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Leishmania major LACK antigen is required for efficient vertebrate parasitization.

Kelly BL, Stetson DB, Locksley RM - J. Exp. Med. (2003)

Bottom Line: We describe the genomic organization of the four lack genes in the L. major diploid genome together with results of selected lack gene targeting.Complementation with an additional lack copy was sufficient to induce robust lesion development, which also occurred using parasites with two lack genes.Conversely, attempts to generate lack- parasites failed, suggesting that LACK is required for parasite viability.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Medicine, University of California San Francisco, San Francisco, CA 94143, USA.

ABSTRACT
The Leishmania major LACK antigen is a key target of the immune response in susceptible BALB/c mice and remains a viable vaccine candidate for human leishmaniasis. We describe the genomic organization of the four lack genes in the L. major diploid genome together with results of selected lack gene targeting. Parasites containing a single lack gene in either the upstream or downstream locus grew comparably to wild-type promastigotes in vitro, but failed to parasitize BALB/c mice efficiently, even in a T cell-deficient environment. The replication of single copy lack mutants as amastigotes was attenuated in macrophages in vitro, and parasites failed to increase in numbers in immunodeficient mice, despite their persistence over months. Complementation with an additional lack copy was sufficient to induce robust lesion development, which also occurred using parasites with two lack genes. Conversely, attempts to generate lack- parasites failed, suggesting that LACK is required for parasite viability. These data suggest that LACK is critical for effective mammalian parasitization and thus represents a potential drug target for leishmaniasis.

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Related in: MedlinePlus

Infection of mouse macrophages with lack-targeted L. major in vitro. BALB/c bone marrow–derived macrophages were incubated with indicated lack-targeted L. major promastigotes using three parasites per macrophage. After 16 h at 35°C, the monolayers were washed to remove extracellular parasites and stained immediately (A), or washed and then incubated for an additional 80 h at 35°C and stained (B). Monolayers were scored for frequency (percent) of macrophages infected with either five or more amastigotes (5+), one to four amastigotes (1–4), or uninfected (0). In C, mean numbers of intracellular amastigotes per 100 macrophages are indicated at 16 and 96 h after infection using the indicated organisms. For each infection, ∼200 macrophages were analyzed. Data are averages from two experiments.
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fig6: Infection of mouse macrophages with lack-targeted L. major in vitro. BALB/c bone marrow–derived macrophages were incubated with indicated lack-targeted L. major promastigotes using three parasites per macrophage. After 16 h at 35°C, the monolayers were washed to remove extracellular parasites and stained immediately (A), or washed and then incubated for an additional 80 h at 35°C and stained (B). Monolayers were scored for frequency (percent) of macrophages infected with either five or more amastigotes (5+), one to four amastigotes (1–4), or uninfected (0). In C, mean numbers of intracellular amastigotes per 100 macrophages are indicated at 16 and 96 h after infection using the indicated organisms. For each infection, ∼200 macrophages were analyzed. Data are averages from two experiments.

Mentions: The finding that single copy lack parasites displayed impaired virulence suggested that LACK might be required for efficient growth in macrophages, the major target cell for parasitization. To address this issue, bone marrow–derived macrophages were prepared and incubated with the various mutant parasites for 16 h. After washing, the numbers of infected macrophages were similar in comparing the various mutant strains, suggesting that the lack mutations do not affect initial entry into macrophages (Fig. 6 A). After 96 h, however, the percentage of infected macrophages, particularly of macrophages containing large numbers of organisms, was reduced using single copy lack parasites, and this was partially overcome using the reconstituted lack++Δ/– parasites (Fig. 6 B). Microscopically counting the numbers of amastigotes present in macrophages after 96 h of in vitro culture confirmed that single copy mutant parasites essentially did not replicate over a period when lack++/−− organisms had increased almost fourfold (Fig. 6 C).


Leishmania major LACK antigen is required for efficient vertebrate parasitization.

Kelly BL, Stetson DB, Locksley RM - J. Exp. Med. (2003)

Infection of mouse macrophages with lack-targeted L. major in vitro. BALB/c bone marrow–derived macrophages were incubated with indicated lack-targeted L. major promastigotes using three parasites per macrophage. After 16 h at 35°C, the monolayers were washed to remove extracellular parasites and stained immediately (A), or washed and then incubated for an additional 80 h at 35°C and stained (B). Monolayers were scored for frequency (percent) of macrophages infected with either five or more amastigotes (5+), one to four amastigotes (1–4), or uninfected (0). In C, mean numbers of intracellular amastigotes per 100 macrophages are indicated at 16 and 96 h after infection using the indicated organisms. For each infection, ∼200 macrophages were analyzed. Data are averages from two experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2194132&req=5

fig6: Infection of mouse macrophages with lack-targeted L. major in vitro. BALB/c bone marrow–derived macrophages were incubated with indicated lack-targeted L. major promastigotes using three parasites per macrophage. After 16 h at 35°C, the monolayers were washed to remove extracellular parasites and stained immediately (A), or washed and then incubated for an additional 80 h at 35°C and stained (B). Monolayers were scored for frequency (percent) of macrophages infected with either five or more amastigotes (5+), one to four amastigotes (1–4), or uninfected (0). In C, mean numbers of intracellular amastigotes per 100 macrophages are indicated at 16 and 96 h after infection using the indicated organisms. For each infection, ∼200 macrophages were analyzed. Data are averages from two experiments.
Mentions: The finding that single copy lack parasites displayed impaired virulence suggested that LACK might be required for efficient growth in macrophages, the major target cell for parasitization. To address this issue, bone marrow–derived macrophages were prepared and incubated with the various mutant parasites for 16 h. After washing, the numbers of infected macrophages were similar in comparing the various mutant strains, suggesting that the lack mutations do not affect initial entry into macrophages (Fig. 6 A). After 96 h, however, the percentage of infected macrophages, particularly of macrophages containing large numbers of organisms, was reduced using single copy lack parasites, and this was partially overcome using the reconstituted lack++Δ/– parasites (Fig. 6 B). Microscopically counting the numbers of amastigotes present in macrophages after 96 h of in vitro culture confirmed that single copy mutant parasites essentially did not replicate over a period when lack++/−− organisms had increased almost fourfold (Fig. 6 C).

Bottom Line: We describe the genomic organization of the four lack genes in the L. major diploid genome together with results of selected lack gene targeting.Complementation with an additional lack copy was sufficient to induce robust lesion development, which also occurred using parasites with two lack genes.Conversely, attempts to generate lack- parasites failed, suggesting that LACK is required for parasite viability.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Medicine, University of California San Francisco, San Francisco, CA 94143, USA.

ABSTRACT
The Leishmania major LACK antigen is a key target of the immune response in susceptible BALB/c mice and remains a viable vaccine candidate for human leishmaniasis. We describe the genomic organization of the four lack genes in the L. major diploid genome together with results of selected lack gene targeting. Parasites containing a single lack gene in either the upstream or downstream locus grew comparably to wild-type promastigotes in vitro, but failed to parasitize BALB/c mice efficiently, even in a T cell-deficient environment. The replication of single copy lack mutants as amastigotes was attenuated in macrophages in vitro, and parasites failed to increase in numbers in immunodeficient mice, despite their persistence over months. Complementation with an additional lack copy was sufficient to induce robust lesion development, which also occurred using parasites with two lack genes. Conversely, attempts to generate lack- parasites failed, suggesting that LACK is required for parasite viability. These data suggest that LACK is critical for effective mammalian parasitization and thus represents a potential drug target for leishmaniasis.

Show MeSH
Related in: MedlinePlus