Virulence attenuation of a UDP-galactose/N-acetylglucosamine beta1,4 galactosyltransferase expressing Leishmania donovani promastigote.
Protozoan parasites of the genus Leishmania are the causative agent of leishmaniasis, a disease whose manifestations in humans range from mild cutaneous lesions to fatal visceral infections. Human visceral leishmaniasis is caused by Leishmania donovani. Long-term culture in vitro leads to the attenuation of the parasite. This loss of parasite virulence is associated with the expression of a developmentally regulated UDP-Galactose/N-acetylglucosamine beta 1-4 galactosyltransferase and galactose terminal glycoconjugates as determined by their agglutination with the pea nut agglutinin (PNA). Thus, all promastigotes passaged for more than 11 times were 100% agglutinated with PNA, and represent a homogeneous population of avirulent parasites. Identical concentrations of PNA failed to agglutinate promastigotes passaged for < or =5 times. These PNA(-) promastigotes were virulent. Promastigotes passaged from 5 to 10 times showed a mixed population. The identity of populations defined by virulence and PNA agglutination was confirmed by isolating PNA(+) avirulent and PNA(-) virulent clones from the 7th passage promastigotes. Only the PNA(+) clones triggered macrophage microbicidal activity. The PNA(+) clones lacked lipophosphoglycan. Intravenous administration of [(14)C] galactose-labeled parasite in BALB/c mice resulted in rapid clearance of the parasite from blood with a concomitant accumulation in the liver. By enzymatic assay and RT-PCR we have shown the association of a UDP-Galactose/N-acetylglucosamine beta1,4 galactosyltransferase with only the attenuated clones. By immunofluorescence we demonstrated that the enzyme is located in the Golgi apparatus. By western blot analysis and SDS-PAGE of the affinity-purified protein, we have been able to identify a 29 KDa galactose terminal protein from the avirulent clones.
Bhaumik
S K
SK
Infectious Disease and Immunology Division, Indian Institute of Chemical Biology, Kolkata, India.
Singh
M
M
Basu
R
R
Bhaumik
S
S
Roychoudhury
K
K
Naskar
K
K
Roy
S
S
De
T
T
D000371:Agglutination / Q000187:drug effects; D000818:Animals; D002846:Chromatography, Affinity; D002999:Clone Cells; D005696:Galactosidases / Q000494:pharmacology; D005700:Galactosyltransferases / Q000378:metabolism*; D042541:Intracellular Space / Q000187:drug effects / Q000201:enzymology; D007893:Leishmania donovani / Q000201:enzymology* / Q000302:isolation & purification / Q000472:pathogenicity*; D007898:Leishmaniasis, Visceral / Q000469:parasitology; D008099:Liver / Q000469:parasitology; D008264:Macrophages / Q000187:drug effects / Q000469:parasitology; D051379:Mice; D008807:Mice, Inbred BALB C; D009573:Nitrites / Q000378:metabolism; D010271:Parasites / Q000187:drug effects / Q000201:enzymology / Q000302:isolation & purification / Q000472:pathogenicity; D019887:Peanut Agglutinin / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D015800:Protozoan Proteins / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D014531:Uridine Diphosphate Galactose / Q000378:metabolism*; D014774:Virulence / Q000187:drug effects
D009573:Nitrites; D019887:Peanut Agglutinin; D015800:Protozoan Proteins; D013481:Superoxides; D014531:Uridine Diphosphate Galactose; D005700:Galactosyltransferases; D005696:Galactosidases