Inorganic Polyphosphate in the Microbial World. Emerging Roles for a Multifaceted Biopolymer

Inorganic Polyphosphate in the Microbial World. Emerging Roles for a Multifaceted Biopolymer Tomás Albi and Aurelio Serrano* Instituto de Bioquímica Vegetal y Fotosíntesis, Centro de Investigaciones Científicas Isla Cartuja, CSIC y Universidad de Sevilla, Av. Américo Vespucio 49, 41092 Sevilla, SPAIN *To whom correspondence should be addressed: Dr. Aurelio Serrano, Institute for Plant Biochemistry and Photosynthesis, CSIC and University of Seville, Av. Américo Vespucio 49, 41092 Seville, SPAIN. Phone: ++ 34 95 4489524. Fax: ++ 34 95 4460165. E-mail: [email protected]. Web: http://www.ibvf.csic.es/en/bioenergetics-phosphate ABSTRACT Inorganic polyphosphates (polyP) are linear polymers of tens to hundreds orthophosphate residues linked by phosphoanhydride bonds. These fairly abundant biopolymers occur in all extant forms of life, from prokaryotes to mammals, and could have played a relevant role in prebiotic evolution. Since the first identification of polyP deposits as metachromatic or volutin granules in yeasts in the 19th century, an increasing number of varied physiological functions have been reported. Due to their “high energy” bonds analogous to those in ATP and their properties as polyanions, polyP serve as microbial phosphagens for a variety of biochemical reactions, as a buffer against alkalis, as a storage of Ca2+ and as a metal-chelating agent. In addition, recent studies have revealed polyP importance in signaling and regulatory processes, cell viability and proliferation, pathogen virulence, as a structural component and chemical chaperone, and as modulator of microbial stress response. This review summarizes the current status of knowledge and future perspectives of polyP functions and their related enzymes in the microbial world. Revised Manuscript Click here to download Manuscript ManuscriptFINALREVISED.docx Click here to view linked References World Journal of Microbiology and Biotechnology - ACCEPTED REVIEW - 5 Nov 2015 KEYWORDS Inorganic polyphosphate; Cation chelator; Nutrient deficiency; Stress Protection; Cell signaling; Chemical chaperone. ACKNOWLEDGMENTS The authors are thankful to all researchers whose papers have been used for this review, as well as to those others that were not cited because of limited space. Part of this work was supported by research grants from the Spanish (BFU2004-00843, BFU2007-61887, BFU2010-15622) and Andalusian Regional (PAIDI group BIO-261) Governments, all of them partially funded by the EU FEDER program. PAIDI group BIO-261 belongs to the CeiA3 and AndaluciaTECH University Campuses of International Excellence. Authors thank Dr. M. R. Gómez-García for helpful suggestions and discussions. PolyP Chemistry and Biochemistry. Enzymes Involved in PolyP Synthesis and Degradation. 1. Structure and Chemical Composition Inorganic polyphosphates, polyP, are polymers of orthophosphate (Pi) residues linked by phosphoanhydride P-O-P bonds . They are often termed as “condensed phosphates” since they are composed by several Pi units (from three up to thousands) connected by oxygen bridges. Contrary to long-chained polyP, which are poorly soluble in water, the majority of polyP are stable in neutral aqueous solutions even at hight temperatures. Considering their chemical structure (Kulaev et al. 2005) polyP are divided into three classes: cyclic condensed phosphates (also referred as metaphosphates, PnO3nn-, whose simplest member is cyclictriphosphate), linear polyphosphates (or linear metaphosphates, PnO3n+1 (n+2)-, whose shortest component is tripolyphosphate), and lastly, the “ultraphosphates” or branched polyphosphates (Fig.1). PolyP is perhaps one of the biopolymers with the highest density of negative charge. Its analogous structure to the RNA and other polyanions leads to comparable reactivity. For instance, both polymers increase fluorescence of DAPI, which can potentially provoke misinterpretations. As a result, the development of new polyP specific sensitive and selective detection techniques, and its application was critical for further progress on polyP research (Angelova et al. 2014). In fact, in spite of their discovery in the end of the 19th century (Babes 1895) and their wide occurrence, polyP was largely dismissed as a “molecular fossil” (Kornberg 1999). Fortunately, these recent studies have revealed the real physiological importance of polyP, starting an emerging interest in polyP research. 