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 \u3e50 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

Characterization of DNA Sequences that Confer Complement Resistance in \u3ci\u3eLeishmania chagasi\u3c/i\u3e

Serial passage of axenically cultured Leishmania chagasi promastigotes results in a progressive diminution in resistance to complement-mediated lysis (CML), whereas high CML resistance is seen in infectious metacyclic promastigotes from the sandfly vector as well as metacyclic-like promastigotes within low-passage cultures at stationary growth phase. As we previously reported, in a screen seeking to identify novel genes involved in CML resistance: (1) a genomic cosmid library derived from DNA of CML-resistant L. chagasi promastigotes was transfected into highpassage (constitutively CML-sensitive) L. chagasi promastigotes; (2) transformants were screened for acquisition of CML-resistance; (3) multiple cosmid-transfectants exhibited partial CML resistance; and (4) the sequence for one of the cosmids (Cosmid 51) was determined. This report extends the analysis of Cosmid 51, and identifies by deletion analysis a subregion of the cosmid insert that is critical to the CML-resistance phenotype of Cosmid 51 transformants. We also report the sequence determination and initial CML-resistance activity of another cosmid that also confers partial resistance to CML

Characterization of DNA Sequences that Confer Complement Resistance in \u3ci\u3eLeishmania chagasi\u3c/i\u3e

Serial passage of axenically cultured Leishmania chagasi promastigotes results in a progressive diminution in resistance to complement-mediated lysis (CML), whereas high CML resistance is seen in infectious metacyclic promastigotes from the sandfly vector as well as metacyclic-like promastigotes within low-passage cultures at stationary growth phase. As we previously reported, in a screen seeking to identify novel genes involved in CML resistance: (1) a genomic cosmid library derived from DNA of CML-resistant L. chagasi promastigotes was transfected into highpassage (constitutively CML-sensitive) L. chagasi promastigotes; (2) transformants were screened for acquisition of CML-resistance; (3) multiple cosmid-transfectants exhibited partial CML resistance; and (4) the sequence for one of the cosmids (Cosmid 51) was determined. This report extends the analysis of Cosmid 51, and identifies by deletion analysis a subregion of the cosmid insert that is critical to the CML-resistance phenotype of Cosmid 51 transformants. We also report the sequence determination and initial CML-resistance activity of another cosmid that also confers partial resistance to CML

Reduced Hamster Usage and Stress in Propagating \u3ci\u3eLeishmania chagasi\u3c/i\u3e Promastigotes Using Cryopreservation and Saphenous Vein Inoculation

Leishmania chagasi, a causal agent of visceral leishmaniasis, requires passage through lab animals such as hamsters to maintain its virulence. Hamster infection is typically accomplished via cardiac puncture or intraperitoneal injection, procedures accompanied by risks of increased animal stress and death. The use of the hamster model also necessitates a regular supply of infected animals, because L. chagasi parasites newly isolated from an infected hamster can be grown in culture for only several weeks before loss of function/phenotype occurs. In an effort to decrease animal usage and animal stress, experiments were performed to assess a more gentle inoculation procedure (saphenous vein inoculation) and the use of cryopreserved parasite cells for research experiments. Of 81 hamsters inoculated by the saphenous vein, 80 became infected as determined ante mortem, by display of clinical symptoms of leishmaniasis (onset of symptoms at 105 ± 22 days post-inoculation), and postmortem by the presence of parasites within the spleen. Splenic parasite load calculated for a subset (n 5 34) of infected hamsters was 124 to 26,177 Leishmania donovani infection units. Cryopreserved, and never-stored, cells were equivalent in all properties evaluated, including developmental changes in morphology during culture, culture growth rates, parasite resistance to serum-mediated lysis, and expression of developmentally regulated surface proteins major surface protease and promastigote surface antigen

Characterization of DNA Sequences that Confer Complement Resistance in \u3ci\u3eLeishmania chagasi\u3c/i\u3e

Serial passage of axenically cultured Leishmania chagasi promastigotes results in a progressive diminution in resistance to complement-mediated lysis (CML), whereas high CML resistance is seen in infectious metacyclic promastigotes from the sandfly vector as well as metacyclic-like promastigotes within low-passage cultures at stationary growth phase. As we previously reported, in a screen seeking to identify novel genes involved in CML resistance: (1) a genomic cosmid library derived from DNA of CML-resistant L. chagasi promastigotes was transfected into highpassage (constitutively CML-sensitive) L. chagasi promastigotes; (2) transformants were screened for acquisition of CML-resistance; (3) multiple cosmid-transfectants exhibited partial CML resistance; and (4) the sequence for one of the cosmids (Cosmid 51) was determined. This report extends the analysis of Cosmid 51, and identifies by deletion analysis a subregion of the cosmid insert that is critical to the CML-resistance phenotype of Cosmid 51 transformants. We also report the sequence determination and initial CML-resistance activity of another cosmid that also confers partial resistance to CML