, severing its protective role from the actin cytoskeleton ^[33][46][13,57]. Collectively, the proteomic studies that focus on encystation and host–pathogen interactions have enriched the understanding of Giardia pathogenesis.

Several post-translational modifications (PTMs) of proteins are reportedly involved in Giardia encystation, namely deacetylation ^[47][58] and phosphorylation ^[48][59]. Consequently, several proteomic studies have aimed to characterise the PTMs of proteins in G. duodenalis ^{[21][49][50][51][52][53]}[4,11,15,60,61,62], such as the role of G. duodenalis DHHC proteins in protein S-palmitoylation during Giardia encystation ^[52][61]. Specifically, Merino et al. (2014) reported that nine DDHC proteins were identified in trophozoites and encysting cells of G. duodenalis, and concluded that the presence of DDHC proteins in the encysting parasites indicates that the protein S-palmitoylation is maintained and involved in cell signalling, protein-sorting, and protein exporting throughout encystation. However, these proteins showed variation in intracellular localization in trophozoites and patterns of cyst wall expression, suggesting that differentially regulated palmitoylation in Giardia encystation enables the parasite to adapt to various environments ^[52][61].

PTMs of Giardia proteins also cause antigenic variation, as seen in the presence of VSP subpopulations across different Giardia assemblages or different strains of the same assemblage ^[21][11]. Müller et al.’s (2020) attempt to characterise the surface antigens of trophozoites from three different strains of G. duodenalis, namely WBC6 and WBA1 (both representing assemblage A), as well as GS/M-83-H7 (classified as assemblage B), showed that VSP5 (GL50803_113793) and VSP44 (GL50803_113450) were identified in strain WBC6, VSPH7 (GSB150963) was identified in strain GS/M-83-H7, and VSPA6 (GL50803_221693), a hypothetical protein, was identified in strain WBA1 ^[54][63]. Using LFQ intensity and iTop3 protein intensities, the group found that only the WBA1 strain had the most “homogenous” trophozoites, while the others yielded mixed populations of trophozoites.

Recently, Emery-Corbin et al. (2021) utilised a chromatin proteomics analysis, using mass spectrometry for histone identification and MaxQuant software for PTM mapping, to generate a molecular map of histone methylation, acetylation, and phosphorylation modifications in this parasite core histone ^[55][64]. The group identified over 50 sites, including sites with established roles in epigenetic regulation, amounting to a total of 56 histone modifications in Giardia that have been identified thus far ^[56][57][9,65]. Additionally, the authors were able to characterise chromatin modifiers by using protein sequence, domain, and structural homology to annotate the networks of putative histone enzymes, and identified 10 histone PTM sites detected by antibodies using immunoblots, thus providing a comprehensive and complete view of the histone PTMs in Giardia ^[55][64].

Another recent study, by Zhu et al. (2021), that utilised a global approach in investigating metabolic conversion mechanisms of G. duodenalis under stress revealed a total of 2999 lysine acetylation (Kac) sites on 956 proteins and 8877 2-hydroxyisobutyrylation (Khib) sites on 1546 proteins when G. duodenalis was under sugar starvation ^[53][62]. The authors noted a temporal reduction in both Kac and Khib proteins when G. duodenalis was cultured under sugar starvation for 72 h, indicating their involvement in energy conversion metabolism. They concluded that the correlation of acetylation and 2-hydroxyisobutyrylation expressed proteins linked to amino acid metabolism, suggesting that Giardia’s regulatory mechanism involves dynamic changes in acetylation modification to supply energy in the absence of glucose ^[53][62].