Perspective. Abundant Microbial Inorganic Polyphosphate, Poly P Kinase Are Underappreciated

Transcription

1 Abundant Microbial Inorganic Polyphosphate, Poly P Kinase Are Underappreciated Although some view poly P as a mere molecular fossil, the author thought that it plays a more prominent role in microorganisms Arthur Kornberg norganic polyphosphate (poly P) molecules, linear chains containing hundreds I of orthophosphate (P i ) residues linked by high-energy phosphoanhydride bonds (Fig. 1A), are abundant in every cell in nature. These molecules are widely distributed among bacteria, including key pathogens. In eukaryotes, poly P is present in organelles, including nuclei, mitochondria, and vesicles. Summary Inorganic polyphosphate (poly P) molecules and the poly P kinase (PPK1 and PPK2) enzymes that make it are widely distributed among microorganisms and appear to be virulence factors for many pathogens. Several bacterial species with mutations in ppk1 or its equivalent are defective in growth and survival, motility, quorum sensing, biofilm formation, and in a variety of responses to stress. Because poly P and PPK1 and PPK2 appear necessary for virulence, they might be targets for the development of novel treatments. Cells of the social slime mold Dictyostelium discoideum produces a bacterial PPK1 like enzyme (DdPPK1) that, if mutated, are defective in development, sporulation, predation, and in the late stages of cytokinesis and cell division. DdPPK2, an unusual poly P kinase from D. discoideum, consists of more than 100 globular units, each a tetramer of three distinctive actinrelated proteins. Formed billions of years ago from phosphate rock by dehydration at elevated temperature, Poly P was likely a catalyst and precursor in prebiotic syntheses. Our approach to learning about the physiological roles of poly P entails identifying and analyzing the enzymes that make and use this material, while manipulating and studying the genes that encode those enzymes. Synthetic Enzymes, Particularly Poly P Kinase 1 and 2, Play Vital Roles in Bacteria Although poly P kinase (PPK1), the enzyme that catalyzes the synthesis of poly P (Fig. 1B), was first identified in Escherichia coli, it is widely conserved in other species of bacteria. A second poly P kinase (PPK2) is also widely distributed. Several bacterial species with mutations in ppk1 or its equivalent are defective in growth and survival, motility, quorum sensing, biofilm formation, and in a variety of responses to stress. For instance, all the ppk1 mutants of gram-negative or grampositive bacteria tested, and even those among simpler eukaryotes such as slime molds, exhibit a severe defect in stress response and growth, either short or long term. Among several pathogens tested, PPK1 mutants proved to be avirulent. These include Pseudomonas aeruginosa, Salmonella sp., Shigella sp., Vibrio cholerae, and Helicobacter pylori. Thus, PPK1 is a potential target for antimicrobial drugs. Arthur Kornberg was Professor of Biochemistry, Emeritus, in the Department of Biochemistry, Stanford University School of Medicine, Stanford, Calif., when this article was written in the fall of Dr. Kornberg died on 26 October Perspective Volume 3, Number 3, 2008 / Microbe Y 119

2 FIGURE 1 Need for Poly P and PPK1 in Virulence PPK1 and poly P appear to be required for virulence. For example, ppk1 mutants of Salmonella spp. show drastically reduced acid tolerance, lowered polymyxin B resistance, and diminished invasiveness in epithelial cells; their survival in macrophage cells also is severely compromised. Similar mutants of Shigella flexneri, which causes bacillary dysentery, become highly acid sensitive and fail the Sereny test, which evaluates bacterial invasiveness by measuring whether bacteria cause conjunctivitis when introduced into guinea pig eyes. Similar mutants of V. cholerae are not only acid-sensitive but also stationary-phase defective. Mutants of Neisseria meningitidis become highly sensitive to killing by human sera compared to wild-type cells; when complemented with intact PPK1, serum resistance is restored. Mycobacterium tuberculosis, in addition to having a PPK1 active site that is virtually identical to those in several other bacterial pathogens, needs PPK and poly P for growth and survival; mutants in PPK1 fail in their responses to a variety of stringencies and stresses. When ppk1 expression in M. tuberculosis is attenuated through expression of the gene in antisense orientation under control of the hsp60 promoter, survival of these mutants decreases in macrophages. Meanwhile, the PPK1 mutant of P. aeruginosa is aberrant in quorum sensing and in biofilm formation and is defective in producing several virulence factors, including elastase, rhamnolipid, and iron-siderophores. These mutants are not virulent when inoculated onto burned mice, whose tissues the mutants, unlike wild-type cells, cannot colonize. Poly P Kinase (PPK1) catalyzes the reversible synthesis of Poly P from ATP. The enzyme is specific for ATP and ADP. Some Details Describing PPK1, PPK2 in Bacteria and Other Microorganisms During the past decade, the list of bacterial species with PPK1 and PPK2 has lengthened to more than 100 and includes many pathogens (Table 1). The structure of PPK1, including its ATP-binding site, was determined at atomic resolution (Fig. 2). The site, which ordinarily binds ATP, is occupied by the analog AMPPNP, and it is in line with the tunnel, in which the growing chain of poly P is held, that is occupied by tripolyphosphate (P 3 ). This structural information should help investigators to design compounds that block this binding site. In addition, a number of inhibitory compounds were identified by screening libraries of compounds. The absence of the PPK1- active site in animal cells makes the toxicity of such inhibitory drugs unlikely. Meanwhile, the PPK2 sequence is also found in more than 100 bacterial species, including major pathogens (Table 1). Among them are examples of bacterial species with both PPK1 and PPK2 sequences along with a few having only PPK2. The sequence is found widely, including in archaea, rhizobia, cyanobacteria, and Streptomyces spp. Little or nothing is known about the putative enzyme encoded by the ppk2 genes in many of these species. In the case of P. aeruginosa, however, PPK2 differs from PPK1 in being able to synthesize poly P from either ATP or GTP; it also has a strong kinetic preference to serve as a nucleoside diphosphate kinase (NDK) with poly P as donor. The PPK2 of P. aeruginosa is noteworthy for another reason this enzyme was first noted in null mutants lacking PPK1 that nonetheless still could make poly P. The isolated PPK2 proved to be a potent poly P-driven generator of GTP because, unlike PPK1, PPK2 accepts GDP as a substrate. PPK2 appears during the stationary phase of growth of P. aeruginosa. Synthesis of GTP from poly P is 75-fold greater than the reverse reaction, which generates poly P from GTP. Both PPK2 and the classic nucleoside diphosphate kinase may generate guanosine precursors 120 Y Microbe / Volume 3, Number 3, 2008

