The biosynthesis of the major acyl carrier Coenzyme A from pantothenic acid (PA) is critical for survival of Plasmodium falciparum within human erythrocytes. Accordingly, a PA analog α-PanAm showed potent activity against blood stage parasites in vitro; however, its efficacy in vivo and its mode of action remain unknown. We developed a new synthesis route for α-PanAm and showed that the compound is highly effective against blood stages of drug-sensitive and -resistant P. falciparum strains, inhibits development of P. berghei in hepatocytes, and at doses up to 100 mg/kg also inhibits blood stage development of P. chabaudi in mice. We used yeast and its pantothenate kinase Cab1 as models to characterize mode of action of α-PanAm and found that α-PanAm inhibits yeast growth in a PA-dependent manner, and its potency increases dramatically in a yeast mutant with defective pantothenate kinase activity. Biochemical analyses using 14C-PA as a substrate demonstrated that α-PanAm is a competitive inhibitor of Cab1. Interestingly, biochemical and mass spectrometry analyses also showed that the compound is phosphorylated by Cab1. Together, these data suggest that α-PanAm exerts its antimicrobial activity by direct competition with the natural substrate PA for phosphorylation by the pantothenate kinase.

Human babesiosis is an emerging zoonotic infectious disease caused by intraerythrocytic protozoan parasites of the genus Most cases of human babesiosis are caused by and often manifest in individuals over the age of 50 years or in patients with a compromised immune system. Patients who develop symptomatic infections usually experience months of asymptomatic infection after the acute infection has resolved. About one-fifth of-infected adults never develop symptoms. These asymptomatically infected individuals sometimes donate blood and thus can transmit through blood transfusion. Current assays for detection of active infections can be used to screen donor blood prior to transfusion, but they rely primarily on microscopy or PCR methods, which have sensitivity and technical limitations. Here we report the development of an antigen capture enzyme-linked immunosorbent assay (BmGPAC) based on a major secreted immunodominant antigen of (BmGPI12/BmSA1), and we provide evidence that this assay is superior for detection of active infections, compared to available microscopy methods and serological assays. The assay has been evaluated using supernatants of -infected erythrocytes cultured, sera from -infected laboratory mice, and sera from wild mice and human patients. Our data suggest that the BmGPAC assay is a reliable assay for detection of active infections and is superior to real-time PCR and antibody assays for diagnosis of acute infections, screening of the blood supply, and epidemiological surveys of humans and animal reservoir hosts.

Polyamines are positively-charged organic molecules that are important for cellular growth and division. Polyamines and their synthesizing enzymes are particularly abundant in rapidly proliferating eukaryotic cells such as parasitic protozoa and cancer cells. Polyamine biosynthesis inhibitors, such as Elfornithine, are now being considered for cancer prevention and have been used effectively against Trypanosoma brucei. Inhibitors of polyamine biosynthesis have caused growth arrest of Plasmodium falciparum blood stages in vitro, but in P. berghei only partial inhibition has been observed. While polyamine biosynthesis enzymes are characterized and conserved in Plasmodium spp., little is known on the biological roles of these enzymes inside malaria parasite hosts. The bifunctional polyamine biosynthesis enzyme S-adenosyl methionine decarboxylase/ornithine decarboxylase (AdoMetDC/ODC) was targeted for deletion in P. yoelii. Deletion of AdoMetDC/ODC significantly reduced blood stage parasitemia but Anopheles transmission was completely blocked. We showed that male gametocytogenesis and male gamete exflagellation were abolished and consequently no ookinetes or oocyst sporozoites could be generated from adometdc/odc(–) parasites. Supplementation of putrescine and spermidine did not rescue the defective phenotypes of male gametocytes and gametes of the knockout parasites. These results highlight the crucial role of polyamine homeostasis in the development and functions of Plasmodium erythrocytic stages in the blood and in the mosquito vector and validate polyamine biosynthesis pathway enzymes as drug targeting candidates for malaria parasite transmission blocking.

Membrane trafficking is essential to eukaryotic life and is controlled by a complex network of proteins that regulate movement of proteins and lipids between organelles. The GBF1/GEA family of Guanine nucleotide Exchange Factors (GEFs) regulates trafficking between the endoplasmic reticulum and Golgi by catalyzing the exchange of GDP for GTP on ADP Ribosylation Factors (Arfs). Activated Arfs recruit coat protein complex 1 (COP-I) to form vesicles that ferry cargo between these organelles. To further explore the function of the GBF1/GEA family, we have characterized a fission yeast mutant lacking one copy of the essential gene gea1 (gea1+/−), the Schizosaccharomyces pombe ortholog of GBF1. The haploinsufficient gea1+/− strain was shown to be sensitive to the GBF1 inhibitor brefeldin A (BFA) and was rescued from BFA sensitivity by gea1p overexpression. No overt defects in localization of arf1p or arf6p were observed in gea1+/− cells, but the fission yeast homolog of the COP-I cargo sac1 was mislocalized, consistent with impaired COP-I trafficking. Although Golgi morphology appeared normal, a slight increase in vacuolar size was observed in the gea1+/− mutant strain. Importantly, gea1+/− cells exhibited dramatic cytokinesis-related defects, including disorganized contractile rings, an increased septation index, and alterations in septum morphology. Septation defects appear to result from altered secretion of enzymes required for septum dynamics, as decreased secretion of eng1p, a β-glucanase required for septum breakdown, was observed in gea1+/− cells, and overexpression of eng1p suppressed the increased septation phenotype. These observations implicate gea1 in regulation of septum breakdown and establish S. pombe as a model system to explore GBF1/GEA function in cytokinesis.

tr (A.S.I.A.) † These authors contributed equally to this work and are presented in alphabetical order.Abstract: The need for a malaria vaccine is indisputable. A single vaccine for Plasmodium pre-erythrocytic stages targeting the major sporozoite antigen circumsporozoite protein (CSP) has had partial success. Additionally, CD8+ T cells targeting liver-stage (LS) antigens induced by live attenuated sporozoite vaccines were associated with protection in human challenge experiments. To further evaluate protection mediated by LS antigens, we focused on exported pre-erythrocytic proteins (exported protein 1 (EXP1), profilin (PFN), exported protein 2 (EXP2), inhibitor of cysteine proteases (ICP), transmembrane protein 21 (TMP21), and upregulated in infective sporozoites-3 (UIS3)) expressed in all Plasmodium species and designed optimized, synthetic DNA (synDNA) immunogens. SynDNA antigen cocktails were tested with and without the molecular adjuvant plasmid IL-33. Immunized animals developed robust T cell responses including induction of antigen-specific liver-localized CD8+ T cells, which were enhanced by the co-delivery of plasmid IL-33. In total, 100% of mice in adjuvanted groups and 71%-88% in non-adjuvanted groups were protected from blood-stage disease following Plasmodium yoelii sporozoite challenge. This study supports the potential of synDNA LS antigens as vaccine components for malaria parasite infection.