Short-chain polyP Research Articles

A Comprehensive Approach Combining Short-Chain Polyphosphate and Bacterial Biostimulants for Effective Nutrient Solubilization and Enhanced Wheat Growth.

Phosphorus constitutes a crucial macronutrient for crop growth, yet its availability often limits food production. Efficient phosphorus management is crucial for enhancing crop yields and ensuring food security. This study aimed to enhance the efficiency of a short-chain polyphosphate (PolyP) fertilizer by integrating it with plant growth-promoting bacteria (PGPB) to improve nutrient solubilization and wheat growth. Specifically, the study investigated the effects of various bacterial strains on wheat germination and growth when used in conjunction with PolyP. To achieve this, a greenhouse experiment was conducted in which the wheat rhizosphere was amended with a short-chain PolyP fertilizer. Based on the morphological aspect, eight bacteria, designated P1 to P8, were isolated and further characterized. Plant growth-promoting traits were observed in all bacterial strains, as they presented the ability to produce Indole Acetic Acid (IAA) in significant amounts ranging from 7.5 ± 0.3 µg/mL to 44.1 ± 2 µg/mL, expressed by B. tropicus P4 and P. soyae P1, respectively. They also produced ammonia, hydrogen cyanide (HCN), and siderophores. Their effect against the plant pathogen Fusarium culmorum was also assessed, with P. reinekei P2 demonstrating the highest biocontrol activity as it presented a total inhibitory effect. Additionally, some strains exhibited the ability to solubilize/hydrolyze phosphorus, potassium, and zinc. In vivo, the initial growth potential of wheat seeds indicated that those inoculated with the isolated strains exhibited elevated germination rates and enhanced root growth. Based on their plant growth-promoting traits and performance in the germination assay, three strains were selected for producing the best results, specifically phosphorus hydrolyzation/solubilization, zinc solubilization, IAA production, HCN, and siderophores production. Wheat seeds were inoculated by drenching in a bacterial suspension containing 1010 CFU/mL of log phase culture, and an in planta bioassay was conducted in a growth chamber using three selected strains (Pseudomonas soyae P1, Pseudomonas reinekei P2, and Bacillus tropicus P4), applied either individually or with PolyP on a P-deficient soil (28 mg/kg of P Olsen). Our findings demonstrated that the combination of Pseudomonas soyae P1 and PolyP achieved the highest shoot biomass, averaging 41.99 ± 0.87 g. Notably, applying P. soyae P1 or Bacillus tropicus P4 alone yielded similar results to the use of PolyP alone. At the heading growth stage, the combination of Bacillus tropicus P4 and PolyP significantly increased the Chlorophyll Content Index (CCI) to 37.02 µmol/m2, outperforming both PolyP alone (24.07 µmol/m2) and the control (23.06 µmol/m2). This study presents an innovative approach combining short-chain PolyP with bacterial biostimulants to enhance nutrient availability and plant growth. By identifying and characterizing effective bacterial strains, it offers a sustainable alternative to conventional fertilizers.

Human Prune Regulates the Metabolism of Mammalian Inorganic Polyphosphate and Bioenergetics.

Inorganic polyphosphate (polyP) is an evolutionarily conserved and ubiquitous polymer that is present in all studied organisms. PolyP consists of orthophosphates (Pi) linked together by phosphoanhydride bonds. The metabolism of polyP still remains poorly understood in higher eukaryotes. Currently, only F0F1-ATP synthase, Nudt3, and Prune have been proposed to be involved in this metabolism, although their exact roles and regulation in the context of polyP biology have not been fully elucidated. In the case of Prune, in vitro studies have shown that it exhibits exopolyphosphatase activity on very short-chain polyP (up to four units of Pi), in addition to its known cAMP phosphodiesterase (PDE) activity. Here, we expand upon studies regarding the effects of human Prune (h-Prune) on polyP metabolism. Our data show that recombinant h-Prune is unable to hydrolyze short (13-33 Pi) and medium (45-160 Pi) chains of polyP, which are the most common chain lengths of the polymer in mammalian cells. Moreover, we found that the knockdown of h-Prune (h-Prune KD) results in significantly decreased levels of polyP in HEK293 cells. Likewise, a reduction in the levels of polyP is also observed in Drosophila melanogaster loss-of-function mutants of the h-Prune ortholog. Furthermore, while the activity of ATP synthase, and the levels of ATP, are decreased in h-Prune KD HEK293 cells, the expression of ATP5A, which is a main component of the catalytic subunit of ATP synthase, is upregulated in the same cells, likely as a compensatory mechanism. Our results also show that the effects of h-Prune on mitochondrial bioenergetics are not a result of a loss of mitochondrial membrane potential or of significant changes in mitochondrial biomass. Overall, our work corroborates the role of polyP in mitochondrial bioenergetics. It also demonstrates a conserved effect of h-Prune on the metabolism of short- and medium-chain polyP (which are the predominant chain lengths found in mammalian cells). The effects of Prune in polyP are most likely exerted via the regulation of the activity of ATP synthase. Our findings pave the way for modifying the levels of polyP in mammalian cells, which could have pharmacological implications in many diseases where dysregulated bioenergetics has been demonstrated.