3 used for making alginate, the exopolysaccharide that contributes to the virulence of P. aeruginosa in the lungs of patients with cystic fibrosis. This prominent role in virulence for P. aeruginosa raises the question of whether the PPK2 of other pathogens such as M. tuberculosis plays a role in making mycolic acid or otherwise is involved in pathogenesis. We do know that poly P is abundant in M. tuberculosis cells. Thus, PPK2 along with PPK1 may provide targets for the development of novel therapeutic drugs. Table 1. PPK1 and PPK2 homologs a among bacterial pathogens Species with PPK1 homologs Species with PPK2 homologs Species with PPK1 and PPK2 homologs Bordetella pertusis Bordetella bronchiseptica Bacillus anthracis Helicobacter pylori Bordetella parapertusis Brucella melitensis Legionella pneumophila Bacillus thuringiensis Mycobacterium tuberculosis Mycobacterium leprae Corynebacteriuim Pseudomonas aeruginosa diphtheriae Neisseria meningitidis Francisella tularensis Staphylococcus haemolyticus Salmonella enterica Neisseria gonorrhoeae Vibrio cholerae spp. typhi Shigella dysenteriae Staphylococcus aureus Yersinia pestis PPK1 and PPK2 Sequences in Dictyostelium discoideum and Other Eukaryotes In the social slime mold Dictyostelium discoideum, the bacterial PPK1 like enzyme (DdPPK1) contains an unusual N-terminal extension that is essential for catalytic activity, determines cellular localization, and sets other physiological functions. Mutants of DdPPK1 are defective in development, sporulation, predation, and in the late stages of cytokinesis and cell division. Meanwhile, many PPK1 and PPK2 sequences that were once thought to be found only among bacterial species, D. discoideum, and plasmodial pathogens, instead appear to be conserved among a wide variety of eukaryotic species (Table 2). Phagocytosis and predation are physiologic features that can affect virulence. Compared to wild-type D. discoideum, DdPPK1 mutants are less effective at taking up and digesting Klebsiella aerogenes cells. In separate experiments, when Dictyostelium is placed on a lawn of P. aeruginosa mutant cells containing a defective PPK1, the slime mold produces clear plaques. However, if the P. aeruginosa cells contain an intact form of PPK1, they consume the Dictyostelium. Dictyostelium mutants that contain defective PPK1 retain significant levels of poly P because those cells continue to produce DdPPK2. The latter enzyme is unusual, consisting of more than 100 globular units, each a tetramer of three distinctive actin-related proteins (Arps) that are similar to the actin molecules in mammalian muscle cells in terms of their size and globular-filamentous a Based on BLAST search with E. coli PPK1 and P. aeruginosa PPK2 of the NCBI microbial protein/genome database for homologs with an E value structural transitions. Actins, a family of widely conserved proteins, interact with members of the myosin family and contribute to cellular motility. The three Arps of DdPPK2 have a 60% amino acid identity with muscle actin. Of those three, Arp1 is the major subunit in the multisubunit dynactin complex, initially identified as a factor that promotes dynein mediated movement of membrane vesicles along microtubules. Arp2 is recognized as part of a multisubunit complex whose role in cell motility involves initiating polymerization of new actin filaments. Little is known about the third Arp in DdPPK2, Act28, which was identified only recently. Mutants of the Arps in DdPPK2 have several phenotypes. For instance, Arp1 is defective in growth, sporulation, and germination; Table 2. PPK1 and PPK2 homologs a among Eukaryota Species with PPK1 homologs Aedes aegypti Anopheles gambiae Caenorhabditis remanei Candida humicola Dictyostelium discoideum Plasmodium yoelii yoelii Strongylocentrotus purpuratus Species with PPK2 homologs Aedes aegypti Anopheles gambiae Bombyx mori Drosophila simulans Plasmodium yoelii yoelii a Based on BLAST search with E. coli PPK1 and P. aeruginosa PPK2 of the NCBI Eukaryota protein/genome database for homologs with an E value Volume 3, Number 3, 2008 / Microbe Y 121

