Immunodominant proteins α-1 giardin and β-giardin are expressed in both assemblages A and B of Giardia lamblia

Feliziani et al. BMC Microbiology 2011, 11:233 http://www.biomedcentral.com/1471-2180/11/233 RESEARCH ARTICLE Open Access Immunodominant proteins a-1 giardin and b-giardin are expressed in both assemblages A and B of Giardia lamblia Constanza Feliziani1, María C Merino1, María R Rivero1, Ulf Hellman2, María C Pistoresi-Palencia3 and Andrea S Rópolo1* Abstract Background: To date, eight assemblages of Giardia lamblia have been described, but only assemblages A and B are known to infect humans. Despite the fact that the genomic, biological, and clinical differences found between these two assemblages has raised the possibility that they may be considered different species, there is relatively limited information on their phenotypic differences. In the present study, we developed monoclonal antibodies against alpha-1 and beta giardin, two immunodominant proteins produced during G. lamblia infection, and studied their expression and localization in WB (assemblage A) and GS trophozoites (assemblage B). Results: The polyclonal antibodies generated against WB trophozoites, particularly those recognizing intracellular proteins as well as the proteins present at the plasma membrane (variable-specific surface proteins), showed crossreactivity with intracellular proteins in GS trophozoites. The use of monoclonal antibodies against beta giardin indicated ventral disc localization, particularly at the periphery in WB trophozoites. Interestingly, although beta giardin was also restricted to the ventral disc in GS trophozoites, the pattern of localization clearly differed in this assemblage. On the other hand, monoclonal antibodies against alpha-1 giardin showed plasma membrane localization in both assemblages with the bare area of GS trophozoites also being distinguished. Moreover, the same localization at the plasma membrane was observed in Portland-1 (Assemblage A) and in P15 (Assemblage E) trophozoites. Conclusions: We found differences in localization of the beta giardin protein between assemblages A and B, but the same pattern of localization of alpha-1 giardin in strains from Assemblages A, B and E. These findings reinforce the need for more studies based on phenotypic characteristics in order to disclose how far one assemblage is from the other. Background Giardia lamblia is a flagellated unicellular microorganism that causes Giardiasis, a generally self-limited clinical illness [1]. Typically, the infection is characterized by diarrhea, abdominal cramps, bloating, weight loss, and malabsorption, although asymptomatic infection also frequently occurs [2]. G. lamblia infection is transmitted by the faecal-oral route and results from the ingestion of cysts through the consumption of contaminated food or * Correspondence: 1 Laboratorio de Microbiología e Inmunología, Instituto de Investigación Médica Mercedes y Martín Ferreyra, INIMEC - CONICET, Friuli 2434, (5000) Córdoba, Argentina Full list of author information is available at the end of the article water or from person-to-person transmission. Giardia is distributed globally and has been detected in nearly all classes of vertebrates, including domestic animals, wildlife and in marine vertebrates [3,4]. Since the 80’s, differences have been observed between different isolates of Giardia, both in isoenzyme studies and in surface-antigen, as well as in the DNA banding pattern after endonuclease restriction analysis, giving rise to the hypothesis that these differences might explain the various clinical manifestations, host responses and treatment efficacy of human Giardiasis [5-7]. Nowadays, advances in molecular epidemiology have enabled specialized genetic groups (i.e., assemblages) to be identified that are relatively species-specific. Among the eight defined genotypes of © 2011 Feliziani et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Feliziani et al. BMC Microbiology 2011, 11:233 http://www.biomedcentral.com/1471-2180/11/233 Giardia, only assemblages A and B are known to infect humans, and these two have shown differences related to axenic in vitro culture conditions [8-10], metabolism, biochemistry, DNA content, and clinical features, among others [4,11-13]. All these biological differences may be explained by genetic as well as genomic differences, such as the presence of isolate-specific proteins, unique patterns of allelic sequence divergence, differences in genome synteny and in the promoter region of encystation-specific genes and differences in VSP repertoires [14]. It has, therefore, been suggested that assemblages A and B could be considered to be two different Giardia species. During the vegetative stage of the parasite, the trophozoite attaches to the intestinal microvilli to colonize and to resist peristalsis. The ventral disc allows the parasite to orient, ventral side down, to biological or inert substrates, and is a concave cytoskeletal structure surrounded by a plasma membrane, composed of 3 distinct features (microtubules that are coiled around a bare area; microribbons that protrude into the cytoplasm; and cross-bridges that connect adjacent microtubules) [15]. Three gene families of giardins generally localize to the ventral disc including: (i) annexins (i.e. a-giardins) that are localized at the outer edges of microribbons [16-21]; (ii) striated fiberassemblins such as b-giardin, which are closely associated with microtubules and δ-giardin (a component of microribbons) [22,23]; and (iii) g-giardin, which is also a microribbon protein [24]. Alpha-giardins form a large class of proteins encoded by 21 different genes (named a-1 to a-19). All of these 21 alpha-giardin genes in WB were found to be conserved in GS along with the genome synteny, although the structural protein alpha-2 giardin was postulated to be an assemblage A-specific protein of human infective G. lamblia [25]. However, in a recent study, Franzén et al. encountered a a-2 giardin-like gene in the assemblage B GS strain, with a 92% aa identity in a syntenic position [14]. Differences occurring in the structural proteins may explain the differences observed in key infection processes such as adhesion and motility between both assemblages. To date, the intracellular localization of giardins in G. lamblia has been performed using rabbit polyclonal antisera or by the use of epitope tagged a-giardins [19,26]. However, both these methods have limitations when attempting to study assemblages A and B because polyclonal antibodies have shown cross-reaction with other proteins, while transfection experiments are difficult to carry out on GS assemblages [27]. Therefore, we developed monoclonal antibodies (mAbs) against the two immunodominant proteins, a-1 giardin and b-giardin, and com (...truncated)