Short-chain polyphosphates: Extraction effects on migration and size estimation of Saccharomyces cerevisiae extracts with polyacrylamide gel electrophoresis.

Polyacrylamide gel electrophoresis is commonly used to characterize the chain length of polyphosphates (polyP), more generally called condensed phosphates. After separation, nonradioactive, optical polyP staining is limited to chain lengths greater than 15 monomers with toluidine blue or 4',6-diamidino-2-phenylindole. PolyP chain lengths longer than 62 monomers were correlated to the shortest DNA ladders. In this study, synthetic linear polyPs (Sigma-Aldrich "Type 45", estimated mean length of 45 monomers), trimetaphosphate (trimetaP: 3 ring), tripolyphosphate (tripolyP), pyrophosphate (PPi ), and inorganic orthophosphate (o-Pi ) were visualized after separation by an in situ hydrolytic degradation process to o-Pi that was subsequently stained with methyl green. Statistically insignificant migration reduction of synthetic short-chain polyP after perchloric acid or phenol-chloroform extraction was confirmed with the Friedman test. 31 P diffusion-ordered NMR spectroscopy confirmed that extraction also reduced PPi diffusivity by <10%. Linear regression between the Rf peak migration value and the logarithm of synthetic polyP molecular weights enabled estimation of extracted polyP chain lengths from 2 to 45 monomers. Linear polyP extracts from Saccharomyces cerevisiae grown in aerobic conditions were generally shorter than extracts cultured in anaerobic conditions. Extractions from both aerobic and anaerobic S. cerevisiae included tripolyP and o-Pi , but no PPi .

Polyphosphate as an antithrombotic target and hemostatic agent.

Polyphosphate (PolyP) is a polymer comprised of linear phosphate units connected by phosphate anhydride bonds. PolyP exists in a diverse range of eukaryotes and prokaryotes with varied chain lengths ranging from six to thousands of phosphate units. Upon activation, human platelets and neutrophils release short-chain PolyP, along with other components, to initiate the coagulation pathway. Long-chain PolyP derived from cellular or bacterial organelles exhibits higher proinflammatory and procoagulant effects compared to short-chain PolyP. Notably, PolyP has been identified as a low-hemorrhagic antithrombotic target since neutralizing plasma PolyP suppresses the thrombotic process without impairing the hemostatic functions. As an inorganic polymer without uniform steric configuration, PolyP is typically targeted by cationic polymers or recombinant polyphosphatases rather than conventional antibodies, small-molecule compounds, or peptides. Additionally, because of its procoagulant property, PolyP has been incorporated in wound-dressing materials to facilitate blood hemostasis. This review summarizes current studies on PolyP as a low-hemorrhagic antithrombotic target and the development of hemostatic materials based on PolyP.

Expression of polyphosphate kinase from Sphingobacterium siyangensis and its application in ATP regeneration system