4 FIGURE 2 The active site of E. coli PPK1, including bound substrate, in this case AMP-PNP, and the putative Poly P tunnel. The blue and red shadings indicate basic and acidic residues/ regions, respectively. P3 indicates the growing chain of Poly P. The data are kindly provided by Dr. Wenqing Xu at the University of Washington, Seattle and Dr. Sam S. K. Lee, then at ICOS Corporation, Bothell, Washington. Arp2 is not viable; Act28 is defective in sporulation and germination. Synthesis of a poly P chain from ATP is concurrent at each step with elongation of the enzyme by one globular subunit and is readily reversible. DdPPK2 is localized in vacuoles, called acidocalcisomes. They are rich in calcium and poly P, and are responsible for the flux of calcium ions in cells. Acidocalcisomes are prominent in pathogenic protozoa such as Trypanosoma cruzi, Leishmania major, and Toxoplasma gondii, as well as the green alga Chlamydomonas. DdPPK2 may provide a target for the treatment of protozoal diseases. Perspective The view that poly P plays a key role in many species is strengthened and extended at every turn. Among these species are bacteria and protozoa that are the agents of major diseases, raising the possibility that poly P and the enzymes that make it are responsible for virulence in some cases and, thus, could be targets for the development of novel treatments. In studying the functions of poly P, we focused on identifying and characterizing the enzymes that make and use it. Except in a single instance so far in which GTP substitutes for ATP, the latter molecule is key to forming poly P. The absence of such an enzyme in yeast and higher eukaryotes, and the lack of a sequence to encode it, raises the possibility that poly P is made by other enzymes. For instance, when an inhibitor of oxidative phosphorylation is added to acidocalcisomes, poly P is no longer made from P i, suggesting that a proton motive force (PMP) may bypass ATP as an intermediate. Poly P was abundant long before other biologic molecules. By virtue of its capacity as a potent phosphorylating agent, poly P is a plausible source of energy and phosphate in the early evolution of our phosphate world. Because it is a potent chelator of metal ions, it might be involved in a variety of reactions that exploit Mg 2, Mn 2,Ca 2,Fe 2, and Co 2, including those that help to maintain ATP levels in cells. And, as a metabolic regulator, poly P could be involved in homeostasis of P i, metals, and essential nutrients in the face of environmental stresses. Poly P plays essential roles in the virulence of major diseases such as dysentery, tuberculosis, and anthrax; in apoptosis; in the proliferative aspects of cancer; and in osteoporosis and aging. However, not only is poly P often absent from texts on biology and chemistry but, even when noticed, tends to be dismissed as a molecular fossil. Biologists should reflect on this attitude and consider how to move poly P from its lonely outpost to a more prominent place along the frontiers of science. ACKNOWLEDGMENT I am grateful to Narayana Rao, Senior Research Scientist, for his skillful searches of databases that disclosed the abundance of poly P kinase sequences cited in Tables 1 and Y Microbe / Volume 3, Number 3, 2008

5 EDITOR S NOTE This article was submitted by Arthur Kornberg shortly before his death at age 89 on 26 October An obituary for Arthur Kornberg appears on p 148. An article on Kornberg s work with poly P is available online at fall/kornberg.html. SUGGESTED READING Brown, M. R. W., and A. Kornberg Inorganic polyphosphate in the origin and survival of species. Proc. Natl. Acad. Sci. USA 101: Kornberg, A., N. N. Rao, and D. Ault-Riché Inorganic polyphosphate: a molecule of many functions. Annu. Rev. Biochem. 68: Kornberg, A., and C. D. Fraley Inorganic polyphosphate: A molecular fossil come to life. ASM News 66: Gómez-García, M. R., and A. Kornberg Formation of an actin-like filament concurrent with the enzymatic synthesis of inorganic polyphosphate. Proc. Natl. Acad. Sci. USA 101: Sureka, K., S. Dey, P. Datta, A. K. Singh, A. Dasgupta, S. Rodrigue, J. Basu, and M. Kundu Polyphosphate kinase is involved in stress-induced mprab-sige-rel signalling in mycobacteria. Mol. Microbiol. 65: Zhang, H., M. R. Gómez-García, X. Shi, N. N. Rao, and A. Kornberg Polyphosphate kinase 1, a conserved bacterial enzyme, in a eukaryote, Dictyostelium discoideum, with a role in cytokinesis. Proc. Natl. Acad. Sci. USA 104: Volume 3, Number 3, 2008 / Microbe Y 123