Metabolism of FAD, FMN and riboflavin (vitamin B2) in the human parasitic blood fluke Schistosoma mansoni.

BACKGROUND: Schistosomiasis is a parasitic disease caused by trematodes of the genus Schistosoma. The intravascular worms acquire the nutrients necessary for their survival from host blood. Since all animals are auxotrophic for riboflavin (vitamin B2), schistosomes too must import it to survive. Riboflavin is an essential component of the coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD); these support key functions of dozens of flavoenzymes. METHODS: Here, using a combination of metabolomics, enzyme kinetics and in silico molecular analysis, we focus on the biochemistry of riboflavin and its metabolites in Schistosoma mansoni (Sm). RESULTS: We show that when schistosomes are incubated in murine plasma, levels of FAD decrease over time while levels of FMN increase. We show that live schistosomes cleave exogenous FAD to generate FMN and this ability is significantly blocked when expression of the surface nucleotide pyrophosphatase/phosphodiesterase ectoenzyme SmNPP5 is suppressed using RNAi. Recombinant SmNPP5 cleaves FAD with a Km of 178 ± 5.9 µM and Kcat/Km of 324,734 ± 36,347 M- 1.S- 1. The FAD-dependent enzyme IL-4I1 drives the oxidative deamination of phenylalanine to produce phenylpyruvate and H2O2. Since schistosomes are damaged by H2O2, we determined if SmNPP5 could impede H2O2 production by blocking IL-4I1 action in vitro. We found that this was not the case; covalently bound FAD on IL-4I1 appears inaccessible to SmNPP5. We also report that live schistosomes can cleave exogenous FMN to generate riboflavin and this ability is significantly impeded when expression of a second surface ectoenzyme (alkaline phosphatase, SmAP) is suppressed. Recombinant SmAP cleaves FMN with a Km of 3.82 ± 0.58 mM and Kcat/Km of 1393 ± 347 M- 1.S- 1. CONCLUSIONS: The sequential hydrolysis of FAD by tegumental ecto-enzymes SmNPP5 and SmAP can generate free vitamin B2 around the worms from where it can be conveniently imported by the recently described schistosome riboflavin transporter SmaRT. Finally, we identified in silico schistosome homologs of enzymes that are involved in intracellular vitamin B2 metabolism. These are riboflavin kinase (SmRFK) as well as FAD synthase (SmFADS); cDNAs encoding these two enzymes were cloned and sequenced. SmRFK is predicted to convert riboflavin to FMN while SmFADS could further act on FMN to regenerate FAD in order to facilitate robust vitamin B2-dependent metabolism in schistosomes.

NAD-catabolizing ectoenzymes of Schistosoma mansoni.

Infection with schistosomes (blood flukes) can result in the debilitating disease schistosomiasis. These parasites survive in their host for many years, and we hypothesize that proteins on their tegumental surface, interacting with the host microenvironment, facilitate longevity. One such ectoenzyme - the nucleotide pyrophosphatase/phosphodiesterase SmNPP5 can cleave ADP (to prevent platelet aggregation) and NAD (likely preventing Treg apoptosis). A second tegumental ectoenzyme, the glycohydrolase SmNACE, also catabolizes NAD. Here, we undertake a comparative biochemical characterization of these parasite ectoenzymes. Both are GPI-linked and exhibit different optimal pH ranges. While SmNPP5 requires divalent cations, SmNACE does not. The KM values of the two enzymes for NAD at physiological pH differ: SmNPP5, KM = 340 µM ± 44; SmNACE, KM = 49 µM ± 4. NAD cleavage by each enzyme yields different products. SmNPP5 cleaves NAD to form nicotinamide mononucleotide (NMN) and AMP, whereas SmNACE cleaves NAD to generate nicotinamide (NAM) and adenosine diphosphate ribose (ADPR). Each enzyme can process the other's reaction product. Thus, SmNACE cleaves NMN (to yield NAM and ribose phosphate) and SmNPP5 cleaves ADPR (yielding AMP and ribose phosphate). Metabolomic analysis of plasma containing adult worms supports the idea that these cleavage pathways are active in vivo. We hypothesize that a primary function of SmNPP5 is to cleave NAD to control host immune cell function and a primary function of SmNACE is to cleave NMN to generate the vital nutrient nicotinamide (vitamin B3) for convenient uptake by the worms. Chemical inhibition of one or both ectoenzymes could upset worm metabolism and control schistosome infection.

