Sequence analysis and prokaryotic expression of Giardia lamblia α-18 giardin gene

Highlights

• We studied the genetic variation of α18 giardin gene of G. lamblia assemblages A and F from different hosts.

• We analyzed the hydrophobicity, transmembrane domain and secondary structure of the α18 giardin.

• The first report on prokaryotic expression of the α18 giardin gene of G. lamblia

Abstract

To study the genetic variation and prokaryotic expression of α18 giardin gene of Giardia lamblia zoonotic assemblage A and host-specific assemblage F, the α18 genes were amplified from G. lamblia assemblages A and F by PCR and sequenced. The PCR product was cloned into the prokaryotic expression vector pET-28a(+) and the positive recombinant plasmid was transformed into Escherichia coli Rosetta (DE3) strain for the expression. The expressed α18 giardin fusion protein was validated by SDS-PAGE and Western blot analysis, and purified by Ni-Agarose resin. The putative sequence of α18 giardin amino acid was analyzed by bioinformatics software. Results showed that the α18 giardin gene was 861 bp in length, encoding 286 amino acids; it was 100% homologous between human-derived and dog-derived G. lamblia assemblage A, but it was 86.8% homologous with G. lamblia assemblage F (cat-derived). Giardin α18 was about 36 kDa in molecular weight, with good reactivity. Prediction based on in silico analyses: it had hydrophobicity, without signal peptide and transmembrane domain, and contained 11 alpha regions, 13 beta sheets, 1 beta turn and 7 random coils in secondary structure. The above information would lay the foundation for research about the subcellular localization and biological function of α18 giardin in G. lamblia.

Introduction

Giardia lamblia (syn. G. intestinalis, G. duodenalis) is a globally distributed intestinal protozoan that causes giardiasis in humans and other mammals, a generally self-limited illness typically characterized by diarrhea, abdominal cramps, bloating, weight loss, and malabsorption; but asymptomatic infection occurs frequently (Thompson and Monis, 2004, Ryan and Cacciò, 2013). Giardiasis has been listed as one of the ten major parasitic diseases that are harmful to human health in the world (WHO, 1996). G. lamblia has a simple life cycle with cyst and trophozoite forms. The infection is transmitted through the fecal–oral route and accidental ingestion of giardia cysts through the consumption of fecal contaminated food or water or through person-to-person (or, to a lesser extent, animal-to-person) transmission (Yoder et al., 2012). The cysts can excyst into trophozoites in the small intestine. Under hostile environment in the small intestine, trophozoites become cysts by secreting a capsule wall, which passes out of the host in its feces, then they can infect new hosts (Adam, 2001, Gardner and Hill, 2001).

Many studies have confirmed that there is apparently a strong link between cytoskeleton and virulence in G. lamblia; the former is constantly required for parasite attachment, detachment, and movement in response to the changing conditions present in the intestine (Elmendorf et al., 2003, Campanati and Souza, 2009). Giardins are unique cytoskeletal components of G. lamblia, which have been divided into α-, β-, γ-, and δ-giardins. G. lamblia possesses 21 different α-giardin encoding genes numbered from α1- to α19-giardin (α7 giardin appears as three variants), and α-giardin proteins are annexin homologs, showing Ca^2 +-dependent association with phospholipids (Morgan and Fernandez, 1995, Weiland et al., 2005). Given that G. lamblia may represent one of the earliest diverging eukaryotes, the occurrence of as many as 21 annexin homologs is surprising, gene duplication is a potential explanation for the large number of giardin variants found to date (Weiland et al., 2005). Several eukaryotes have no or only two to four annexin homologs e.g. Saccharomyces cerevisiae, Dictyostelium discoideum, Neurospora crassa and Caenorhabditis elegans (Gerke and Moss, 2002). This diversity of α-giardins is suggestive of functional importance in the parasite. Major α-giardins have been shown to localize to cytoskeletal components such as the flagella, the adhesive disk and the plasma membrane, and may be active participants in the highly organized membrane and cytoskeletal rearrangements during excystation and encystation (Weiland et al., 2005, Vahrmann et al., 2008, Saric et al., 2009, Wei et al., 2010, Feliziani et al., 2011, Kim et al., 2013). Many α-giardins are also highly immuno-reactive during acute human giardiasis, and they may be used as a potential target for immune diagnostic antigens and vaccines (Palm et al., 2003, Jenikova et al., 2011). So far, only subcellular localizations of α7.1, α12, α13 and α18 giardins in G. lamblia trophozoite are still unknown and little is known about α18 giardin.

Therefore, we cloned the α18 giardin coding sequence from the genomic DNAs of G. lamblia zoonotic assemblage A (human-derived and dog-derived) and host-specific assemblage F (cat-derived) and analyzed the sequence variation. Bioinformatics analysis was performed to predict the biological features of α18 giardin. The α18 giardin fusion protein was expressed in Escherichia coli and purified by Ni-Agarose resin. These studies will be expected to provide a basis for studying subcellular localization and biological function of α18 giardin in G. lamblia.