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Drosophila melanogaster-based screening for multihost virulence factors of Pseudomonas aeruginosa PA14 and identification of a virulence-attenuating factor, HudA.

ABSTRACT: Pseudomonas aeruginosa is an important opportunistic human pathogen that interacts with phylogenetically diverse nonmammalian hosts, including plants, nematodes, and insects. Here, we exploited the P. aeruginosa-induced killing of the fruit fly Drosophila melanogaster as an assay system to screen for virulence-attenuated mutants of P. aeruginosa PA14. Fifteen nonredundant mutants were isolated from 4,018 random transposon (TnphoA) insertion clones, and 13 out of them (86.7%) displayed significantly reduced virulence in a murine peritonitis model as well. The TnphoA insertion sites of the 15 mutants were determined; already known virulence genes (dsbA, pvdI, fhlB, pilF, and wspF) and new virulence genes such as PA0253 (hudR), PA0369, PA2077, PA0272, PA2113, PA2965 (fabF1), and PA2002 were identified; one insertion was located at the intergenic region between PA1928 and PA1929; and the other two insertions were located in the genes (PA14_35740 and PA14_36000) within a putative genomic island, indicating a potential pathogenicity island of PA14. Further characterization of hudR, a virulence gene which encodes a MarR/SlyA family transcription factor, revealed that elevated expression of PA0254 (hudA [homologous to UbiD]) was necessary and sufficient for the virulence attenuation of the hudR mutant. The HudR protein repressed the hudAR operon by directly binding to its upstream promoter region. Collectively, these results validate the relevance of the D. melanogaster model for the high-throughput identification of new virulence factors involved in the multihost pathogenesis of P. aeruginosa.

SUBMITTER: Kim SH

PROVIDER: S-EPMC2519450 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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Drosophila melanogaster-based screening for multihost virulence factors of Pseudomonas aeruginosa PA14 and identification of a virulence-attenuating factor, HudA.

Kim Seol-Hee SH Park Shin-Young SY Heo Yun-Jeong YJ Cho You-Hee YH

Infection and immunity 20080630 9

Pseudomonas aeruginosa is an important opportunistic human pathogen that interacts with phylogenetically diverse nonmammalian hosts, including plants, nematodes, and insects. Here, we exploited the P. aeruginosa-induced killing of the fruit fly Drosophila melanogaster as an assay system to screen for virulence-attenuated mutants of P. aeruginosa PA14. Fifteen nonredundant mutants were isolated from 4,018 random transposon (TnphoA) insertion clones, and 13 out of them (86.7%) displayed significan ...[more]

PMID: 18591226

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Project description:Pseudomonas aeruginosa commonly infects hospitalized patients and the lungs of individuals with cystic fibrosis. This species is known for forming biofilms, which are communities of bacterial cells held together and encapsulated by a self-produced extracellular matrix. The matrix provides extra protection to the constituent cells, making P. aeruginosa infections challenging to treat. We previously identified a gene, PA14_16550, which encodes a DNA-binding TetR-type repressor and whose deletion reduced biofilm formation. Here, we assessed the transcriptional impact of the 16550 deletion and found six differentially regulated genes. Among them, our results implicated PA14_36820 as a negative regulator of biofilm matrix production, while the remaining 5 had modest effects on swarming motility. We also screened a transposon library in a biofilm-impaired ΔamrZ Δ16550 strain for restoration of matrix production. Surprisingly, we found that disruption or deletion of recA increased biofilm matrix production, both in biofilm-impaired and wild-type strains. Because RecA functions both in recombination and in the DNA damage response, we asked which function of RecA is important with respect to biofilm formation by using point mutations in recA and lexA to specifically disable each function. Our results implied that loss of either function of RecA impacts biofilm formation, suggesting that enhanced biofilm formation may be one physiological response of P. aeruginosa cells to loss of either RecA function. IMPORTANCE Pseudomonas aeruginosa is a notorious human pathogen well known for forming biofilms, communities of bacteria that protect themselves within a self-secreted matrix. Here, we sought to find genetic determinants that impacted biofilm matrix production in P. aeruginosa strains. We identified a largely uncharacterized protein (PA14_36820) and, surprisingly, RecA, a widely conserved bacterial DNA recombination and repair protein, as negatively regulating biofilm matrix production. Because RecA has two main functions, we used specific mutations to isolate each function and found that both functions influenced matrix production. Identifying negative regulators of biofilm production may suggest future strategies to reduce the formation of treatment-resistant biofilms.

Dataset Information

Drosophila melanogaster-based screening for multihost virulence factors of Pseudomonas aeruginosa PA14 and identification of a virulence-attenuating factor, HudA.

Publications

Drosophila melanogaster-based screening for multihost virulence factors of Pseudomonas aeruginosa PA14 and identification of a virulence-attenuating factor, HudA.

Similar Datasets

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