Polyphosphate kinase plays an important role in the catalytic synthesis of ATP in vitro. In order to find a polyphosphate kinase that can efficiently synthesize ATP using short-chain polyphosphate (polyP) as substrate, the polyphosphate kinase 2 (PPK2) from Sphingobacterium siyangensis was cloned and expressed in Escherichia coli BL21(DE3). As an enzyme for ATP regeneration, PPK2 was used in combination with l-amino acid ligase (YwfE) to produce l-alanyl-l-glutamine (Ala-Gln). The length of ppk2 of S. siyangensis is 810 bp, encoding 270 amino acids. The SDS-PAGE showed that PPK2 was expressed correctly and its molecular weight was 29.7 kDa as expected. The reaction conditions of PPK2 were optimized. PPK2 could maintain good activity in the range of 22-42 ℃ and pH 7-10. The highest enzyme activity was observed at 37 ℃, pH 7, 30 mmol/L magnesium ion (Mg2+), 5 mmol/L ADP and 10 mmol/L sodium hexametaphosphate, and the yield of ATP reached 60% of the theoretical value in 0.5 hours at this condition. When used in combination with YwfE to produce Ala-Gln, the PPK2 showed a good applicability as an ATP regeneration system, and the effect was similar to that of direct addition of ATP. The PPK2 from S. siyangensis shows good performance in a wide range of temperature and pH, synthesizes ATP with cheap and readily available short chain polyP as substrate. The PPK2 thus provides a new enzyme source for ATP dependent catalytic reaction system.

Biomineralization and AHLs-guided quorum sensing enhanced phosphorus recovery in the alternating aerobic/anaerobic biofilm system under metal ion stress

The alternating aerobic/anaerobic biofilm system had been applied for phosphorus (P) enrichment and recovery because of the advantage of low energy consumption and high efficiency. The metal ions and N-acyl-L-homoserine lactones (AHLs) in system were studied to better clarify the mechanism of P uptake/release under metal ion stress. The results indicated that the increase of metal ions stimulated the release of AHLs, and AHLs-guided quorum sensing (QS) enhanced P uptake. Moreover, biomineralization could stimulate the increase of P content in biofilm (Pbiofilm). Meanwhile, some ortho-p was converted to short-chain poly-p in extracellular polymer substance (EPS), and others were transferred into cell through EPS to synthesize poly-p. With the Pbiofilm increased, more P could be absorbed/released due to the shift in the metabolic model of polyphosphate accumulating organisms (PAOs). The release of AHLs between microorganisms was also inhibited when PAOs reached the state of P saturation (75.6 ± 2.5 mg/g SS), which meant that the effect of signaling function would tend to stabilize, and the 169.2 ± 2.6 mg/L P concentration in the enriched solution was obtained due to the P release was inhibited. Moreover, P was rapidly transferred to the new enriched solution after the P was recovered, and PAOs restored its capability of P uptake/release. In addition, 31P-NMR analysis demonstrated that EPS played a major role in PAOs compared to cell, and inorganic phosphorus (IP) played an essential role in the uptake/release of P compared to organic phosphorus (OP). Furthermore, the microbiological analysis showed that Candidatus Accumulibacter was positively correlated with AHLs (P < 0.05). This study provided essential support for clarifying the P metabolism mechanism of PAOs.

In vitro biosynthesis of ATP from adenosine and polyphosphate

Adenosine triphosphate (ATP) acts as a crucial energy currency in vivo, and it is a widely used energy and/or phosphate donor for enzyme-catalyzed reactions in vitro. In this study, we established an in vitro multi-enzyme cascade system for ATP production. Using adenosine and inorganic polyphosphate (polyP) as key substrates, we combined adenosine kinase and two functionally distinct polyphosphate kinases (PPKs) in a one-pot reaction to achieve chain-like ATP regeneration and production. Several sources of PPK were screened and characterized, and two suitable PPKs were selected to achieve high rates of ATP production. Among these, Sulfurovum lithotrophicum PPK (SlPPK) exhibited excellent activity over a wide pH range (pH 4.0–9.0) and synthesized ATP from ADP using short-chain polyP. Furthermore, it had a half-life > 155.6 h at 45 °C. After optimizing the reaction conditions, we finally carried out the coupling-catalyzed reaction with different initial adenosine concentrations of 10, 20, and 30 mM. The highest yields of ATP were 76.0, 70.5, and 61.3%, respectively.Graphical

NUDIX hydrolases with inorganic polyphosphate exo- and endopolyphosphatase activities in the glycosome, cytosol and nucleus of Trypanosoma brucei.

