Polyphosphate is an extracellular signal that can facilitate bacterial survival in eukaryotic cells

Polyphosphate is a linear chain of phosphate residues and is present in organisms ranging from bacteria to humans. Pathogens such as Mycobacterium tuberculosis accumulate polyphosphate, and reduced expression of the polyphosphate kinase that synthesizes polyphosphate decreases their survival. How polyphosphate potentiates pathogenicity is poorly understood. Escherichia coli K-12 do not accumulate detectable levels of extracellular polyphosphate and have poor survival after phagocytosis by Dictyostelium discoideum or human macrophages. In contrast, Mycobacterium smegmatis and Mycobacterium tuberculosis accumulate detectable levels of extracellular polyphosphate, and have relatively better survival after phagocytosis by D. discoideum or macrophages. Adding extracellular polyphosphate increased E. coli survival after phagocytosis by D. discoideum and macrophages. Reducing expression of polyphosphate kinase 1 in M. smegmatis reduced extracellular polyphosphate and reduced survival in D. discoideum and macrophages, and this was reversed by the addition of extracellular polyphosphate. Conversely, treatment of D. discoideum and macrophages with recombinant yeast exopolyphosphatase reduced the survival of phagocytosed M. smegmatis or M. tuberculosis. D. discoideum cells lacking the putative polyphosphate receptor GrlD had reduced sensitivity to polyphosphate and, compared to wild-type cells, showed increased killing of phagocytosed E. coli and M. smegmatis. Polyphosphate inhibited phagosome acidification and lysosome activity in D. discoideum and macrophages and reduced early endosomal markers in macrophages. Together, these results suggest that bacterial polyphosphate potentiates pathogenicity by acting as an extracellular signal that inhibits phagosome maturation.

In metazoans, cells such as macrophages use phagocytosis to obtain nutrients, remove cell debris, and engulf and kill pathogens (1). Phagocytosis begins by recognition of particles by cell-surface receptors and engulfment of the ingested particle to form a phagosome. Ingested material in the phagolysosome is then degraded by lysosomal acid, enzymes, and oxygen radicals (2, 3). Many successful pathogens, including Mycobacterium tuberculosis, Legionella pneumophila, Neisseria gonorrhoeae, and Streptococcus pyogenes, have evolved countermeasures to evade phago-lysosomal killing, allowing the pathogen to live in the arrested phagosome (4–8). One commonly used countermeasure is to inhibit phagosome acidification and fusion with lysosomes (9–11). How pathogens inhibit this process is poorly understood.Polyphosphate is a linear chain of phosphate residues present in all kingdoms of life (12). Polyphosphate metabolism is associated with the virulence of pathogens such as Burkholderia mallei, M. tuberculosis, Salmonella enterica, Shigella flexneri, and Pseudomonas aeruginosa (13–17). In a wide range of bacteria, including pathogens, polyphosphate is synthesized from ATP by an essential enzyme polyphosphate kinase (PPK), and polyphosphate levels are maintained by exopolyphosphatase (PPX), an enzyme that degrades polyphosphate by removing terminal phosphate residues (18–22). PPK mutants of many pathogens display decreased growth, reduced sensitivity to stress and starvation, decreased survival, reduced invasiveness, defects in quorum sensing, and defects in other features associated with virulence (13, 14, 23, 24). However, the role of PPK in the survival of pathogenic bacteria is not clearly understood.The eukaryotic social amoeba D. discoideum uses phagocytosis to uptake nutrients such as bacteria (25). D. discoideum cells accumulate extracellular polyphosphate, and, as the local cell density increases, the extracellular polyphosphate concentration increases (26). To anticipate starvation when a colony of cells is about to overgrow its food supply, the concomitant high extracellular polyphosphate concentration inhibits proliferation (26). The G-protein–coupled receptor glutamate receptor-like protein D (GrlD) binds, and helps cells sense, polyphosphate (26, 27).Since not digesting nutrients might be a way to inhibit D. discoideum proliferation, we examined whether polyphosphate might inhibit phagosome maturation in D. discoideum and human macrophages. In this report, we show that both exogenous polyphosphate and polyphosphate released from bacteria inhibit phagosome maturation in D. discoideum and that this effect is conserved in human macrophages.