Development of a genetic assay to distinguish between Leishmania viannia species on the basis of isoenzyme differences.

BACKGROUND: Tegumentary leishmaniasis in Latin America is caused mainly by Leishmania viannia braziliensis complex parasites. L. braziliensis and Leishmania viannia peruviana are the 2 predominant Leishmania species in Peru. L. braziliensis is more virulent, because it can cause mucocutaneous leishmaniasis, known as espundia, that results in severe facial destruction. Early identification of the species that causes the initial cutaneous infection would greatly help to prevent mucocutaneous leishmaniasis, because it would allow more aggressive treatment and follow-up. However, because of the close genetic similarity of L. braziliensis and L. peruviana, there currently exists no simple assay to distinguish between these species. METHODS: We cloned the mannose phosphate isomerase gene from both L. braziliensis and L. peruviana. It is the only known isoenzyme capable of differentiating between L. braziliensis and L. peruviana in multilocus enzyme electrophoresis. Interestingly, only a single nucleotide polymorphism was found between the mannose phosphate isomerase genes from L. braziliensis and L. peruviana, resulting in an amino acid change from threonine to arginine at amino acid 361. A polymerase chain reaction assay was developed to distinguish the single nucleotide polymorphism of the mannose phosphate isomerase gene to allow for the specific identification of L. braziliensis or L. peruviana. RESULTS: This assay was validated with 31 reference strains that were previously typed by multilocus enzyme electrophoresis, successfully applied to patient biopsy samples, and adapted to a real-time polymerase chain reaction assay. CONCLUSIONS: This innovative approach combines new genetic knowledge with traditional biochemical fundamentals of multilocus enzyme electrophoresis to better manage leishmaniasis in Latin America.

Inhibition of type I and type II phosphomannose isomerases by the reaction intermediate analogue 5-phospho-D-arabinonohydroxamic acid supports a catalytic role for the metal cofactor.

The phosphomannose isomerases (PMI) comprise three families of proteins: type I, type II, and type III PMIs. Members of all three families catalyze the reversible isomerization of D-mannose 6-phosphate (M6P) and D-fructose 6-phosphate (F6P) but share little or no sequence identity. Because (1) PMIs are essential for the survival of several microorganisms, including yeasts and bacteria, and (2) the PMI enzymes from several pathogens do not share significant sequence identity to the human protein, PMIs have been considered as potential therapeutic targets. Elucidation of the catalytic and regulatory mechanisms of the different types of PMIs is strongly needed for rational species-specific drug design. To date, inhibition and crystallographic studies of all PMIs are still largely unexplored. As part of our research program on aldose-ketose isomerases, we report in this paper the evaluation of two new inhibitors of type I and type II PMIs from baker's yeast and Pseudomonas aeruginosa, respectively. We found that 5-phospho-D-arabinonohydroxamic acid (5PAH), which is the most potent inhibitor of phosphoglucose isomerase (PGI), is by far the best inhibitor ever reported of both type I and type II PMI-catalyzed isomerization of M6P to F6P. 5PAH, which has an inhibition constant at least 3 orders of magnitude smaller than that of previously reported PMI inhibitors, may be the first high-energy intermediate analogue inhibitor of the enzymes. We also tested the related molecule 5-phospho-D-arabinonate (5PAA), which is a strong competitive inhibitor of PGI, and found that it does not inhibit either PMI. All together, our results are consistent with a catalytic role for the metal cofactor in PMI activity.

Sequence relationships in the legume lectin fold and other jelly rolls.

Distant sequence relationships in proteins containing the beta jelly-roll fold were investigated using sensitive sequence comparison methods, including PSI-BLAST and Hidden Markov Models. A relationship was identified between the rmlC-like and phosphomannose isomerase SCOP (version 1.53) superfamilies, which were merged in the most recent SCOP release. No other distant sequence relationships linking jelly roll superfamilies were found.

Aedes aegypti in Tahiti and Moorea (French Polynesia): isoenzyme differentiation in the mosquito population according to human population density.

Genetic differences at five polymorphic isoenzyme loci were analyzed by starch gel electrophoresis for 28 Aedes aegypti samples. Considerable (i.e., high Fst values) and significant (i.e., P values >10(-4)) geographic differences were found. Differences in Ae. aegypti genetic structure were related to human population densities and to particularities in mosquito ecotopes in both Tahiti and Moorea islands. In highly urbanized areas (i.e., the Papeete agglomeration), mosquitoes were highly structured. Recurrent extinction events consecutive to insecticidal treatments during dengue outbreaks tend to differentiate mosquito populations. In less populated zones (i.e., the east coast of Moorea and Tahiti), differences in ecotope characteristics could explain the lack of differentiation among mosquitoes from rural environments such as the east coast of Tahiti where natural breeding sites predominate. When the lowest populated zones such as Tahiti Iti and the west coast of Moorea are compared, mosquito are less differentiated in Moorea. These results will be discussed in relation to the recent findings of variation in mosquito infection rates for dengue-2 virus.