LACK, a RACK1 ortholog, facilitates cytochrome c oxidase subunit expression to promote Leishmania major fitness.

Leishmania are kinetoplastid parasites that cause the sandfly-transmitted disease leishmaniasis. To maintain fitness throughout their infectious life cycle, Leishmania must undergo rapid metabolic adaptations to the dramatically distinct environments encountered during transition between sandfly and vertebrate hosts. We performed proteomic and immunoblot analyses of attenuated L. major strains deficient for LACK, the Leishmania ortholog of the mammalian receptor for activated c kinase (RACK1), that is important for parasite thermotolerance and virulence. This approach identified cytochrome c oxidase (LmCOX) subunit IV as a LACK-dependent fitness protein. Consistent with decreased levels of LmCOX subunit IV at mammalian temperature, and in amastigotes, LmCOX activity and mitochondrial function were also impaired in LACK-deficient L. major under these conditions. Importantly, overexpression of LmCOX subunit IV in LACK-deficient L. major restored thermotolerance and macrophage infectivity. Interestingly, overexpression of LmCOX subunit IV enhanced LmCOX subunit VI expression at mammalian temperature. Collectively, our data suggest LACK promotes Leishmania adaptation to the mammalian host environment by sustaining LmCOX subunit IV expression and hence energy metabolism in response to stress stimuli such as heat. These findings extend the repertoire of RACK1 protein utility to include a role in mitochondrial function.

Metabolism of FAD, FMN and riboflavin (vitamin B2) in the human parasitic blood fluke Schistosoma mansoni.

Schistosomiasis is a parasitic disease caused by trematode worms of the genus Schistosoma. The intravascular worms acquire the nutrients necessary for their survival from host blood. Since all animals are auxotrophic for riboflavin (vitamin B2), schistosomes too must import it to survive. Riboflavin is an essential component of the coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD); these support key functions of dozens of flavoenzymes. In this work we focus on the biochemistry of riboflavin and its metabolites in Schistosoma mansoni. We show that when schistosomes are incubated in murine plasma, levels of FAD decrease over time while the levels of FMN increase. We show that live schistosomes can cleave exogenous FAD to generate FMN and this ability is significantly blocked when expression of the surface ectoenzyme SmNPP5 is suppressed using RNAi. Recombinant SmNPP5 cleaves FAD with a Km of 178 ± 5.9 µM. The FAD-dependent enzyme IL-4I1 drives the oxidative deamination of phenylalanine to produce phenylpyruvate and H2O2 in the extracellular environment. Since schistosomes can be damaged by H2O2, we determined if SmNPP5 could impede H2O2 production by blocking IL-4I1 action in vitro. We found that this was not the case, suggesting that covalently bound FAD on IL-4I1 is inaccessible to SmNPP5. We also report here that live schistosomes can cleave exogenous FMN to generate riboflavin and this ability is significantly impeded when expression of a second surface ectoenzyme (alkaline phosphatase, SmAP) is suppressed. Recombinant SmAP cleaves FMN with a Km of 3.82 ± 0.58 mM. Thus, the sequential hydrolysis of FAD by tegumental ecto-enzymes SmNPP5 and SmAP can generate free vitamin B2 around the worms from where it can be conveniently imported by, we hypothesize, the recently described schistosome riboflavin transporter SmaRT. In this work we also identified in silico schistosome homologs of enzymes that are involved in intracellular vitamin B2 metabolism. These are riboflavin kinase (SmRFK) as well as FAD synthase (SmFADS); cDNAs encoding these two enzymes were cloned and sequenced. SmRFK is predicted to convert riboflavin to FMN while SmFADS could further act on FMN to regenerate FAD in order to facilitate robust vitamin B2-dependent metabolism in schistosomes.