2. Natural Occurrence of PolyP Polyphosphate is ubiquitous in living beings having being found in archaea, bacteria, algae, fungi, protists, plants, insects and mammals (Brown and Kornberg 2004; Docampo et al. 2005a; Rao et al. 2009). PolyP reserves were formely discovered in bacteria and unicellylar eukaryotes being dennoted as metachromatic or volutin granules due to their metachromatic effect - they appear red when stained with methylene blue. Later, once polyP was proven as one of its main components, they were also referred as polyphosphate granules o acidocalcisomes (Docampo et al. 2005a). Besides this widely distribution among living organisms, the amount and chemical structure of polyP reserves may vary depending the species and the particular growth conditions. In general, prokaryotes and protists are able to accumulate polyP at higher rates than multicellular eukaryotes. 3. Enzymes Involved in PolyP Synthesis Synthesis of PolyP in Prokaryotic Microorganisms: Polyphosphate Kinase Most studies concerning proteins involved in polyP biosynthesis have been focused on microorganisms, namely bacteria, including pathogenic and phosphate-accumulating strains, yeasts and parasitic protists. Based on these findings, some orthologs have been identified in microorganisms of other taxonomic groups. Nevertheless, to date there are still numerous organisms with no archetypical orthologs identifed so far, in spite of being able to accumulate high polyP levels. Consequently, it is deduced that they should have alternative pathways for polyP synthesis. In prokaryotes -and in some microbial eukaryotes as well (Zhang et al. 2007)- polyP is mainly sinthesized by polyphosphate kinase 1 (PPK1; polyphosphate:ADP phosphotransferase, EC 2.7.4.1), which catalyzes the reversible transfer of the energy-rich γ-phosphate from ATP to enlongate the polyP chain. PolyPn + ATP ↔ PolyPn+1 + ADP PPK1 (Pfam PF02503) is a member of the phosphotransferases superfamily, and exhibits other enzymatic activities including ATP synthesis from polyP, nucleoside-diphosphate kinase, guanosine 5′-tetraphosphate synthesis and autophosphorylation (Tzeng and Kornberg 2000). A genomes screening using the BLAST engine revealed ppk1 homologs in more than 354 prokaryotes (Tzeng and Kornberg 1998). However, no ppk1 homologs have been identified so far in higher eukaryotes, both higher plants and animals. Moreover, various studies have proved the importance of ppk1, and PPK1 has been shown to be an essential enzyme. Lack of PPK1 severely compromised cell viability of many bacteria under stationary-growth phase and their effective responses to a wide range of stress factors, such as heat, UV light, pH, antibiotics, etc. Similarly, bacterial mutants lacking PPK1 are defective in cell motility, quorum sensing, biofilm formation and virulence, and show ultrastructural defects (Brown and Kornberg 2008; Fraley et al. 2007; Rashid et al. 2000b; Sanyal et al. 2013). As a result, ppk1 has been proposed as a novel target for next generation antibiotics. Nonetheless, PPK1 is not the sole enzyme responsible for polyP synthesis. In particular, high Mm polyP were identified in ppk1-lacking null mutants of Pseudomonas aeruginosa (Ishige et al. 2002). The alternative enzyme was called PPK2 (Pfam PF03976). Similarly to ppk1, ppk2 is absent in plants and metazoans and has been claimed to have a role in virulence of bacterial pathogens, in connection with alginate synthesis and biofilm formation, being then considered as an attractive target for antibiotics. However, PPK2 is frequently a polyP-degrading enzyme since its capacity to use polyP for GTP synthesis is 75-fold greater than its Poly P synthetic activity from GTP (Ishige et al. 2002). PPK2 can also serve as a PolyP:AMP phosphotransferase (EC 2.7.4.B2) and PolyP:ADP phosphotransferase (Ishige and Noguchi 2000): PolyPn + AMP ↔ PolyPn-1 + ADP Conversely to PPK1, PPK2 is no strictly specific for ATP and it is able to efficiently use either GTP or ATP. Many microbial genomes encode multiple ppk2 paralogs (Zhang et al. 2002). In fact, there are probably three subfamilies of PPK2 enzymes containing a single or two homologous PPK2 domains. Thus, whereas class I PPK2 is monodomain and catalyzes NTP synthesis from NDP, classes