Trypanosoma brucei, a protist parasite that causes African trypanosomiasis or sleeping sickness, relies mainly on glycolysis for ATP production when in its mammalian host. Glycolysis occurs within a peroxisome-like organelle named the glycosome. Previous work from our laboratory reported the presence of significant amounts of inorganic polyphosphate (polyP), a polymer of three to hundreds of orthophosphate units, in the glycosomes and nucleoli of T. brucei. In this work, we identified and characterized the activity of two Nudix hydrolases (NHs), T. brucei Nudix hydrolase (TbNH) 2 and TbNH4, one located in the glycosomes and the other in the cytosol and nucleus, respectively, which can degrade polyP. We found that TbNH2 is an exopolyphosphatase with higher activity on short chain polyP, while TbNH4 is an endo- and exopolyphosphatase that has similar activity on polyP of various chain sizes. Both enzymes have higher activity at around pH 8.0. We also found that only TbNH2 can dephosphorylate ATP and ADP but with lower affinity than for polyP. Our results suggest that NHs can participate in polyP homeostasis and therefore may help control polyP levels in glycosomes, cytosol and nuclei of T. brucei.

Dual role of inorganic polyphosphate in cardiac myocytes: The importance of polyP chain length for energy metabolism and mPTP activation

We have previously demonstrated that inorganic polyphosphate (polyP) is a potent activator of the mitochondrial permeability transition pore (mPTP) in cardiac myocytes. PolyP depletion protected against Ca2+-induced mPTP opening, however it did not prevent and even exacerbated cell death during ischemia-reperfusion (I/R). The central goal of this study was to investigate potential molecular mechanisms underlying these dichotomous effects of polyP on mitochondrial function. We utilized a Langendorff-perfused heart model of I/R to monitor changes in polyP size and chain length at baseline, 20 min no-flow ischemia, and 15 min reperfusion. Freshly isolated cardiac myocytes and mitochondria from C57BL/6J (WT) and cyclophilin D knock-out (CypD KO) mice were used to measure polyP uptake, mPTP activity, mitochondrial membrane potential, respiration and ATP generation. We found that I/R induced a significant decrease in polyP chain length. We, therefore, tested, the ability of synthetic polyPs with different chain length to accumulate in mitochondria and induce mPTP. Both short and long chain polyPs accumulated in mitochondria in oligomycin-sensitive manner implicating potential involvement of mitochondrial ATP synthase in polyP transport. Notably, only short-chain polyP activated mPTP in WT myocytes, and this effect was prevented by mPTP inhibitor cyclosprorin A and absent in CypD KO myocytes. To the contrary, long-chain polyP suppressed mPTP activation, and enhanced ADP-linked respiration and ATP production. Our data indicate that 1) effect of polyP on cardiac function strongly depends on polymer chain length; and 2) short-chain polyPs (as increased in ischemia-reperfusion) induce mPTP and mitochondrial uncoupling, while long-chain polyPs contribute to energy generation and cell metabolism.

Short chain polyphosphates as a strategic colloidal source of phosphate for parenteral admixtures

Short chain sodium polyphosphates – (NaPO3)9 – PolyP – were added to 2-in-1 pediatric parenteral nutrition (PN) formulations as new alternative phosphate sources. Solutions of PolyP and Ca2+ in the presence of Mg2+, glucose, amino acids and trace elements were analyzed by electronic absorption and 31P NMR spectroscopies, as well as by pH measurements. Additionally, to verify the biocompatibility of short chain PolyP solutions, red blood cell (RBCs) units were evaluated by analyzing hemoglobin, hematocrit, and hemolysis. The blood clotting action of the PolyP was determined through Prothrombin time. We observed that the short chain PolyP anions did not interfere in the quality control parameters for RBCs units or in the blood extrinsic coagulation pathway. The spectral changes observed in the 31P NMR confirm the association of Mg2+ and Ca2+ with PolyP in the admixtures. Compatibility diagrams showed that the PolyP-Ca/Mg systems reflect a gel behavior based on the PolyP chains interaction with the incoming cations maintaining a supramolecular equilibrium until a limiting concentration. We observed that the greater the PolyP concentration, the greater the amount of Ca2+ that could be added to the admixture without phase separation and that the increased complexity of species (glucose, amino acid, trace elements and Mg2+) in the admixture favored a still higher final Ca2+ concentration. The overall colloidal behavior of short chain PolyPs shows that these polyanions function not only as alternative and safe phosphate sources in PN formulations but also as polyanions that increase the admixture stability against phase separation by efficiently sequestering metal cations present.

Screening a Protein Array with Synthetic Biotinylated Inorganic Polyphosphate To Define the Human PolyP-ome.