Transgenic overexpression of heat shock protein (HSP83) enhances protein kinase A activity, disrupts GP63 surface protease expression and alters promastigote morphology in Leishmania amazonensis.

Leishmania parasites undergo morphological changes during their infectious life cycle, including developmental transitions within the sandfly vector, culminating in metacyclic stages that are pre-adapted for infection. Upon entering vertebrate host phagocytes, Leishmania differentiate into intracellular amastigotes, the form that is ultimately transmitted back to the vector to complete the life cycle. Although environmental conditions that induce these cellular transitions are well-established, molecular mechanisms governing Leishmania morphologic differentiation in response to these cues remain largely uncharacterized. Previous studies indicate a key role for HSP83 in both promastigote metacyclogenesis and amastigote differentiation. To further elucidate HSP83 functions in the Leishmania lifecycle, we examined the biological impact of experimentally elevating HSP83 gene expression in Leishmania. Significantly, HSP83 overexpression was associated with altered metacyclic morphology, increased protein kinase A (PKA) activity and decreased expression of the Leishmania major surface protease, GP63. Corroborating these findings, overexpression of the L. amazonensis PKA catalytic subunit resulted in a largely similar phenotype. Our findings demonstrate for the first time in Leishmania, a functional link between HSP83 and PKA in the control of Leishmania gene expression, replication and morphogenesis.

Previous exposure to a low infectious dose of Leishmania major exacerbates infection with Leishmania infantum in the susceptible BALB/c mouse.

The geographic distribution of Leishmania major overlaps with several other species of Leishmania. This study seeks to examine what effect previous exposure to L. major has on the outcome of infection with Leishmania infantum, the agent of virulent visceral leishmaniasis. The L. major immune response is well characterized by a strong Th1 response leading to resolution and protection against subsequent re-infection. A contrasting Th2 immune response leads to disseminated disease, while the role Th17 cytokines may play in Leishmania infection is still being explored. The cytokine profile, antibody titer, and parasite burden were evaluated in the susceptible BALB/c mouse after L. infantum infection in either naïve mice or those previously infected with a low/self-healing dose of L. major. Only IL-4 expression in mice previously exposed to L. major was found to be significantly increased over controls, a cytokine with an ambiguous role in L. infantum infection. However, disease exacerbation, with a notably higher parasite burden, was observed in the L. major exposed mice compared to the L. infantum only. Cross-reactive antibodies were seen in both groups of infected mice regardless of their immune history. Studies have shown a role for opsonizing antibodies leading to increased disease in visceral leishmaniasis. We speculate that cross-reactive antibodies may be playing a role in augmenting visceral disease in mice with immunological memory to L. major.

Schistosoma mansoni and the purinergic halo.

Intravascular schistosomes may control immune and hemostatic responses by regulating the nature and amount of selected host purinergic signaling molecules - such as adenosine triphosphate (ATP), adenosine diphosphate (ADP), and nicotinamide adenine dinucleotide (NAD) - surrounding them. Such metabolites are collectively known as the worm's 'purinergic halo'. Host-interactive, membrane-bound, tegumental ectonucleotidases, notably SmATPDase1, SmNPP5, SmAP and SmNACE, can degrade proinflammatory, prothrombotic and immunomodulatory purinergic metabolites like those listed. A common catabolic product is the anti-inflammatory metabolite adenosine that can additionally be taken in by the worms as food. We envision the tegumental ectonucleotidases as having a twofold role at the worm surface: first, they degrade potentially harmful host signaling molecules, and second, they generate vital nutrients around the worms from where these can be conveniently imported.

Schistosomes Impede ATP-Induced T Cell Apoptosis In Vitro: The Role of Ectoenzyme SmNPP5.

