Comparisons of Mutants Lacking the Golgi UDP-Galactose or GDP-Mannose Transporters Establish that Phosphoglycans Are Important for Promastigote but Not Amastigote Virulence in Leishmania major

Abstract

Abundant surface Leishmania phosphoglycans (PGs) containing [Gal(␤1,4)Man(␣1-PO 4 )]-derived repeating units are important at several points in the infectious cycle of this protozoan parasite. PG synthesis requires transport of activated nucleotide-sugar precursors from the cytoplasm to the Golgi apparatus. Correspondingly, null mutants of the L. major GDP-mannose transporter LPG2 lack PGs and are severely compromised in macrophage survival and induction of acute pathology in susceptible mice, yet they are able to persist indefinitely and induce protective immunity. However, lpg2 ؊ L. mexicana amastigotes similarly lacking PGs but otherwise normal in known glycoconjugates remain able to induce acute pathology. To explore this further, we tested the infectivity of a new PG-null L. major mutant, which is inactivated in the two UDP-galactose transporter genes LPG5A and LPG5B. Surprisingly this mutant did not recapitulate the phenotype of L. major lpg2 ؊ , instead resembling the L. major lipophosphoglycan-deficient lpg1 ؊ mutant. Metacyclic lpg5A ؊ /lpg5B ؊ promastigotes showed strong defects in the initial steps of macrophage infection and survival. However, after a modest delay, the lpg5A ؊ /lpg5B ؊ mutant induced lesion pathology in infected mice, which thereafter progressed normally. Amastigotes recovered from these lesions were fully infective in mice and in macrophages despite the continued absence of PGs. This suggests that another LPG2-dependent metabolite is responsible for the L. major amastigote virulence defect, although further studies ruled out cytoplasmic mannans. These data thus resolve the distinct phenotypes seen among lpg2 ؊ Leishmania species by emphasizing the role of glycoconjugates other than PGs in amastigote virulence, while providing further support for the role of PGs in metacyclic promastigote virulence.

The Role of the Mitochondrial Glycine Cleavage Complex in the Metabolism and Virulence of the Protozoan Parasite Leishmania major

“…4, A and B). In previous studies of other mutant L. major, we showed that the delayed lesion phenotype similar to that seen with gcvP Ϫ metacyclic promastigotes infections arose from stage-specific effects (27,(45)(46)(47). In such mutants, the ability of the metacyclic promastigote to establish infections in macrophages and/or mice was compromised but not its ability to replicate and induce pathology as the amastigote stage.…”

“…WT and gcvP Ϫ /ϩGCVP parasites persisted at similar levels (3,300 Ϯ 7,100 WT parasites per lesion, n ϭ 5; 2,200 Ϯ 2,700 gcvP Ϫ /ϩGCVP parasites per lesion, n ϭ 8), whereas gcvP Ϫ were found at somewhat higher levels (9,300 Ϯ 14,000 parasites per lesion, n ϭ 9), although this difference was not statistically significant. gcvP Ϫ Amastigotes Show Attenuated Virulence-In previous studies of other mutant L. major, we showed that a "delayed lesion" phenotype similar to that seen with gcvP Ϫ metacyclic promastigote infections arose from a defect in the virulence of metacyclic promastigotes but not of amastigotes (27,(45)(46)(47). This model was tested here by purifying amastigotes from gcvP Ϫ -infected mice showing significant pathology and inoculating them directly back into mice as amastigotes (Fig.…”

Leishmania majorPhosphoglycans Influence the Host Early Immune Response by Modulating Dendritic Cell Functions

“…However, our recent studies of another PG-deficient L. major mutant, obtained through inactivation of the UDP-Gal transporters encoded by the LPG5A and LPG5B genes (lpg5A Ϫ lpg5B Ϫ ), question this assumption. Unexpectedly, the lpg5A Ϫ lpg5B Ϫ mutant resembled the lpg1 Ϫ rather than the lpg2 Ϫ parasites, by displaying a virulence defect when tested as promastigotes but showing normal virulence as amastigotes (7,8). This suggests that a loss of LPG2-dependent glycoconjugates, other than PGs, may underlie the virulence defect of lpg2 Ϫ L. major.…”

“…Ϫ , lpg2 AB, and lpg5A Ϫ lpg5B Ϫ mutants were derivatives of the WT L. major strain LV39 clone 5 (Rho/SU/59/P) and were made by homologous gene targeting as described previously (7,8,43,45). Parasites were cultured at 26°C in M199 medium (HyClone, Logan, UT) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Cansera, Mississauga, Ontario, Canada), 2 mM L-glutamine, 100 U/ml penicillin, and 100 g/ml streptomycin (Invitrogen Life Technologies, Burlington, Ontario, Canada).…”

“…However, those parasites that go on to survive can form amastigotes that are still virulent in vivo (43), consistent with the observation that amastigotes do not express LPG. In contrast, lpg2 Ϫ parasites, generated by deleting the LPG2 gene encoding the transporter required for GDP mannose uptake into the Golgi lumen, lack both LPG and other PG-containing molecules (27) and other potential LPG2-dependent metabolites (8). These parasites are highly attenuated in vivo, showing deficiencies in the early steps of promastigote survival, and fail to induce any overt pathology in infected mice (45).…”