Phenotypes are established by tight regulation on protein functions. This regulation can be mediated allosterically, through protein binding, and covalently, through post-translational modification (PTM). The integration of an ever-increasing number of PTMs into regulatory networks enables and defines the proteome complexity. Protein PTMs can occur enzymatically and nonenzymatically. Polyphosphorylation, which is a recently discovered PTM that belongs to the latter category, is the covalent attachment of the linear ortho-phosphate polymer called inorganic polyphosphate (polyP) to lysine residues. PolyP, which is ubiquitously present in nature, is also known to allosterically control protein function. To date, lack of reagents has prevented the systematic analysis of proteins covalently and/or allosterically associated with polyP. Here, we report on the chemical synthesis of biotin-modified monodisperse short-chain polyP (bio-polyP8-bio) and its subsequent use to screen a human proteome array to identify proteins that associate with polyP, thereby starting to define the human polyP-ome.

Inorganic polyphosphate in methylotrophic yeasts.

Inorganic polyphosphate (polyP) is a significant regulatory and metabolic compound in yeast cells. We compared polyP content and localization, polyphosphatase activities, and transcriptional profile of polyP-related genes in industrially important methylotrophic yeasts, Hansenula polymorpha and Pichia pastoris. The increased need for phosphate, the decrease of long-chain polyP level, the accumulation of short-chain polyP, and enhanced endopolyphosphatase activity in the crude membrane fraction were observed in methanol-grown cells compared with glucose-grown cells of both species. Transcriptome analysis revealed notable differences in the expression patterns of key genes encoding proteins related to polyP metabolism. In methanol-grown cells, the genes encoding endopolyphosphatases and phosphate transporters were upregulated. The changes in polyP metabolism are probably related to the peculiarities of bioenergetics of methanol-grown cells.

Identification and Characterization of Glycosomal and Cytosolic Nudix Hydrolases with Polyphosphate Hydrolyzing Activity in Trypanosoma brucei

Trypanosoma brucei is a protozoan parasite that causes the neglected tropical disease African trypanosomiasis, also known as sleeping sickness. T. brucei infection in humans leads to death if untreated and in cattle causes a disease called Nagana, which has considerable socioeconomic impact in developing nations of sub‐Saharan Africa. T. brucei is well adapted to fight the host immune response as well as various stress conditions it endures in its various hosts. The parasite relies only on glycolysis for ATP production when inside the mammalian host. It activates oxidative phosphorylation once in the fly vector. We are interested in understanding the metabolism and functions of the inorganic phosphate polymer polyphosphate (polyP) in trypanosomes. Previous work from our laboratory reported the importance of polyP for parasite homeostasis and survival. In this work, we identified and characterized the activity of two Nudix hydrolases, NH2 and NH4 that can degrade polyP. We found that NH2 is an exopolyphosphatase with higher activity on short chain polyP, while NH4 is an endopolyphosphatase that has similar activity on polyP of various chain sizes. Both enzymes have higher activity at around pH 8.0. NH2 has a glycosomal targeting signal and it was found previously in the proteome of glycosomes, peroxisome‐related organelles where glycolysis occurs. We confirmed NH2 localization using specific antibodies while endogenously tagged NH4 was localized to the cytosol. We also found that only NH2 can dephosphorylate ATP and ADP. However, NH2 has a higher affinity for polyP, which suggests that polyP could be relevant for glycosomal function. Additionally, NH2 could also participate in regulation of ATP/ADP levels. Furthermore, using the polyP‐binding domain (PPDB) of yeast exopolyphosphatase we localized long chain polyP to the glycosomes of trypanosomes. Our results suggest that Nudix hydrolases can participate in polyP homeostasis and therefore may help control polyP levels in glycosomes and cytosol.Support or Funding InformationNIH (5R01AI077538‐09)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Localization of Short-Chain Polyphosphate Enhances its Ability to Clot Flowing Blood Plasma

Short-chain polyphosphate (polyP) is released from platelets upon platelet activation, but it is not clear if it contributes to thrombosis. PolyP has increased propensity to clot blood with increased polymer length and when localized onto particles, but it is unknown whether spatial localization of short-chain polyP can accelerate clotting of flowing blood. Here, numerical simulations predicted the effect of localization of polyP on clotting under flow, and this was tested in vitro using microfluidics. Synthetic polyP was more effective at triggering clotting of flowing blood plasma when localized on a surface than when solubilized in solution or when localized as nanoparticles, accelerating clotting at 10–200 fold lower concentrations, particularly at low to sub-physiological shear rates typical of where thrombosis occurs in large veins or valves. Thus, sub-micromolar concentrations of short-chain polyP can accelerate clotting of flowing blood plasma under flow at low to sub-physiological shear rates. However, a physiological mechanism for the localization of polyP to platelet or vascular surfaces remains unknown.