Schistosomes (blood flukes) can survive in the bloodstream of their hosts for many years. We hypothesize that proteins on their host-interactive surface impinge on host biochemistry to help ensure their long-term survival. Here, we focus on a surface ectoenzyme of Schistosoma mansoni, designated SmNPP5. This ~53 kDa glycoprotein is a nucleotide pyrophosphatase/phosphodiesterase that has been previously shown to: (1) cleave adenosine diphosphate (ADP) and block platelet aggregation; and (2) cleave nicotinamide adenine dinucleotide (NAD) and block NAD-induced T cell apoptosis in vitro. T cell apoptosis can additionally be driven by extracellular adenosine triphosphate (ATP). In this work, we show that adult S. mansoni parasites can inhibit this process. Further, we demonstrate that recombinant SmNPP5 alone can both cleave ATP and impede ATP-induced T cell killing. As immunomodulatory regulatory T cells (Tregs) are especially prone to the induction of these apoptotic pathways, we hypothesize that the schistosome cleavage of both NAD and ATP promotes Treg survival and this helps to create a less immunologically hostile environment for the worms in vivo.

The essential schistosome tegumental ectoenzyme SmNPP5 can block NAD-induced T cell apoptosis.

Infection with intravascular platyhelminths of the genus Schistosoma can result in the debilitating disease schistosomiasis. Schistosomes (blood flukes) can survive in the host for many years. We hypothesize that proteins on their host-interactive surface modify the worm's external environment to help insure worm survival. Previously, we have shown that a surface ectoenzyme of Schistosoma mansoni, SmNPP5 - a nucleotide pyrophosphatase/phosphodiesterase - can cleave ADP and block platelet aggregation in vitro. In this work, we show that both adult schistosomes and recombinant SmNPP5 can cleave the exogenous purinergic signaling molecule nicotinamide adenine dinucleotide (NAD). In doing so, worms and rSmNPP5 can prevent NAD-induced apoptosis of T cells in vitro. Since regulatory T cells (Tregs) are especially prone to such NAD-induced cell death (NICD), we hypothesize that schistosome cleavage of NAD promotes Treg survival which creates a more immunologically hospitable environment for the worms in vivo. In addition to SmNPP5, schistosomes express another host-interactive NAD-degrading enzyme, SmNACE. We successfully suppressed the expression of SmNPP5 and SmNACE (singly or together) using RNAi. Only SmNPP5-suppressed worms, and not SmNACE-suppressed worms, were significantly impaired in their ability to cleave exogenous NAD compared to controls. Therefore, we contend that ectoenzyme SmNPP5 on the surface of the worm is primarily responsible for extracellular NAD cleavage and that this helps modulate the host immune environment by preventing Treg cell death.

Schistosome migration in the definitive host.

Schistosomes are parasitic blood flukes that infect >200 million people around the world. Free-swimming larval stages penetrate the skin, invade a blood vessel, and migrate through the heart and lungs to the vasculature of the liver, where maturation and mating occurs. From here, the parasite couples migrate to their preferred egg laying sites. Here, we compare and contrast what is known about the migration patterns within the definitive host of the three major species of human schistosome: Schistosoma mansoni, S. japonicum, and S. haematobium. We conclude that intravascular schistosomes are inexorable colonizers whose migration and egg laying strategy is profligate; all three species (and their eggs) can be found throughout the mesenteric venules, the rectal venous plexus, and, to a greater or lesser extent, the urogenital venous plexuses. In addition, it is common for parasite eggs to be deposited in locations that lack easy access to the exterior, further demonstrating the relentless exploratory nature of these intravascular worms.

Schistosomes can hydrolyze proinflammatory and prothrombotic polyphosphate (polyP) via tegumental alkaline phosphatase, SmAP.

