RESUMO
Pseudomonas aeruginosa plasmid pUM505 possesses a pathogenicity island that contains the pumAB genes that encode products with sequence similarity to Toxin-Antitoxin (TA) modules. RT-PCR assays on the overlapping regions of the pumAB genes generated a bicistronic messenger RNA, suggesting that they form an operon. When the pumAB genes were cloned into the pJET vector, recombinant plasmid pJET-pumAB was maintained under nonselective conditions in Escherichia coli cells after six daily subcultures, whereas pJET without pumAB genes was lost. These data indicate that pumAB genes confer post-segregational plasmid stability. In addition, overexpression of the PumA protein in the E. coli BL21 strain resulted in a significant growth inhibition, while BL21 co-expressing the PumA and PumB proteins did not show growth inhibition. These results indicate that pumAB genes encode a TA system where the PumB protein counters the toxic effects of the PumA toxin. Furthermore, P. aeruginosa PAO1 transformants with the pumA gene increased Caenorhabditis elegans and mouse mortality rate and improved mouse organ invasion, effects neutralized by the PumB protein. Moreover, purified recombinant His-PumA protein decreased the viability of C. elegans, indicating that the PumA protein could acts as a toxin. These results indicate that PumA has the potential to promoter the PAO1 virulence against C. elegans and mice when is expressed in absence of PumB. This is the first description, to our knowledge, of a plasmid-encoded TA system that confers plasmid stability and encoded a toxin with the possible ability to increase the P. aeruginosa virulence.
Assuntos
Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidade , Genes Bacterianos/genética , Plasmídeos/genética , Pseudomonas aeruginosa/genética , Sistemas Toxina-Antitoxina/genética , Fatores de Virulência/genética , Animais , Antitoxinas/genética , Proteínas de Bactérias/genética , Sequência de Bases , Caenorhabditis elegans/efeitos dos fármacos , Modelos Animais de Doenças , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Vetores Genéticos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Óperon/genética , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/mortalidade , Pseudomonas aeruginosa/patogenicidade , RNA Bacteriano/análise , Proteínas Recombinantes/genética , Análise de Sequência , Virulência/genéticaRESUMO
The pUM505 plasmid was isolated from a clinical strain of Pseudomonas aeruginosa. This plasmid contains a genomic island with sequence similar to islands found in chromosomes of virulent P. aeruginosa clinical isolates. The objective of this work was to determine whether pUM505 increases the virulence of P. aeruginosa and to identify the genes responsible for this property. First, using the lettuce-leaf model, we found that pUM505 significantly increases the virulence of P. aeruginosa reference strain PAO1. pUM505 also increased the PAO1 virulence in a murine model and increased cytotoxicity of this strain toward HeLa cells. Thus, we generated a pUM505 gene library of 103 clones in the pUCP20 binary vector. The library was transferred to Escherichia coli TOP10 and P. aeruginosa PAO1 to identify genes. The lettuce-leaf model allowed us to identify three recombinant plasmids that increased the virulence of both E. coli and P. aeruginosa strains. These recombinant plasmids also increased the virulence of the PAO1 strain in mice and induced a cytotoxic effect in HeLa cells. Eleven genes were identified in the virulent transformants. Of these genes, only the pUM505 ORF 2 has homology with a gene previously implicated in virulence. These results indicate that pUM505 contains several genes that encode virulence factors, suggesting that the plasmid may contribute directly to bacterial virulence.
