CHAPTER 1: LITERATURE REVIEW
2.1 Introduction
African trypanosomosis affects both humans (HAT) and animals (AAT) in rural central Africa. The human infective parasites use wild and domestic animals as a reservoir (Njiokou et al., 2006; Cordon-Obras et al., 2009; Anderson et al., 2011). Thus, the control of AAT is critical for the elimination of HAT, as control of the insect vector is an ineffective strategy (Rotureau and Van Den Abbeele, 2013). The indiscriminate use of trypanocides and the lack of new chemotherapies, resulted in the emergence of drug resistance, and there is consequently a need for new drugs (Field et al., 2017).
Molecular targets for the development of novel chemotherapies are those which are essential for the parasite’s survival in the host (Hölzmuller et al., 2008). These targets, also known as virulence factors, are used in an anti-disease strategy rather than an anti-parasite strategy (Antoine-Moussiaux et al., 2009) as the development of a vaccine is unlikely due to antigenic variation (La Greca and Magez, 2011). The anti-disease strategy focusses on targeting of factors which are essential to the growth and survival of the parasite (Stuart et al., 2008).
Homologues of the metazoan caspases are the metacaspases (MCA) which are found in all kingdoms except that of the metazoan (Uren et al., 2000). Apoptosis is a controversial process in unicellular organisms; however, evidence in its favour is mounting (Deponte, 2008; Kaczanowski et al., 2011). Morphological and biochemical features, which are similar to those seen during apoptosis, have been described in T. brucei (Welburn et al., 2006b), T. cruzi (Ameisen et al., 1995), Leishmania spp.
(Gannavaram and Debranbant, 2012), Giardia lamblia and Plasmodium falciparum (Bruchhaus et al., 2007). Due to the roles played by caspases in apoptosis and non-apoptotic events, it is thought that the MCAs may function in a manner similar to that of the caspases. As such, the MCAs are considered to be virulence factors as well as attractive drug targets due to their absence in their mammalian hosts.
Despite the conserved caspase-haemoglobinase fold (Aravind and Koonin, 2002), the caspases and MCA are distinctly different in their substrate specificity, activation
29 mechanisms, calcium dependency and control of peptidolytic activity. In order to determine the role and processes they are involved in, the MCAs need to be identified, functionally characterised and their roles in parasite homeostasis determined.
A number of kinetoplastid MCAs have been studied to date, including the multicopy MCAs, MCA1 (Szallies et al., 2002), MCA2 (Helms et al., 2006; Moss et al., 2007;
McLuskey et al., 2012; Machado et al., 2013), MCA3 (Helms et al., 2006), MCA4 (Szallies et al., 2002; Proto et al., 2011) and MCA5 (Helms et al., 2006) from T. b. brucei, the MCA3 and -5 from T. cruzi (Kosec et al., 2006; Laverrière et al., 2012), and the single copy MCAs from L. major (Gonzáles et al., 2007; Zalila et al., 2011;
Castanys-Muῆoz et al., 2012; Casanova et al., 2015), L. donovani (Lee et al., 2007;
Raina and Kaur, 2012) and L. mexicana (Castanys-Muῆoz et al., 2012). Most studies focused on the native MCA function in vitro when cell death has been induced in the parasites. Very few studies focus on the characterisation of the recombinant and native enzymes themselves.
Analysis of the phylogenetic relatedness of the multi- and single copy MCA peptidases in Trypanosoma spp. T. cruzi, Leishmania spp. and the single MCA from Saccharomyces cerevisiae (YCA1), shows a clear division between the multi- and single copy groups, shown in yellow and blue, respectively, in Fig. 2.1. Within the multicopy group, a high sequence identity exists between the MCA2 and -3 of T. b. brucei, T. b. gambiense and T. evansi. The MCA2 and -3 of the animal infective T. congolense and T. vivax species, are not as related to their human infective counterparts. The MCA1, -4 and -5 of T. b. brucei, T. b. gambiense and T. evansi share a 100% sequence identity, and therefore, reference will only be made to those from T. b. brucei.
Pseudopeptidases have substitutions for the catalytic residues and have been shown to be inactive (Reynolds and Fischer, 2015). The MCA1s (Fig. 2.1, shown in red) have both the catalytic His and Cys substituted with Tyr and Ser, respectively, whilst the MCA4s (Fig. 2.1, shown in purple) all possess the catalytic His, but have a Ser substitution for the catalytic Cys. The substitution of catalytic residues are common with some pseudopeptidases being shown to play key regulatory roles (Pils and Schultz, 2004; Reynolds and Fischer, 2015). It has been reported that TbbMCA3 processes TbbMCA4, releasing TbbMCA4 which plays a role in cell cycle and parasite virulence (Proto et al., 2011).
The focus of the current study is the MCA5 from the animal infective T. congolense and T. vivax (Uilenberg and Boyt, 1998). These MCAs differ from the multicopy gene
30 products, as they possess a long Pro-, Gln-, Tyr-rich C-terminal domain. This extended domain is present in each of the single copy MCAs in both Trypanosoma spp. and Leishmania spp. (Appendix A3) and is thought to play an important role in protein-protein interactions (Kay et al., 2000). One such example is the apoptotic-like response in T. cruzi parasites, in which the TcrMCA5 lacking the C-terminal domain, was overexpressed (Laverrière et al., 2012).
Figure 2.1: Molecular phylogenetic analysis of the kinetoplastid MCAs and the single MCA from S. cerevisiae. The bootstrap consensus tree from 500 replicates (Felsenstein, 1985) utilising the maximum likelihood method, was used to deduce the evolutionary history of 26 MCA protein sequences (Jones et al., 1992) with MEGA7 (Kumar et al., 2016). The protein sequences were obtained from TriTrypDB (Aslett et al., 2010); TbbMCA from T. b. brucei (927), TbgMCA from T. b. gambiense (DAL972), TviMCA from T. vivax (Y486), TevMCA from T. evansi (STIB 805), TcoMCA from T. congolense (IL3000), TcrMCA from T. cruzi (Sylvio X10/1), LmjMCA from L. major (Friedlin), LmxMCA from L. mexicana (MHOM/GT/2001/U1103), LdnMCA from L. donovani (BPK282A1) and were compared to the YCA1 from S. cerevisiae (UniProt Q08601). The single copy MCAs are grouped in yellow and the multicopy MCAs in blue. The multicopy MCAs with mutations of the catalytic Cys (purple) and both catalytic His and Cys (red) were blocked in their respective colours. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.
31 In the work described in this chapter, the MCA2 gene from T. b. brucei and MCA5 gene from T. congolense were cloned, to include a N-terminal 6xHis tag, and were expressed in the soluble and insoluble fractions using Escherichia coli. After solubilisation, on column refolding and purification using nickel affinity chromatography, purified recombinant TviMCA5 was used to produce antibodies in chickens. The chicken antibodies produced against both TcoMCA5 and TviMCA5 were separately coupled to hydrazide resin to purify the respective MCAs present in the soluble expression fractions.