Schistosoma mansoni is a long-lived intravascular trematode parasite that can infect humans causing the chronic debilitating disease, schistosomiasis. We hypothesize that the action of host-interactive proteins found at the schistosome surface allows the worms to maintain a safe, anti-thrombotic and anti-inflammatory environment around them in the bloodstream. One such protein is the ˜60 kDa alkaline phosphatase SmAP which is known to be expressed in the outer tegument of all intravascular life stages. We demonstrate in this work that the parasites (schistosomula as well as adult males and females) can hydrolyze polyphosphate (polyP) - an anionic, linear polymer of inorganic phosphates that is produced and released by immune cells as well as by activated platelets and that induce proinflammatory and prothrombotic pathways. Purified recombinant SmAP can likewise cleave polyP and with a Km of 6.9 ±â€¯1 mM. Finally, parasites whose SmAP gene has been suppressed by RNAi are significantly impaired in their ability to hydrolyze polyP. SmAP-mediated cleavage of polyP may contribute to the armamentarium of schistosomes that promotes their survival in the hostile intravascular habitat. This is the first report of any pathogen cleaving this bioactive metabolite.

Disruption of the Putative Ribosome-Binding Motif of a Scaffold Protein Impairs Cytochrome c Oxidase Subunit Expression in Leishmania major.

During their parasitic life cycle, through sandflies and vertebrate hosts, Leishmania parasites confront strikingly different environments, including abrupt changes in pH and temperature, to which they must rapidly adapt. These adaptations include alterations in Leishmania gene expression, metabolism, and morphology, allowing them to thrive as promastigotes in the sandfly and as intracellular amastigotes in the vertebrate host. A critical aspect of Leishmania metabolic adaptation to these changes is maintenance of efficient mitochondrial function in the hostile vertebrate environment. Such functions, including generation of ATP, depend upon the expression of many mitochondrial proteins, including subunits of cytochrome c oxidase (COX). Significantly, under mammalian temperature conditions, expression of Leishmania major COX subunit IV (LmCOX4) and virulence are dependent upon two copies of LACK, a gene that encodes the ribosome-associated scaffold protein, LACK (Leishmania ortholog of RACK1 [receptor for activated C kinase]). Targeted replacement of an endogenous LACK copy with a putative ribosome-binding motif-disrupted variant (LACKR34D35G36→LACKD34D35E36) resulted in thermosensitive parasites that showed diminished LmCOX4 expression, mitochondrial fitness, and replication in macrophages. Surprisingly, despite these phenotypes, LACKD34D35E36 associated with monosomes and polysomes and showed no major impairment of global protein synthesis. Collectively, these data suggest that wild-type (WT) LACK orchestrates robust LmCOX4 expression and mitochondrial fitness to ensure parasite virulence, via optimized functional interactions with the ribosome.IMPORTANCELeishmania parasites are trypanosomatid protozoans that persist in infected human hosts to cause a spectrum of pathologies, from cutaneous and mucocutaneous manifestations to visceral leishmaniasis caused by Leishmania donovani The latter is usually fatal if not treated. Persistence of L. major in the mammalian host depends upon maintaining gene-regulatory programs to support essential parasite metabolic functions. These include expression and assembly of mitochondrial L. major cytochrome c oxidase (LmCOX) subunits, important for Leishmania ATP production. Significantly, under mammalian conditions, WT levels of LmCOX subunits require threshold levels of the Leishmania ribosome-associated scaffold protein, LACK. Unexpectedly, we find that although disruption of LACK's putative ribosome-binding motif does not grossly perturb ribosome association or global protein synthesis, it nonetheless impairs COX subunit expression, mitochondrial function, and virulence. Our data indicate that the quality of LACK's interaction with Leishmania ribosomes is critical for LmCOX subunit expression and parasite mitochondrial function in the mammalian host. Collectively, these findings validate LACK's ribosomal interactions as a potential therapeutic target.

Structural basis for species-selective targeting of Hsp90 in a pathogenic fungus.