II and III are bi-domain PPK2 enzymes which catalyse the synthesis of NMP, or both NMP and NDP, respectively (Motomura et al. 2014). Likewise ppk1, ppk2 widely occur among prokaryotic microorganisms, and hundreds of ppk2 homologs have been identified to date. However, many bacteria should synthesize polyP by unknown enzymes, since one-third of bacterial species known so far lack both ppk1 and ppk2 (Whitehead et al. 2014). Synthesis of PolyP in Protists: Arp and VTC Proteins In the slime mold Dictyostellium discoideum a new type of PPK, named DdPPK2, was identified. This enzyme is a complex of three actin-related proteins (Arp), which can polymerize into an actin-like filament concurrently with the reversible synthesis of polyP chain from ATP (Gomez-Garcia and Kornberg 2004; Spudich 2004). In yeast and trypanosomes (Lander et al. 2013) an alternative pathway responsible for polyP synthesis that involves VTC4, a subunit of the vacuolar transport chaperone (VTC) complex, has been described. VTC4 is a member of the Conserved Protein Domain family VTC (Pfam PF09359), which belongs to the CYTH-like phosphatases superfamily (cl11964). S. cerevisiae VTC complex is also involved in several other cellular processes, like vacuolar-membrane fusion (Hothorn et al. 2009; Ogawa et al. 2000a; Uttenweiler et al. 2007), microautophagy (Cohen et al. 1999; Hothorn et al. 2009; Muller et al. 2002; Ogawa et al. 2000a; Uttenweiler et al. 2007). Homologs of VTC4 have been inferred in the genomes of apicomplexan protists, fungi and microalgae (Aksoy et al. 2014). Chlamydomonas reinhardtii VTC1 is required for polyP synthesis and polyP granule accumulation in acidocalcisomes. A deficient acidocalcisome formation in protistan cells deprived of N, P, or mainly S, may impact various function associated with energetics, trafficking of periplasmic proteins and regulation of cellular processes (Aksoy et al. 2014; Moreno and Docampo 2013). Other Enzymes for PolyP Synthesis The dolichyl diphosphate:polyphosphate phosphotransferase (EC 2.7.4.20) was related to the synthesis of the small fraction of polyP associated with the vacuolar membrane of Saccharomyces cerevisiae (Schomburg and Stephan 1997), and performs the following reaction: Dolichyl diphosphate + PolyPn → Dolichyl phosphate + PolyPn-1 Lastly, an 3-phospho-D-glycerol-phosphate:polyphosphate phosphotransferase (EC 2.7.4.17) was found in the fungus Neurospora crassa (Kukaev et al. 1971). The enzyme, which has not been purified and needs further investigations, catalyzes the following reaction: 3-Phospho-D-glycerol-1-phosphate + PolyPn → 3-Phosphoglycerate + PolyPn+1 4. Enzymes that Degrade PolyP Exopolyphosphatase and Guanosine Pentaphosphate Hydrolase The main enzyme responsible for polyP usage in microorganisms is the exopolyphosphatase (PPX; Polyphosphate phosphohydrolase, EC 3.6.1.11). PPX hydrolyzes and processively splits Pi from the end of the polyP chain: PolyPn + H2O → PolyPn-1 + Pi Two major non-homologous classes of PPX are defined based on their primary structure. A first PPX class is established by the archetypical exopolyphosphatase PPX1, first identified in Saccharomyces cerevisiae, and their orthologues later described in yeasts, other fungi and protists. PPX1 belongs to the superfamily of DHHDHHA2 phosphoesterases (Pfam PF02833), which also includes the prokaryotic family II pyrophosphatases (Young et al. 1998) and the Nudix hydrolase family (Lonetti et al. 2011). Some of these Nudix proteins, such as the human protein h-prune, a binding protein of the metastasis suppressor nm23-H1, have been proved to efficiently hydrolyze polyP (Tammenkoski et al. 2008). The human protein h-prune and the yeast PPX1 proteins share a high sequence identity (27%). PPX1 is an extremely active phoshohydrolase which can hydrolyze polyP, adenosine tetraphosphate and GPT; but does not hydrolyze PPi or NTPs. A second exopolyphosphatase class includes the Ppx-GppA polyphosphatases (Pfam PF02541) which belong to the sugar kinase/actin/hsp 70 superfamily. Ppx-GppA exopolyphosphatases are widely distributed among bacteria and archaea, processively hydrolyse linear polyP of 3 up to thousands of Pi residues, and also have nucleoside triphosphatase (NTPase) activity (Albi and Serrano 2014). Thus, prokaryotic PPXs and eukaryotic (fungal/protistan) PPXs belong to different families of polyphosphatases and do not