The goal of this project is to identify molecular determinants associated with virulence in L. braziliensis strains and clones. Previous studies have revealed an unexpected degree of karyotype heterogeneity in L. b. panamensis strains isolated from Colombian patients residing in the Pacific coast region, where at least two subspecies of L. braziliensis and multiple zymodemes are present. This heterogeneity and the finding of strains with distinct karyotypes in the recurrent lesions of the same patient prompt us to examine the possible molecular bases of this chromosomal variability. The stability or liability of karyotupe and schizodeme, and occurrence of sexual recombination will be investigated by infecting experimental invertebrate and vertebrate hosts with cloned, molecularly typed organisms, followed by reisolation and comparison of the chromosonal constitution and kDNA RFLP patterns. Pulsed field gradient gel and transverse alternating field electrophoresis have revealed extra chromosomal DNA in some L. b. panamensis strains. The nature and possible correlation of these plasmid- like bands with drug resistance (in vivo and in vitro) will be investigated in this project. Probes for these elements will be prepared in order to detect the corresponding sequences in strains with and without evidence for extrachromosomal DNA. This approach should reveal whether the extrachromosomal sequences are unique or shared, present, absent or integrated within chromosomes. Reactivity patterns with a panel of monoclonal antibodies that define epitopes expressed on members of the L. braziliensis complex have shown that the absence of subspecies-specific determinants for L. b. braziliensis is more frequently associated with mucosal strains than cutaneous strains of this subspecies. Two-dimensional protein profiles have provided for L. b. guyanensis. Fractionation procedures (Triton X 114, FPLC) and immunoblotting will be used in combination with 2-D electrophoresis to identify gene products associated with clinical and experimental virulence behaviors.
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