The host plant Pinus pinaster exerts specific effects on phosphate efflux and polyphosphate metabolism of the ectomycorrhizal fungus Hebeloma cylindrosporum: a radiotracer, cytological staining and 31P NMR spectroscopy study

Ectomycorrhizal (ECM) association can improve plant phosphorus (P) nutrition. Polyphosphates (polyP) synthesized in distant fungal cells after P uptake may contribute to P supply from the fungus to the host plant if they are hydrolyzed to phosphate in ECM roots then transferred to the host plant when required. In this study, we addressed this hypothesis for the ECM fungus Hebeloma cylindrosporum grown in vitro and incubated without plant or with host (Pinus pinaster) and non-host (Zea mays) plants, using an experimental system simulating the symbiotic interface. We used 32 P labelling to quantify P accumulation and P efflux and in vivo and in vitro nuclear magnetic resonance (NMR) spectroscopy and cytological staining to follow the fate of fungal polyP. Phosphate supply triggered a massive P accumulation as newly synthesized long-chain polyP in H. cylindrosporum if previously grown under P-deficient conditions. P efflux from H. cylindrosporum towards the roots was stimulated by both host and non-host plants. However, the host plant enhanced 32 P release compared with the non-host plant and specifically increased the proportion of short-chain polyP in the interacting mycelia. These results support the existence of specific host plant effects on fungal P metabolism able to provide P in the apoplast of ectomycorrhizal roots.

TcPho91 is a contractile vacuole phosphate sodium symporter that regulates phosphate and polyphosphate metabolism in Trypanosoma cruzi.

We have identified a phosphate transporter (TcPho91) localized to the bladder of the contractile vacuole complex (CVC) of Trypanosoma cruzi, the etiologic agent of Chagas disease. TcPho91 has 12 transmembrane domains, an N-terminal regulatory SPX (named after SYG1, Pho81 and XPR1) domain and an anion permease domain. Functional expression in Xenopus laevis oocytes followed by two-electrode voltage clamp showed that TcPho91 is a low-affinity transporter with a Km for Pi in the millimolar range, and sodium-dependency. Epimastigotes overexpressing TcPho91-green fluorescent protein have significantly higher levels of pyrophosphate (PPi ) and short-chain polyphosphate (polyP), suggesting accumulation of Pi in these cells. Moreover, when overexpressing parasites were maintained in a medium with low Pi , they grew at higher rates than control parasites. Only one allele of TcPho91 in the CL strain encodes for the complete open reading frame, while the other one is truncated encoding for only the N-terminal domain. Taking advantage of this characteristic, knockdown experiments were performed resulting in cells with reduced growth rate as well as a reduction in PPi and short-chain polyP levels. Our results indicate that TcPho91 is a phosphate sodium symporter involved in Pi homeostasis in T. cruzi.

Clotting activity of polyphosphate-functionalized silica nanoparticles.

We present a silica nanoparticle (SNP) functionalized with polyphosphate (polyP) that accelerates the natural clotting process of the body. SNPs initiate the contact pathway of the blood-clotting system; short-chain polyP accelerates the common pathway by the rapid formation of thrombin, which enhances the overall blood-clotting system, both by accelerating fibrin generation and by facilitating the regulatory anticoagulation mechanisms essential for hemostasis. Analysis of the clotting properties of bare SNPs, bare polyP, and polyP-functionalized SNPs in plasma demonstrated that the attachment of polyP to SNPs to form polyP-SNPs creates a substantially enhanced synergistic effect that lowers clotting time and increases thrombin production at low concentrations. PolyP-SNP even retains its clotting function at ambient temperature. The polyP-SNP system has the potential to significantly improve trauma-treatment protocols and outcomes in hospital and prehospital settings.