have structural similarity. In addition, bacteria posses another Ppx-GppA exopolyphosphatase sharing ca 40% sequence similarity with its archetypical prokaryotic paralog and catalytically less efficient than the latter, the guanosine pentaphosphate phosphohydrolase (GppA, EC 3.6.1.40) which also catalyzes the following reaction: Guanosine 5’-triphosphate, 3’-diphosphate → Guanosine 5’-diphosphate,3’-diphosphate + Pi Endopolyphosphatase Besides PPX1 exopolyphosphatase, yeasts, fungi and protists also posses an endopolyphosphatase enzyme. The archetypical endopolyphosphatase of S. cerevisiae (PPN1; Polyphosphate depolymerase, EC 3.6.1.10) is a transmembrane bitopic protein which belong to the Calcineurin-like phosphoesterase superfamily (Pfam PF00149), and cleaves long polyP into shorter polyP molecules without releasing Pi (Sethuraman et al. 2001): PolyPn + H2O → oligopolyphosphates In rich growth conditions, yeast PPN1 acts as an endopolyphosphatase in the presence of Mg2+. However, under certain stress conditions, such as toxic heavy-metals, PPN1 shifts to an Co2+-dependent exopolyphosphatase activity (Andreeva et al. 2015). Functions of PolyP in Microorganisms 1. PolyP as a Structural Component One of the most exciting chemical features of polyP is its strong ability to interact with a variety of inorganic and organic compounds resulting in ternary complexes. Thus, polyP (with and average length of 150 Pi residues) can form complexes with poly-β-hydroxybutyrate (mean size of 170) an Ca2+ ions. In the proposed structure, poly-β-hydroxybutyrate (PHB) corresponds to the outer layer which is directly in contact with the lipid membrane. Inside, PHB ester groups are bonded to polyP by ionic interactions and to Ca2+ by ion dipoles (Reusch and Sadoff 1988). The polyP/Ca2+/PHB complex has been detected in the plasma membrane of many naturally competent bacteria, and in various subcellular compartments of eukaryotes (Reusch 1989). This ternary complex constitutes a transient channel increasing membrane permeability - selective for Ca2+ ions - which probably plays an important physiological role in competence for DNA entry and transformation (Castuma et al. 1995). Further researches in mammals support this assumption (Dedkova and Blatter 2014). It has been recently review that polyP may be a strong activator of the mitochondrial permeability transition pore in cardiomycetes, heart muscle cells, playing a structural role in their mitochondria membrane systems (Dedkova and Blatter 2014; Seidlmayer et al. 2012a; Seidlmayer et al. 2012b). 2. PolyP as a Substitute for ATP As phosphorylated compound with a Gibbs free energy of hydrolysis similar to ATP (-30.5 kJ mol−1), polyP may act as a substitute for ATP in diverse enzymatic reactions (Kornberg et al. 1999). Donor for Sugars: PolyP-gluco(manno)kinase Polyphosphate gluco(manno)kinase (PPGK; Polyphosphate-glucose phosphotransferase, EC 2.7.1.63), a member of the ROK (Repressor-ORF-Kinase) superfamily (Pfam PF00480), catalyzes the phosphorylation of monosaccharides –glucose, mannose, and in some cases fructose– using polyP or ATP as a phosphoryl donor. PolyPn + D-glucose → PolyPn-1 + D-glucose-6-phosphate This enzymatic activity has been reported in a variety of phylogenetically different bacteria, including important pathogens (Szymona and Ostrowski 1964; Szymona and Szymona 1978), bacteria of activated sludge (Tanaka et al. 2003), and N2-fixing cyanobacteria (Albi and Serrano 2015). Donor for Adenylate Kinases: NAD Kinase NAD kinase ((polyP)/ATP:NAD 2’-phosphotransferase, EC 2.7.1.23; Pfam PF01513) catalyzes the phosphorylation of NAD to yield NADP. In some prokaryotes, NAD kinases use either ATP or polyP as phosphoryl donors: ATP + NAD → ADP + NADP PolyPn + NAD → PolyPn-1 + NADP PolyP have been postulated as the precursor of ATP and the primitive energy donor in the origin of life (Kornberg 1995; Lipmann 1965). Similarly to ATP, polyP is composed by high-energy phosphate groups and it was likely present on prebiotic earth. Moreover, polyP formed by high pressure and desiccation (phosphate condensation) might have been naturally abundant (sedimentary rocks, hydrothermal vents, volcanic exudates) in the early earth (Lohrmann and Orgel 1968; Miller and Parris 1964). Based on their comparable chemical properties and its ubiquity, we should not exclude a role for polyP in energy