New strategies are needed to counter the escalating threat posed by drug-resistant fungi. The molecular chaperone Hsp90 affords a promising target because it supports survival, virulence and drug-resistance across diverse pathogens. Inhibitors of human Hsp90 under development as anticancer therapeutics, however, exert host toxicities that preclude their use as antifungals. Seeking a route to species-selectivity, we investigate the nucleotide-binding domain (NBD) of Hsp90 from the most common human fungal pathogen, Candida albicans. Here we report structures for this NBD alone, in complex with ADP or in complex with known Hsp90 inhibitors. Encouraged by the conformational flexibility revealed by these structures, we synthesize an inhibitor with >25-fold binding-selectivity for fungal Hsp90 NBD. Comparing co-crystals occupied by this probe vs. anticancer Hsp90 inhibitors revealed major, previously unreported conformational rearrangements. These insights and our probe's species-selectivity in culture support the feasibility of targeting Hsp90 as a promising antifungal strategy.

Genetic Characterization of Plasmodium Putative Pantothenate Kinase Genes Reveals Their Essential Role in Malaria Parasite Transmission to the Mosquito.

The metabolic machinery for the biosynthesis of Coenzyme A (CoA) from exogenous pantothenic acid (Vitamin B5) has long been considered as an excellent target for the development of selective antimicrobials. Earlier studies in the human malaria parasite Plasmodium falciparum have shown that pantothenate analogs interfere with pantothenate phosphorylation and block asexual blood stage development. Although two eukaryotic-type putative pantothenate kinase genes (PanK1 and PanK2) have been identified in all malaria parasite species, their role in the development of Plasmodium life cycle stages remains unknown. Here we report on the genetic characterization of PanK1 and PanK2 in P. yoelii. We show that P. yoelii parasites lacking either PanK1 or PanK2 undergo normal asexual stages development and sexual stages differentiation, however they are severely deficient in ookinete, oocyst and sporozoite formation inside the mosquito vector. Quantitative transcriptional analyses in wild-type and knockout parasites demonstrate an important role for these genes in the regulation of expression of other CoA biosynthesis genes. Together, our data provide the first genetic evidence for the importance of the early steps of pantothenate utilization in the regulation of CoA biosynthesis and malaria parasite transmission to Anopheles mosquitoes.

Genotype diversity of Trypanosoma cruzi in small rodents and Triatoma sanguisuga from a rural area in New Orleans, Louisiana.

BACKGROUND: Chagas disease is an anthropozoonosis caused by the protozoan parasite Trypanosoma cruzi that represents a major public health problem in Latin America. Although the United States is defined as non-endemic for Chagas disease due to the rarity of human cases, the presence of T. cruzi has now been amply demonstrated as enzootic in different regions of the south of the country from Georgia to California. In southeastern Louisiana, a high T. cruzi infection rate has been demonstrated in Triatoma sanguisuga, the local vector in this area. However, little is known about the role of small mammals in the wild and peridomestic transmission cycles. METHODS: This study focused on the molecular identification and genotyping of T. cruzi in both small rodents and T. sanguisuga from a rural area of New Orleans, Louisiana. DNA extractions were prepared from rodent heart, liver, spleen and skeletal muscle tissues and from cultures established from vector feces. T. cruzi infection was determined by standard PCR using primers specific for the minicircle variable region of the kinetoplastid DNA (kDNA) and the highly repetitive genomic satellite DNA (satDNA). Genotyping of discrete typing units (DTUs) was performed by amplification of mini-exon and 18S and 24Sα rRNA genes and subsequent sequence analysis. RESULTS: The DTUs TcI, TcIV and, for the first time, TcII, were identified in tissues of mice and rats naturally infected with T. cruzi captured in an area of New Orleans, close to the house where the first human case of Chagas disease was reported in Louisiana. The T. cruzi infection rate in 59 captured rodents was 76%. The frequencies of the detected DTUs in such mammals were TcI 82%, TcII 22% and TcIV 9%; 13% of all infections contained more than one DTU. CONCLUSIONS: Our results indicate a probable presence of a considerably greater diversity in T. cruzi DTUs circulating in the southeastern United States than previously reported. Understanding T. cruzi transmission dynamics in sylvatic and peridomestic cycles in mammals and insect vectors will be crucial to estimating the risk of local, vector-borne transmission of T. cruzi to humans in the United States.