Trypanosoma brucei Vacuolar Transporter Chaperone 4 (TbVtc4) Is an Acidocalcisome Polyphosphate Kinase Required for in Vivo Infection

Polyphosphate (polyP) is an anionic polymer of orthophosphate groups linked by high energy bonds that typically accumulates in acidic, calcium-rich organelles known as acidocalcisomes. PolyP synthesis in eukaryotes was unclear until it was demonstrated that the protein named Vtc4p (vacuolar transporter chaperone 4) is a long chain polyP kinase that localizes to the yeast vacuole. Here, we report that TbVtc4 (Vtc4 ortholog of Trypanosoma brucei) encodes, in contrast, a short chain polyP kinase that localizes to acidocalcisomes. The subcellular localization of TbVtc4 was demonstrated by fluorescence and electron microscopy of cell lines expressing TbVtc4 in its endogenous locus fused to an epitope tag and by purified polyclonal antibodies against TbVtc4. Recombinant TbVtc4 was expressed in bacteria, and polyP kinase activity was assayed in vitro. The in vitro growth of conditional knock-out bloodstream form trypanosomes (TbVtc4-KO) was significantly affected relative to the parental cell line. This mutant had reduced polyP kinase activity and short chain polyP content and was considerably less virulent in mice. The wild-type phenotype was recovered when an ectopic copy of the TbVtc4 gene was expressed in the presence of doxycycline. The mutant also exhibited a defect in volume recovery under osmotic stress conditions in vitro, underscoring the relevance of polyP in osmoregulation.

Yolk hydrolases in the eggs of Anticarsia gemmatalis hubner (Lepidoptera: Noctuidae): A role for inorganic polyphosphate towards yolk mobilization

Despite being the main insect pest on soybean crops in the Americas, very few studies have approached the general biology of the lepidopteran Anticarsia gemmatalis and there is a paucity of studies with embryo formation and yolk mobilization in this species. In the present work, we identified an acid phosphatase activity in the eggs of A. gemmatalis (agAP) that we further characterized by means of biochemistry and cell biology experiments. By testing several candidate substrates, this enzyme proved chiefly active with phosphotyrosine; in vitro assays suggested a link between agAP activity and dephosphorylation of egg yolk phosphotyrosine. We also detected strong activity with endogenous and exogenous short chain polyphosphates (PolyP), which are polymers of phosphate residues involved in a number of physiological processes. Both agAP activity and PolyP were shown to initially concentrate in small vesicles clearly distinct from typically larger yolk granules, suggesting subcellular compartmentalization. As PolyP has been implicated in inhibition of yolk proteases, we performed in vitro enzymatic assays with a cysteine protease to test whether it would be inhibited by PolyP. This cysteine protease is prominent in Anticarsia egg homogenates. Accordingly, short chain PolyP was a potent inhibitor of cysteine protease. We thereby suggest that PolyP hydrolysis by agAP is a triggering mechanism of yolk mobilization in A. gemmatalis.

Factor XII promotes blood coagulation independent of factor XI in the presence of long‐chain polyphosphates

Inorganic polyphosphates (polyP), which are secreted by activated platelets (short-chain polyP) and accumulate in some bacteria (long-chain polyP), support the contact activation of factor XII (FXII) and accelerate the activation of FXI. The aim of the present study was to evaluate the role of FXI in polyP-mediated coagulation activation and experimental thrombus formation. Pretreatment of plasma with antibodies that selectively inhibit FXI activation by activated FXII (FXIIa) or FIX) activation by activated FXI (FXIa) were not able to inhibit the procoagulant effect of long or short-chain polyP in plasma. In contrast, the FXIIa inhibitor, corn trypsin inhibitor, blocked the procoagulant effect of long and short polyP in plasma. In a purified system, long polyP significantly enhanced the rate of FXII and prekallikrein activation and the activation of FXI by thrombin but not by FXIIa. In FXI-deficient plasma, long polyP promoted clotting of plasma in an FIX-dependent manner. In a purified system, the activation of FXII and prekallikrein by long polyP promoted FIX activation and prothombin activation. In an ex vivo model of occlusive thrombus formation, inhibition of FXIIa with corn trypsin inhibitor but not of FXI with a neutralizing antibodies abolished the prothrombotic effect of long polyP. We propose that long polyP promotes FXII-mediated blood coagulation bypassing FXI. Accordingly, some polyp-containing pathogens may have evolved strategies to exploit polyP-initiated FXII activation for virulence, and selective inhibition of FXII may improve the host response to pathogens.