Characterization of a compensatory mutant of Leishmania major that lacks ether lipids but exhibits normal growth, and G418 and hygromycin resistance.
Ether glycerolipid biosynthesis in Leishmania major initiates with the acylation of dihydroxyacetonephosphate by the glycosomal dihydroxyacetonephosphate acyltransferase LmDAT. We previously reported that a null mutant of LmDAT is severely affected in logarithmic growth, survival during stationary phase, and in virulence in mice. In addition, it lacks all ether glycerolipids, produces altered forms of the ether-lipid based virulence factors lipophosphoglycan and increased levels of GPI-anchored protein gp63. Here, we describe the characterization of a compensatory mutant of a null strain of LmDAT, Δlmdat/Δlmdat(rev). Similarly to the null mutant, the Δlmdat/Δlmdat(rev) strain formed altered forms of lipophosphoglycan and increased levels of gp63, and was avirulent in mice infection. Further, dihydroxyacetonephosphate acyltransferase activity was absent in the revertant clone, indicating that a mutation in another acyltransferase gene did not confer dihydroxyacetonephosphate specificity. In contrast, the revertant grew normally but still exhibited poor survival during stationary phase. In addition, agarose gel analysis of its genomic DNA failed to detect any amplified DNA. Surprisingly, its sensitivity to aminoglycoside based antibiotics G418 and hygromycin was lower than that of the null mutant, wild type and complemented line.
Department of Biological Sciences, St. John's University, 8000 Utopia Parkway, Jamaica, NY 11439, USA. zufferer@stjohns.edu
Rachel
R
Zufferey
Stergios S
SS
Bibis
Tongtong
T
Zhu
Subbhalakshmi
S
Dhalladoo
D000217:Acyltransferases / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology*; D002934:Cinnamates / Q000494:pharmacology*; D004351:Drug Resistance / Q000235:genetics; D005784:Gene Amplification; D005839:Gentamicins / Q000494:pharmacology*; D006028:Glycosphingolipids / Q000378:metabolism; D006921:Hygromycin B / Q000031:analogs & derivatives* / Q000494:pharmacology; D018320:Leishmania major / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008666:Metalloendopeptidases / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D009154:Mutation; D010742:Phospholipid Ethers / Q000378:metabolism*; D011509:Proteoglycans / Q000378:metabolism; D015800:Protozoan Proteins / Q000378:metabolism; D014774:Virulence / Q000235:genetics
D000981:Antiprotozoal Agents; D002934:Cinnamates; D005839:Gentamicins; D006028:Glycosphingolipids; D008565:Membrane Proteins; D010742:Phospholipid Ethers; C400527:Ppg1 protein, Leishmania; D011509:Proteoglycans; D015800:Protozoan Proteins; C008290:lipophosphonoglycan; D006921:Hygromycin B; C026273:hygromycin A; C010680:antibiotic G 418; D000217:Acyltransferases; C020051:glycerone-phosphate O-acyltransferase; D008666:Metalloendopeptidases; C055146:glycoprotein gp63, Leishmania