Hemoglobin degradation in intraerythrocytic malaria parasites is a vast process that occurs in an acidic digestive vacuole. Proteases that participate in this catabolic pathway have been defined. Studies of protease biosynthesis have revealed unusual targeting and activation mechanisms. Oxygen radicals and heme are released during proteolysis and must be detoxified by dismutation and polymerization, respectively. The quinoline antimalarials appear to act by preventing sequestration of this toxic heme. Understanding the disposition of hemoglobin has allowed identification of essential processes and metabolic weakpoints that can be exploited to combat this scourge of mankind.

Hemoglobin metabolism in the malaria parasite Plasmodium falciparum.

http://tmp.wustl.edu/archives/TMPFall2006/10-4-06SSPonpuak-1.pdf

Identification and characterization of falcilysin, a metallopeptidase involved in hemoglobin catabolism within the malaria parasite Plasmodium falciparum.

Proteases from a variety of protozoan parasites have been characterized at the molecular and cellular levels, and the many roles that proteases play in these organisms are coming into focus. Central roles have been proposed for proteases in diverse processes such as host cell invasion and egress, encystation, excystation, catabolism of host proteins, differentiation, cell cycle progression, cytoadherence, and both stimulation and evasion of host immune responses. Detailed structural and functional characterization of parasite proteases has led to novel insights into the workings of these fascinating catalytic machines. The possibility of developing selective inhibitors of key proteases of pathogenic parasites into novel chemotherapeutic strategies is being vigorously explored.

Biological roles of proteases in parasitic protozoa.

The search for novel antimalarial drug targets is urgent due to the growing resistance of Plasmodium falciparum parasites to available drugs. Proteases are attractive antimalarial targets because of their indispensable roles in parasite infection and development, especially in the processes of host erythrocyte rupture/invasion and hemoglobin degradation. However, to date, only a small number of proteases have been identified and characterized in Plasmodium species. Using an extensive sequence similarity search, we have identified 92 putative proteases in the P. falciparum genome. A set of putative proteases including calpain, metacaspase, and signal peptidase I have been implicated to be central mediators for essential parasitic activity and distantly related to the vertebrate host. Moreover, of the 92, at least 88 have been demonstrated to code for gene products at the transcriptional levels, based upon the microarray and RT-PCR results, and the publicly available microarray and proteomics data. The present study represents an initial effort to identify a set of expressed, active, and essential proteases as targets for inhibitor-based drug design.

/pdf/data-mining-approaches-reveal-hidden-families-of-proteases-4sncjr1lck.pdf

Data-Mining Approaches Reveal Hidden Families of Proteases in the Genome of Malaria Parasite

Recent developments in the design and synthesis of hybrid molecules based on aminoquinoline ring and their antiplasmodial evaluation.

. Using fluorogenic substrates and polyacrylamide gels we detected in cell-free extracts of Plasmodium falciparum, Plasmodium chabaudi chabaudi and Plasmodium berghei only a single aminopeptidase. A comparative study of the aminopeptidase activity in each extract revealed that the enzymes have similar specificities and kinetics, a near-neutral pH optima of 7.2 and are moderately thermophilic. Each has an apparent molecular weight of 80,000 ± 10,000, determined by high performance liquid chromatography on a calibrated SW500 column. Whilst the P. c. chabaudi and P. berghei activity co-migrate in native polyacrylamide gels, that of P. falciparum migrates more slowly. The three enzymes can be selectively inhibited by ortho-phenanthroline and are thus metallo-aminopeptidases; however, in contrast to other aminopeptidases the metal co-factor does not appear to be Zn2+.

Aminopeptidases from Plasmodium falciparum, Plasmodium chabaudi chabaudi and Plasmodium berghei

Abstract Antibiotic treatments have detrimental effects on the microbiome and lead to antibiotic resistance. To develop a phage therapy against a diverse range of clinically relevant Escherichia coli , we screened a library of 162 wild-type (WT) phages, identifying eight phages with broad coverage of E. coli , complementary binding to bacterial surface receptors, and the capability to stably carry inserted cargo. Selected phages were engineered with tail fibers and CRISPR–Cas machinery to specifically target E. coli . We show that engineered phages target bacteria in biofilms, reduce the emergence of phage-tolerant E. coli and out-compete their ancestral WT phages in coculture experiments. A combination of the four most complementary bacteriophages, called SNIPR001, is well tolerated in both mouse models and minipigs and reduces E. coli load in the mouse gut better than its constituent components separately. SNIPR001 is in clinical development to selectively kill E. coli , which may cause fatal infections in hematological cancer patients. 

/pdf/engineered-phage-with-antibacterial-crispr-cas-selectively-2jokvltd.pdf

Alice Troy

Papers

Aminopeptidases from Plasmodium falciparum, Plasmodium chabaudi chabaudi and Plasmodium berghei

Engineered phage with antibacterial CRISPR–Cas selectively reduce E. coli burden in mice