Genetic complementation to identify DNA elements that influence complement resistance in Leishmania chagasi.
Past studies showed that Leishmania spp. promastigotes exhibit differential sensitivity to complement mediated lysis (CML) during development in vitro and in vivo. Leishmania chagasi promastigotes in cultures during logarithmic and stationary growth phases are CML-sensitive or CML-resistant when exposed to human serum, respectively, but only in cultures recently initiated with parasites from infected animals; serially passaged cultures become constitutively CML-sensitive regardless of growth phase. Building on these observations, a genetic screen was conducted to identify novel complement resistance factors of L. chagasi. A cosmid library containing genomic DNA was transfected into a promastigote line previously subjected to >50 serial passages. Selection with human serum for CML resistance yielded 12 transfectant clones. Cosmids isolated from 7 of these clones conferred CML resistance when transfected into an independent, high-passage promastigote culture; at 12% human serum, the mean survival of transfectants was 37% (+/- 11.6%), and that of control transfectants was about 1%. Inserts within the 7 cosmids were unique. Determination of the complete DNA sequence for 1 cosmid indicated that its 32-kilobase insert was 89% identical (overall) to a 31-kilobase region of Leishmania major chromosome 36, which is predicted to encode 6 genes, all of which encode hypothetical proteins.
Dahlin-Laborde
R R
RR
Department of Entomology, Iowa State University, Ames 50011-1250, USA.
Yu
T P
TP
Beetham
J K
JK
D000818:Animals; D003165:Complement System Proteins / Q000276:immunology*; D003360:Cosmids / Q000737:chemistry / Q000235:genetics; D006224:Cricetinae; D016054:DNA, Protozoan / Q000032:analysis*; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D006801:Humans; D018314:Leishmania infantum / Q000235:genetics* / Q000276:immunology*; D008647:Mesocricetus; D015183:Restriction Mapping; D012692:Serial Passage; D014162:Transfection
D016054:DNA, Protozoan; D003165:Complement System Proteins