protometabolism of primordial cells (Achbergerova and Nahalka 2011; Brown and Kornberg 2004). Furthermore, subsequent biochemical studies on bacterial NAD kinases and glucokinases revealed that some of them are active with polyP. In particular, polyP can really substitute for ATP, and some enzymes are even strictly dependent on polyP. This hypothesis is substantiated by several later observations from the biochemical properties of NAD kinases and glucokinases: most bacterial enzymes are able to use both phosphoryl donors with an observed progressive decrease in the preference for polyP in the phylogenetically newer taxa (Albi and Serrano 2015). stress responses (Maciag et al. 2011). σ38 also controls the alginate production as well as swimming and twitching motility in Pseudomonas aeruginosa. Similarly, the σE regulon in Mycobacterium spp. determines the response to oxidative and phosphate-limiting stresses and is also required for virulence and persistence of this pathogen (Manganelli et al. 2001). 9. Stress Protection Microbial strategies in response to oxidative and other stresses comprise several mechanisms on posttranslational control, redox-regulated adjustment of cellular metabolism, and the activation of specific molecular chaperones. Recently, it was reported that polyP exerts as a functional protein-protective chemical chaperone at physiological levels (Gray et al. 2014). Thus, polyP is able to stabilize a broad range of proteins maintaining their competent conformations, preventing them from unfolding and aggregation. In addition, bacterial ppk-null mutants suffer from higher protein damage than the wild-type strain upon a similar proteotoxic stress. Besides, polyP may have some advantages compared to other chaperones since it does not react with oxidants, such as HClO, and does not require ATP hydrolysis for its protective activity (Kampinga 2014). On the other hand, polyP enhance Vibrio cholerae cells tolerance to environmental stresses in Pi-limiting conditions (Jahid et al. 2006), and ppk-null mutants of Lactobacillus spp. (Alcantara et al. 2014) and E. coli (Gray et al. 2014), which are not able to produce polyP, show reduced growth or are more sensitive to acidic pH, ethanol, heat, high-salt and oxidative stresses. Furthermore, it has been reported that polyP production by bacteria of the human gastrointestinal tract protects the intestinal epithelia from oxidative stress (Segawa et al. 2011). To summarize, these results corroborate the important contribution of polyP to the regulation of growth, cell survival and stress tolerance of many microorganisms. PROSPECT Despite the identification of polyP at the end of the nineteenth century and its extensive occurrence covering the whole evolutionary lineages, polyP was largely dismissed and forgotten during decades. Recently, there is an increasing interest in this polymer, which goes hand-in-hand with the revelation of its unexpected and intriguing involvement in critical cellular functions in prokaryotes and eukaryotes. Thanks to these studies, it has become evident an active association between polyP and many physiological processes of paramount importance for microorganisms, such as multilayer metabolic regulation, stress responses, pathogen resistance, etc. In a broader context, polyP was also recently reported to be involved in a variety of biological processes related to human health, such as cardiac ischaemia, blood coagulation, apoptosis and stress-induced cell death (Dedkova and Blatter 2014; Hernandez-Ruiz et al. 2006; Seidlmayer et al. 2012b), suggesting its therapeutic use. 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Representative schematic structures of the three main polyPs structural classes. (a) Linear polyphosphates, (b) cyclic polyphosphates (also named metaphosphates), and (c) branched polyphosphates (also named ultraphosphates). Figure 2. Functions of polyPs in prokaryotic and eukaryotic microorganisms. In most cases the indicated functions have been reported for both prokaryotes (bacteria, archaea) and eukaryotes (fungi, microalgae and parasitic and free-living heterotrophic protists). Fig. 1 P O P O O O O P O O O O O n Linear polyphosphate (a) P O P O O P O O O O O P O O O P Branched polyphosphate (ultraphosphate) (c) Cyclic tripolyphosphate (metaphosphate) (b) P O O O P P O O O O O O Figure 1 Click here to download Figure Figure 1.pdf