In order to control giardial infections, cellular and humoral immunity is required; the production of CD8+ T lymphocytes correlates in particular with the parasite clearance (Faubert, 2000). To escape the host humoral immune response, several protozoan pathogens can undergo antigenic variation. In Giardia, this evasion is achieved by the on-off switching of the expression genes encoding VSPs.
VSPs are cysteine-rich proteins covering the entire surface of the parasite, including flagella; the VSP family comprises a repertoire of approximately 200 genes in both human-infecting assemblages, A and B (Ankarklev et al., 2010, Adam et al., 2010, Nash, 2002, Adam, 2001). No identical VSPs have been found in the two assemblages (Franzen et al., 2009). The vsp coding regions comprise about 3 % of Giardia total genome; it can be raised to 4 % with the inclusion of the upstream
intergenic areas (which may be tasked with control of the vsp genes expression) (Adam et al., 2010).
The VSPs size ranges from 20 to 200 kDa; VSPs have variable amino termini (CXXC) and semi-conserved carboxyl termini (containing GGCY motifs). They also have a conserved hydrophilic cytoplasmic tail (CRGKA). Immediately adjacent to this CRGKA domain is a hydrophobic domain that forms the protein membrane anchor (Fig 1.15) (Ankarklev et al., 2010, Adam et al., 2010). The entire surface of Giardia is covered with VSPs; the expression of VSPs at the surface is mutually exclusive, except during differentiation and switching when several VSPs are simultaneously expressed (Prucca et al., 2011). Switching has been reported to happen every 6 to 13 generations; however, it depends on the growth conditions, Giardia isolate and the specific VSP expressed (Ankarklev et al., 2010). According to Singer et al, there is a selection for and against VSPs in immunodeficient hosts which demonstrates that VSPs may play a key role in the host-parasite interaction. In this study, it was also suggested that clear preferences existed for specific VSPs in different hosts.
Antigenic variation may allow the parasite to express VSPs that enhance survival in many different hosts and environmental conditions explaining the large repertoire of VSPs present in Giardia genome(Singer et al., 2001). However, the differences between hosts that may affect VSP selection have yet to be determined.
In addition to being involved in the immune evasion and host-parasite interaction, VSPs are also components of cellular signalling. Indeed, some VSPs are specifically palmitoylated on the cysteine residue in the conserved CRGKA carboxyl -terminal motif, located in the cytoplasmic tail (Fig 1.15). This palmitoylation helps to regulate the segregation of the proteins that are detergent resistant to domains on the plasma membrane (so-called lipid rafts) (Touz et al., 2005, Hiltpold et al., 2000). It was also suggested that complement-independent antibody-mediated cytotoxicity was due to specific changes or signalling mediated by the palmitoyl ated CRGKA cytoplasmic tail (Touz et al., 2005). Other VSPs can be citrullinated on the CRGKA arginine residue by arginine deiminase (Fig 1.15). Citrullation has an important role in the VSP switching mechanism; a mutation of this residue affects the switching frequency (Touz et al., 2008).
Fig 1. 15: Variant Surface Proteins (VSPs) schematic representation (Based on Ankarklev et al., 2010 with permission). VSPs produce a dense coat by covering the whole surface of the trophozoite. Only one type of VSP is found on the parasite surface (except during differentiation and the switching which occurs every 6 to 13 generations). One VSP usually dominates in a population of parasites; however, a few express other VSPs. VSPs contain about 11-12 % of cysteine AA, most of which is found numerous CXXC ( ) that build up disulfide bonds. The amino terminus is the most variable portion and is called variable domain; this domain seems to be at the interface between the parasite and its environment. The extracellular domain close to the plasma membrane is a semi-conserved domain and contains one or two GGCY motifs ( ). All VSPs have a conserved hydrophilic cytoplasmic tail composed of the amino acids CRGKA. This motif can be modified by palmitoylation of the cysteine residue and by citrullination of the arginine residue.
Our knowledge of the molecular mechanisms involved in the regulation of Giardia antigenic variation is still limited. There is no evidence of (i) gene rearrangements, (ii) DNA modifications along with the presence of expression-linked copies, (iii) sequence alterations of the DNA, (iv) telomere-expression-linked transcription requirements. All of these regulation mechanisms have been linked to antigenic variation in others protozoan parasites (such as Trypanosoma brucei and Plasmodium falciparum) (Lopez-Rubio et al., 2007). However, Kulakova et al suggested that epigenetic mechanisms control VSP regulation. They demonstrated that the expression of vsp was not because of special DNA rearrangement. They also showed that antigenic variation occurred in the absence of vsp gene movement but was associated with in situ chromosome changes in the immediate upstream sequences of the expressed VSP (Kulakova et al., 2006). This suggests that some epigenetic mechanisms may be involved in vsp gene transcription activation (Ankarklev et al., 2010, Kulakova et al., 2006). Other studies proposed an alternative hypothesis: vsp genes post-transcriptional silencing occurs via a micro RNA (miRNA)-mediated mechanism (Saraiya and Wang, 2008, Prucca et al., 2008).
During their investigation, Prucca et al showed that the silencing machinery in Giardia processes specifically vsp RNAs in vitro. Hence, it is possible that chromatin modification and post-transcriptional processes collaborate to regulate VSP expression.
Their transport to the surface represents a vital trafficking pathway in Giardia. An immunoelectron microscopy study showed the presence of VSPs in the ER, at the plasma membrane and in PVs, but not in ESVs of encysting cells (McCaffery et al., 1994). However, no intermediate compartments containing VSP cargo protein in the export pathway between the ER and the plasma membrane were identified. It was suggested that, because of a C-terminal transmembrane anchor and a short five AA cytoplasmic domain (CRGKA), these proteins were in direct contact with the cytoplasm and thus potentially able to mediate their own export from the ER. Marti et al showed that it was possible to generate an inducible chimeric reporter that trafficked to the plasma membrane by combining the exodomain of a Toxoplasma gondii surface antigen (SAG1) and the conserved C-terminus of a VSP under the control of the CWP1 promoter. This indicates that the
C-terminus of VSPs may be sufficient to direct a foreign antigen to the plasma membrane (Marti and Hehl, 2003). On the other hand, by using the entire VSP exodomain in a reporter domain, another study showed that the transport of VSP to the plasma membrane despite the absence of the cytoplasmic domain (Touz et al., 2003). However, due to the fact that a control experiment using a heterologous exodomain was not included in this study, it remained unclear whether the reporter lacking a C-terminus targeting signal was exported to the plasma membrane simply by co-transport through interaction with endogenous VSPs. A number of reporter proteins with different heterologous components were unable to exit the ER and progress along the secretory pathway without a VSP C-terminus which indicates that the presence of a general default pathway seems unlikely (Hehl and Marti, 2004, Marti and Hehl, 2003). It was suggested that coatomer proteins II (COPII) were recruited directly via the conserved cytoplasmic tail for the export of VSPs (Hehl and Marti, 2004). Once VSPs have left the ER, their transport to the plasma appears to follow a direct pathway with fast kinetics (Marti and Hehl, 2003).
VSPs are the most studied of Giardia-pathogenic factors; they are the main mechanism for Giardia evasion of the host humoral immune response. Therefore, how exactly antigenic variation occurs in Giardia in absence of any immune pressure has to be determined. How a VSP is replaced by another during antigenic switching requires also further investigations, as regulation mechanisms of VSPs expression.