Abstract
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.
Similar content being viewed by others
References
Abadi AT, Mobarez AM, Bonten MJ, Wagenaar JA, Kusters JG (2014) Clinical relevance of the cagA, tnpA and tnpB genes in Helicobacter pylori. BMC Gastroenterol 9:14–33
Battle SE, Meyer F, Rello J, Kung VL, Hauser AR (2008) Hybrid pathogenicity island PAGI-5 contributes to the highly virulent phenotype of a Pseudomonas aeruginosa isolate in mammals. J Bacteriol 190:7130–7140
Battle S, Rello J, Hauser A (2009) Genomic islands of Pseudomonas aeruginosa. Microbiology 290:70–78
Cervantes C, Ohtake H, Chu L, Misra TK, Silver S (1990) Cloning, nucleotide sequence and expression of the chromate resistance determinant of Pseudomonas aeruginosa plasmid pUM505. J Bacteriol 172:287–291
Dong X, Mindrinos M, Davis KR, Ausubel FM (1991) Induction of Arabidopsis defense genes by virulent and avirulent Pseudomonas syringae strains and by a cloned virulence gene. Plant Cell 3:61–72
Fajardo A, Martínez M, Mercadillo M, Galán J, Ghysels B, Matthijs S, Cornelis P, Wiehlmann L, Tümmler B, Baquero F, Martínez J (2008) The neglected intrinsic resistome of bacterial pathogens. PLoS ONE 3(2):e1619
Feltman H, Schulert G, Khan S, Jain M, Peterson L, Hauser AR (2001) Prevalence of type III secretion genes in clinical and environmental isolates of Pseudomonas aeruginosa. Microbiology 147:2659–2669
Green MR, Sambrook J (2012) Molecular cloning: a laboratory manual, 4th edn. Cold Spring Harbor Laboratory Press, New York
Harrison E, Carter M, Luck S, Ou H, He X, Deng Z, O´Callaghan C, Kadioglu A, Rajakumarkumar K (2010) Pathogenicity Islands PAPI-1 and PAPI-2 contribute individually and synergistically to the virulence of Pseudomonas aeruginosa strain PA14. Infect Immun 78:1437–1446
He J, Baldini RL, Déziel E, Saucier M, Zhang Q, Liberati NT, Lee D, Urbach J, Goodman HM, Rahme LG (2004) The broad host range pathogen Pseudomonas aeruginosa strain PA14 carries two pathogenicity islands harboring plant and animal virulence genes. Proc Natl Acad Sci USA 101:2530–2535
Hester CM, Lutkenhaus J (2007) Soj (ParA) DNA binding is mediated by conserved arginines and is essential for plasmid segregation. Proc Natl Acad Sci USA 18:20326–20331
Lee DG, Urbach JM, Wu G, Liberati NT, Feinbaum RL, Li L, Grills G, Montgomery K, Kucherlapati R, Rahme L, Ausubel F (2006) Genomic analysis reveals that Pseudomonas aeruginosa virulence is combinatorial. Genome Biol 7:R90
Li LL, Malone JE, Iglewski BH (2007) Regulation of the Pseudomonas aeruginosa quorum-sensing regulator VqsR. J Bacteriol 189:4367–4374
Liebert CA, Hall RM, Summers AO (1999) Transposon Tn21, flagship of the floating genome. Microbiol Mol Biol Rev 63:507–522
Rahme L, Stevens E, Wolfort S, Shao J, Tompkins R, Ausubel F (1995) Common virulence factors for bacterial pathogenicity in plants and animals. Science 268:1899–1902
Rahme LG, Tan MW, Le L, Wong SM, Tompkins RG, Calderwood SB, Ausubel FM (1997) Use of model plant hosts to identify Pseudomonas aeruginosa virulence factors. Proc Natl Acad Sci USA 94:13245–13250
Ramírez-Díaz MI, Díaz-Pérez C, Vargas E, Riveros-Rosas H, Campos-García J, Cervantes C (2008) Mechanisms of bacterial resistance to chromium compounds. Biometals 21:321–332
Ramírez-Díaz MI, Díaz-Magaña A, Meza-Carmen V, Johnstone L, Cervantes C, Rensing C (2011) Nucleotide sequence of Pseudomonas aeruginosa conjugative plasmid pUM505 containing virulence and heavy-metal resistance genes. Plasmid 66:7–18
Secretaría de Agricultura, Ganadería, Desarrollo Rural, Pesca y Alimentación (SAGARPA) (2001) NORMA Oficial Mexicana NOM-062-ZOO-1999. Especificaciones técnicas para la producción, cuidado y uso de los animales de laboratorio. Diario Oficial de la Federación 2:107–167 (In Spanish)
Siddavattam D, Khajamohiddin S, Manavathi B, Pakala SB, Merrick M (2003) Transposon-like organization of the plasmid-borne organophosphate degradation (opd) gene cluster found in Flavobacterium sp. Appl Environ Microbiol 69:2533–2539
Silby MW, Winstanley C, Godfrey SA, Levy SB, Jackson RW (2011) Pseudomonas genomes: diverse and adaptable. FEMS Microbiol Rev 35:652–680
Sousa AM, Pereira MO (2014) Pseudomonas aeruginosa diversification during infection development in cystic fibrosis lungs-a review. Pathogens 18:680–703
Vázquez-Rivera D, González O, Guzmán-Rodríguez J, Díaz-Pérez AL, Ochoa-Zarzosa A, López-Bucio J, Meza-Carmen V, Campos-García J (2015) Cytotoxicity of cyclodipeptides from Pseudomonas aeruginosa PAO1 leads to apoptosis in human cancer cell lines. Biomed Res Int. doi:10.1155/2015/197608
West SE, Schweizer HP, Hricová K, Uvízl R, Neiser J, Blahut L, Urbánek K (1994) Construction of improved Escherichia-Pseudomonas shuttle vectors derived from pUC18/19 and sequence of the region required for their replication in Pseudomonas aeruginosa. Gene 148:81–86
Wolfgang MC, Kulasekara BR, Liang X, Boyd D, Wu K, Yang Q, Miyada CG, Lory S (2003) Conservation of genome content and virulence determinants among clinical and environmental isolates of Pseudomonas aeruginosa. Proc Natl Acad Sci USA 100:8484–8489
Acknowledgments
This work was supported by grants from Coordinación de la Investigación Científica (UMSNH; 2.6, 2.35), IDCA-PRODEP (9305) and Consejo Nacional de Ciencia y Tecnología (CONACYT; 181747, 167071). ERA, KCHR, ADM and SPDP were supported by postgraduate fellowships from CONACYT.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Rodríguez-Andrade, E., Hernández-Ramírez, K.C., Díaz-Peréz, S.P. et al. Genes from pUM505 plasmid contribute to Pseudomonas aeruginosa virulence. Antonie van Leeuwenhoek 109, 389–396 (2016). https://doi.org/10.1007/s10482-015-0642-9
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10482-015-0642-9