A 63 kDa VSP9B10A-like protein expressed in a C-8 Giardia duodenalis Mexican clone.
BACKGROUND
It is well documented that Giardia duodenalis undergoes surface antigenic variation both in vivo and in vitro. Proteins involved have been characterized and referred to as VSP (variable surface protein).


METHODS
Two cloned cDNA inserts of 0.45 and 1.95 kb were obtained from G. duodenalis expression library and sequenced. Comparison sequence analyses were made against Genbank. PCR analysis was performed on G. duodenalis isolates to identify isolates bearing genes encoding such a peptide. Specific antiserum was prepared against 450-bp encoded peptide and tested by Western blot, immunofluorescence, and inhibition of adhesion of G. duodenalis to target cells.


RESULTS
We cloned and characterized a G. duodenalis 450-bp DNA fragment; its DNA sequence analysis revealed that this fragment displayed 99% identity with vsp9B10A gene. Predicted amino acid sequence for this fragment also had significant (99%) identity to VSP9B10A. A second 1.95-kb insert, which encompassed the 450-bp cDNA fragment, was also isolated; its DNA and amino acid sequence displayed 99.5% identity with vsp9B10A gene and 99.2% with the corresponding inferred protein, respectively. This inferred protein contained 24 Cys-X-X-Cys motifs and long ORF of 642 aminoacids. PCR analysis showed that DNA sequence encoding a fragment of this gene was present in P1, CIEA:0487:2-C-8 clone and in INP:180800-B2 G. duodenalis human isolates, while it was absent in sheep isolate of G. duodenalis INP:150593-J10.


CONCLUSIONS
Immunofluorescence analysis using antibodies raised against the peptide encoded by 450-bp fragment showed that expression of this epitope varies on trophozoite surface of the C-8 Mexican clone and is involved in parasite adhesion to target epithelial cells.
Bermúdez-Cruz
Rosa María
RM
Departamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico National (Cinvestav del IPN), Mexico City, Mexico.
Ortega-Pierres
Guadalupe
G
Ceja
Víctor
V
Coral-Vázquez
Ramón
R
Fonseca
Rocío
R
Cervantes
Lourdes
L
Sánchez
Alejandra
A
Depardón
Francisco
F
Newport
George
G
Montañéz
Cecilia
C
D000595:Amino Acid Sequence; D000818:Animals; D000953:Antigens, Protozoan / Q000737:chemistry; D001483:Base Sequence; D015153:Blotting, Western; D002448:Cell Adhesion; D002460:Cell Line; D002675:Child, Preschool; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004285:Dogs; D000939:Epitopes / Q000737:chemistry; D015723:Gene Library; D014644:Genetic Variation; D005872:Giardia / Q000378:metabolism*; D005873:Giardiasis / Q000276:immunology; D006801:Humans; D007700:Kinetics; D008800:Mexico; D051379:Mice; D008807:Mice, Inbred BALB C; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012756:Sheep; D013997:Time Factors
D000953:Antigens, Protozoan; D018076:DNA, Complementary; D000939:Epitopes; D010455:Peptides; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D004247:DNA