Virulence attenuation of a UDP-galactose/N-acetylglucosamine β1,4 galactosyltransferase expressing Leishmania donovani promastigote

Abstract

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.

UDP-Gal: N-acetylglucosamine β 1–4 galactosyltransferase expressing live attenuated parasites as vaccine for visceral leishmaniasis

“…L. donovani strain AG83 (MHOM/IN/83/AG83) was maintained in golden hamsters as previously described [16]. Promastigotes obtained after transforming amastigotes from infected spleen were maintained in M199 (Invitrogen Life Technologies) as previously described [16]. For infection, 4-to 6-week-old mice were inoculated with 2×10 7 , second passage promastigotes in 0.2 mL saline through the intra-cardiac route (i.c.).…”

“…Cloning of parasites by limiting dilution Parasites from a mixed population of seventh passage were cloned by the limiting dilution method essentially as described previously [16]. Avirulent clones were selected on the basis of PNA agglutination and GalT4 expression [16].…”

“…A-LD parasites were biosynthetically labeled with 14 [C] galactose and virulent LD clones (V-LD) with 3 [H] mannose as previously mentioned [16]. Single cell suspension of splenocytes from BALB/c mice were prepared after Ficoll density gradient centrifugation and then suspended in complete RPMI 1640.…”

“…Measurement of reactive oxygen species (RO) and nitric oxide (NO) ROS and NO was measured essentially as described earlier [16]. Adherent macrophages (M) were incubated in presence or absence of inhibitors as mentioned above.…”

“…However, in the absence of a clear genetic profile of any avirulent organisms available, their use for human vaccination would be unacceptable, because of the risk of reversion to a virulent phenotype [15]. Our earlier work has established that the loss of virulence of LD promastigotes on longitudinal propagation was associated with the expression of a developmentally regulated galactosyltransferase (GalT4) and the stage specific expression of galactose terminal glycoconjugates in the attenuated parasite clones [16]. These attenuated clonal parasites are genetically defined and did not show any reversion to virulent phenotype when kept in culture for 30 passages.…”