Replication of Plasmodium in reticulocytes can occur without hemozoin formation, resulting in chloroquine resistance

Most studies on malaria-parasite digestion of hemoglobin (Hb) have been performed using P. falciparum maintained in mature erythrocytes, in vitro. In this study, we examine Plasmodium Hb degradation in vivo in mice, using the parasite P. berghei, and show that it is possible to create mutant parasites lacking enzymes involved in the initial steps of Hb proteolysis. These mutants only complete development in reticulocytes and mature into both schizonts and gametocytes. Hb degradation is severely impaired and large amounts of undigested Hb remains in the reticulocyte cytoplasm and in vesicles in the parasite. The mutants produce little or no hemozoin (Hz), the detoxification by-product of Hb degradation. Further, they are resistant to chloroquine, an antimalarial drug that interferes with Hz formation, but their sensitivity to artesunate, also thought to be dependent on Hb degradation, is retained. Survival in reticulocytes with reduced or absent Hb digestion may imply a novel mechanism of drug resistance. These findings have implications for drug development against human-malaria parasites, such as P. vivax and P. ovale, which develop inside reticulocytes

Cloning and analysis of the pepV dipeptidase gene of Lactococcus lactis MG1363

The gene pepV, encoding a dipeptidase from Lactococcus lactis subsp. cremoris MG1363, was identified in a genomic library in pUC19 in a peptidase-deficient Escherichia coli strain and subsequently sequenced. PepV of L. lactis is enzymatically active in E. coli and hydrolyzes a broad range of dipeptides but no tri-, tetra-, or larger oligopeptides. Northern (RNA) and primer extension analyses indicate that pepV is a monocistronic transcriptional unit starting 24 bases upstream of the AUG translational start codon. The dipeptidase oft. lactis was shown to be similar to the dipeptidase encoded by pepV of L. delbrueckii subsp. lactis, with 46% identity in the deduced amino acid sequences. A PepV-negative mutant of L. lactis was constructed by single-crossover recombination. Growth of the mutant strain in milk was significantly slower than that of the wild type, but the strains ultimately reached the same final cell densities