RESUMEN
The opportunistic pathogen Pseudomonas aeruginosa is a leading cause of nosocomial infections, which are becoming increasingly difficult to treat due to antibiotic resistance. Polyphosphate (polyP) plays a key role in P. aeruginosa virulence, stress response, and antibiotic tolerance, suggesting an attractive drug target. Here, we show that the small molecule gallein disrupts polyphosphate homeostasis by inhibiting all members of both polyphosphate kinase (PPK) families (PPK1 and PPK2) encoded by P. aeruginosa, demonstrating dual-specificity PPK inhibition for the first time. Inhibitor treatment phenocopied ppk deletion to reduce cellular polyP accumulation and attenuate biofilm formation, motility, and pyoverdine and pyocyanin production. Most importantly, gallein attenuated P. aeruginosa virulence in a Caenorhabditis elegans infection model and synergized with antibiotics while exhibiting negligible toxicity toward the nematodes or HEK293T cells, suggesting our discovery of dual-specificity PPK inhibitors as a promising starting point for the development of new antivirulence therapeutics. IMPORTANCE Many priority bacterial pathogens such as P. aeruginosa encode both PPK1 and PPK2 enzymes to maintain polyphosphate homeostasis. While PPK1 and PPK2 have distinct structures and catalytic mechanisms, they are both capable of synthesizing and consuming polyphosphate; thus, PPK2 enzymes can compensate for the loss of PPK1 and vice versa. In this study, we identified the small molecule gallein as a dual-specificity inhibitor of both PPK1 and PPK2 enzyme families in P. aeruginosa. Inhibitor treatment reduced cellular polyP in wild-type (WT), Δppk1, and Δppk2 strains to levels that were on par with the Δppk1 Δppk2A Δppk2B Δppk2C knockout control. Treatment also attenuated biofilm formation, motility, toxin production, and virulence to a similar extent, thereby elucidating a hitherto-undocumented role of PPK2 enzymes in P. aeruginosa virulence phenotypes. This work therefore establishes PPK2s, in addition to PPK1, as valuable drug targets in P. aeruginosa and provides a favorable starting molecule for future inhibitor design efforts.
Asunto(s)
Antibacterianos/farmacología , Inhibidores Enzimáticos/farmacología , Fosfotransferasas (Aceptor del Grupo Fosfato)/antagonistas & inhibidores , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/patogenicidad , Xantenos/farmacología , Animales , Antibacterianos/uso terapéutico , Caenorhabditis elegans/efectos de los fármacos , Caenorhabditis elegans/microbiología , Inhibidores Enzimáticos/uso terapéutico , Células HEK293 , Humanos , Fenotipo , Fosfotransferasas (Aceptor del Grupo Fosfato)/clasificación , Infecciones por Pseudomonas/tratamiento farmacológico , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/enzimología , Virulencia/efectos de los fármacos , Xantenos/uso terapéuticoRESUMEN
We designed novel quantitative real-time polymerase chain reaction (qPCR) primers for the polyphosphate kinase 1 (ppk1) gene, targeting eight individual "Candidatus Accumulibacter" (referred to as Accumulibacter) clades. An evaluation of primer sets was conducted regarding the coverage, specificity, and PCR efficiency. (i) All primer sets were designed to cover all available sequences of the target clade. (ii) The phylogenetic analysis of the sequences retrieved from the qPCR products by each primer set demonstrated a high level of specificity. (iii) All calibration curves presented high PCR efficiencies in the range of 85-112% (R(2) = 0.962-0.998). In addition, the possible interference of non-target amplicons was individually examined using the qPCR assay for 13 Accumulibacter clades, which were either undetected or showed negligible detection. With the primers designed by other research groups, a highly selective and sensitive qPCR-based method was developed to quantify all Accumulibacter clades, with the exception of Clade IE, in one assay, which enables more comprehensive insights into the community dynamics. The applicability to environmental samples was demonstrated by profiling the Accumulibacter clades in activated sludge samples of nine full-scale wastewater treatment plants.
Asunto(s)
Fosfotransferasas (Aceptor del Grupo Fosfato)/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Cartilla de ADN/química , Cartilla de ADN/metabolismo , ADN Bacteriano/aislamiento & purificación , ADN Bacteriano/metabolismo , Bases de Datos Factuales , Biblioteca de Genes , Funciones de Verosimilitud , Fosfotransferasas (Aceptor del Grupo Fosfato)/clasificación , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Filogenia , Microbiología del SueloRESUMEN
Polyphosphate (poly P) is a polymer of up to several hundred phosphate residues and is important to a variety of cell processes. The main poly P synthetic enzyme in many bacteria is poly P kinase 1 (PPK1), which until recently had been detected among eukaryotes in some protists only. There is now evidence for the presence in several other eukaryotes of PPK1 homologues and also a second bacteria-type enzyme, PPK2. The latest genome databases reveal that the 'Kornberg' enzyme complex of three actin-related proteins, termed DdPPK2 in Dictyostelium discoideum, might also be ubiquitous in eukaryotes. Owing to the intimate association of poly P synthesis with the formation of structural fibres, this ubiquity indicates a central role for this molecule in the evolution of eukaryotic cells.